Plant Alkaloid Engineering

Plant Alkaloid Engineering
Fumihiko Sato, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; Graduate School of Science, Osaka Prefecture University,
Sakai, Japan
© 2019 Elsevier Inc. All rights reserved.
1 Outline 5
2 Introduction 5
2.1 Instrumental and Analytical Advances 6
2.1.1 Mass spectrometry (MS) 6
2.1.2 Stable isotope labeling 7
2.1.3 Mass spectrometry imaging (MSI) 7
2.1.4 Nuclear magnetic resonance (NMR) 7
2.2 Outlines of Metabolic Engineering and Synthetic Biology 7
3 Isolation and Identification of Molecular Tools in Pathway Engineering 8
3.1 General Background 8
3.2 Coexpression Analysis to Isolate Candidate Genes in Alkaloid Biosynthesis 8
3.2.1 Transcriptome analysis of metabolite producing and nonproducing tissues/cells or plant varieties
with different metabolite accumulation 8
3.2.2 Induced expression of biosynthetic enzyme genes in secondary metabolism for gene isolation 9
3.3 Comparative Genomics and Gene Cluster Analysis Based on the Sequence Homology 9
3.3.1 Homology search based on the transcriptome data 9
3.3.2 Coexpression analysis and comparative genomics based on genome sequence 10
3.3.3 Gene family analyses based on genome sequence 10
3.4 Key Enzymes in Alkaloid Biosynthesis 10
3.4.1 Amine–aldehyde condensation by Pictet-Spengler catalyzing enzymes 11
3.4.2 Berberine bridge enzyme (BBE) and flavin-binding proteins 13
3.4.3 Cytochrome P450s (P450s) 13
3.4.4 Carbocation-mediated cyclization; iridoid synthase (IS) 15
3.4.5 Glucosidases 15
3.4.6 Scaffold modifying methyltransferases (MTs) 16
3.4.7 Dioxygenases 17
3.5 Additional Molecular Option for Pathway Engineering 17
3.5.1 Molecular tools obtained from nonalkaloid producing organisms 17
3.5.2 Protein engineering based on the 3D-structure of enzyme and/or with mutagenesis 18
3.6 Transcription Factors 19
3.6.1 APETALA2 (AP2)/ethylene response factors (ERFs) 19
3.6.2 Basic helix-loop-helix (bHLH) family; MYC2 and non-MYC2 type 20
3.6.3 WRKY 20
3.6.4 Posttranslational regulation of TFs in secondary metabolism 20
3.7 Genes for Transport, Accumulation and Storage 20
3.7.1 ABC transporters 20
3.7.2 Antiporter and multidrug and toxic compound extrusion protein (MATE) 21
3.7.3 Nitrate/peptide family transporter (NPF) 21
3.7.4 Purine permease (PUP) 21
3.8 Progresses in Engineering Methods 21
3.8.1 General strategies for metabolic engineering; over-expression or silencing of gene using stable transformation 21
3.8.2 Transient system to evaluate the possibility of metabolic engineering 23
3.8.3 Genome editing 24
3.8.4 Heterologous expression of biosynthetic enzyme genes in microbe 24
3.9 Synthetic Biology 25
3.9.1 Strategy for synthetic biology; pathway identification, metabolic modeling, pathway engineering, metabolic control
network, and drug discovery 25
3.9.2 Metabolon and design of gene fusion protein 25
3.10 Feeding of Substrate and Combination with Chemical Strategy 26
4 Engineering for Alkaloid Production 26
4.1 Engineering in Isoquinoline Alkaloid (IQA) Biosynthesis 26
4.1.1 Biosynthetic enzymes and their genes in the pathway 26
4.1.2 Cell specific expression 30
Comprehensive Natural Products III: Chemistry and Biology https://doi.org/10.1016/B978-0-12-409547-2.14696-7 1

2 Plant Alkaloid Engineering
4.1.3 Transport and accumulation 31

4.1.4 Metabolic engineering 31
4.1.5 Metabolic engineering with transcription factor 32
4.1.6 Synthetic biology 32
4.1.7 Enzymatic production; single or stepwise conversion 35
4.2 Engineering in Terpenoid Indole Alkaloid Biosynthesis 36
4.2.1 Outline of biosynthesis 36
4.2.2 Cell specific expression and transcription factors 38
4.2.4 Synthetic biology 39
4.3 Nicotine and Tropane Alkaloids 39
4.3.1 Enzymes, transcription factors and other components involved in biosynthesis 40
4.4 Purine Derived Alkaloids 43
4.4.1 Biosynthetic enzymes and genes 43
4.4.2 Metabolic engineering and synthetic biology 44
4.5 Steroidal Glycoalkaloids 44
4.5.1 Biosynthetic enzymes and metabolic engineering 45
4.5.2 Steroidal glycoalkaloids from Liliaceae 45
5 Future Perspectives 45
6 Summary 46
Acknowledgments 46
References 46
Abbreviations
10OMT 10-hydroxydihydrobenzophenanthridine alkaloid 10-O-methyltransferase
12OMT 12-hydroxydihydrobenzophenanthridine alkaloid 12-O-methyltransferase
16T3O 16-methoxytabersonine 3-oxygenase
2ODDs 2-oxoglutarate/Fe(II)-dependent dioxygenases
3,4-DHBA 3,4-dihydroxybenzaldehyde
3,4-DHPAA 3,4-dihydroxyphenylacetaldehyde
40 OMT N-methylcoclaurine 40 -O-methyltransferase
4HPAA 4-hydroxyphenylacetaldehyde
5bR Progesterone 5b-reductase
6OMT Norcoclaurine 6-O-methyltransferase
7OMT Reticuline 7-O-methyltransferase
ADC Arginine decarboxylase
AKR Aldo-keto reductase
AMT Accurate mass-time
AO Aspartate oxidase
AP2 APETALA2
AT Acetyl transferase
BA Benzyladenine
BAC Bacterial artificial chromosome
BBE Berberine bridge enzyme
BBL BBE-like
BGCs Biosynthetic gene clusters
bHLH Basic helix-loop-helix
BIA Benzylisoquinoline alkaloid
BIS1/2 bHLH Iridoid synthesis 1 and 2
bp Base pairs
CaMV35S Cauliflower mosaic virus 35S
CAS or CYP719A1 Canadine synthase
CDS Coding sequence
CEX Carboxylesterase
CheSyn or CYP719A5 Cheilanthiforine synthase
Cj Coptis japonica
Plant Alkaloid Engineering 3
CM/PDH Chorismate mutase/prephenate dehydrogenase

CNMT Coclaurine N-methyltransferase
CODM Codeine O-demethylase
COR Codeinone reductase
CPRs Cytochrome P450 oxidoreductases
CPT Camptothecin
Cr or C. roseus Catharunthus roseus
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
CYP Cytochrome P450
CYP719A2/A3 Stylopine synthase/canadine synthase
CYP80B1 N-methyl norcoclaurine hydroxylase
D4H Desacetoxyvindoline-4-hydroxylase
DAHPS 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase
DAO Diamine oxidase
DAT Acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase
DB10H Dihydrobenzophenanthridine alkaloid 10-hydroxylase
DB12H Dihydrobenzophenanthridine alkaloid 12-hydroxylase
DBOX Dihydrobenzophenanthridine alkaloid oxidase
DHBO Copper-dependent dihydrobenzophenanthridine oxidase
DIMBOA 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
DLH Deoxyloganic acid hydroxylase
DMAPP Dimethylallyl pyrophosphate
DODC DOPA specific decarboxylase
E. coli Escherichia coli
EC Enzyme Commission
Ec Eschscholzia californica
ERFs Ethylene Response Factors
ESI-FT-ICR Electro Spray Ionization-Fourier transform-ion cyclotron resonance
EST Expressed sequence tag
fbr Feedback inhibition resistant
FPP Farnesyl pyrophosphate
G8H Geraniol 8-hydroxylase
GABA g-aminobutyric acid
GAME9 Glycoalkaloid metabolism 9
GES Geraniol synthase
GO Gene ontology
GOR 8-hydroxygeraniol oxidoreductase
GPP Geranyl pyrophosphate
H6H Hyoscyamine 6b-hydroxylase
HMGR HMG CoA reductase
IDI1 IPP:DMAPP isomerase
IMP Inosine-50 -monophosphate
IO 7-deoxyloganetic acid synthase/iridoid oxidase
IPP isopentenyl pyrophoshate
IQA Isoquinoline alkaloids
IRS Iridodial synthase
IS Iridoid synthase
JAT Jasmonate (JA)-inducible alkaloid transporter
JID JAZ-interacting domain
KEGG Kyoto Encyclopedia of Genes and Genomes
LAMT Loganic acid methyltransferase
LC Liquid chromatography
m/z Mass-to charge ratio
MAO Monoamine oxidase
MAT Minovincine 19-O-acetyltransferase
MATE Multidrug and toxic compound extrusion protein
MDR Multidrug resistance
MeJA Methyl jasmonate
MIAs Monoterpenoid indole alkaloids
MND Morphinan N-demethylase

MPO N-methylputrescine oxidase
MS Mass spectrometry
MSH or CYP82N5 N-methylstylopine 14-hydroxylase
MSI Mass spectrometry imaging
N Unknown nucleotide
N40 OMT Norbelladine 40 -O-methyltransferase
NBS Norbelladine synthase
NCS Norcoclaurine synthase
NEPS Nepetalactol-related short-chain dehydrogenase
NGS Next-generation sequencing
NISO Neopinone isomerase
NMCH N-methylcoclaurine N-methyltransferase
NMR Nuclear magnetic resonance
NMTs N-methyltransferases
NPF Nitrate/peptide family transporter
NR Noroxomaritidine reductase
NUP Nicotine uptake permease
ODC Ornithine decarboxylase
OMT O-methyltransferase
P450 Cytochrome P450
P6H or CYP82N2v2 Protopine 6-hydroxylase
P7ODM Papaverine 7-O-demethylase
PAC Henylacetylcarbinol
PCD Pterin carbinolamine dehydratase
PCR Polymerase chain reaction
PE Paired-end
PEG Polyethylene glycol
PEPS Phosphoenolpyruvate synthetase
PGA Potato glycoalkaloid biosynthesis
PIP Pinoresinol-lariciresinol reductase/isoflavone reductase/phenylcoumaran benzylic ether reductase
PMT Putrescine N-methyltransferase
PNPs Plant natural products
Ps Papaver somniferum
PTPS 6-Pyruvoyl-tetrahydropterin synthase
PUP Purine permeases
PYKS Polyketide synthase
QDHPR Quinonoid dihydropteridine reductase
QPT Quinolinic acid phosphoribosyl transferase
QS Quinolinate synthase
RNAi RNA interference
RT Reverse transcription
SAT or SalAT Acetylcoenzyme A:salutaridinol-7-O-acetyltransferase
SAM S-adenosyl methionine
SAR or SalR Salutaridine:NADPH 7-oxidoreductase
SAS, SalS, or CYP719B1 Salutaridine synthase
SBE Sarpagan bridge enzyme
SDR Short-chain alcohol dehydrogenase
SepR Sepiapterin reductase
SGAs Steroidal glycoalkaloids
SGD Strictosidine b-glucosidase
SLS Secologanin synthase
SMT or SOMT Scoulerine 9-O-methyltransferase
SPDS Spermidine synthase
SSR2 Sterol side chain reductase 2
STORR or REPI [(S)- to (R)-reticuline] or reticuline epimerase
STOX (S)-tetrahydroprotoberberine oxidase
STR Strictosidine synthase
StySyn or CYP719A2/A3 Stylopine synthase
T16H Tabersonine 16-hydroxylase

T19H Tabersonine 19-hydroxylase
T3O Tabersonine 3-oxidase
TALEN Transcription activator-like effector nuclease
TAm Transaminase
TAs Tropane alkaloids
TAT Tabersonine derivative 19-O-acetyltransferase
TDC Tryptophan decarboxylase
TEX Tabersonine 6,7-epoxidase
TH Tyrosine hydroxylase
THBO Tetrahydroberberine oxidase
THS Thebaine synthase
TIA Terpenoid indole alkaloid
TKT Transketolase
T6ODM Thebaine 6-O-demethylase
TNMT Tetrahydroprotoberberine N-methyltransferase
TPI Triosephoshate isomerase
TPT Catharanthine transporter
TR Tropinone reductase
TRV Tobacco rattle virus
TYDC Tyrosine/dopa decarboxylase
TYR Tyrosinase
TyrAT Tyrosine aminotransferase
VIGS Virus-induced gene silencing
WHO World Health Organization
ZCT Zinc-finger C. roseus TF
1 Outline
Plant alkaloids are rich resources for the pharmaceuticals. Characterization and identification of biosynthetic enzymes and enzyme
genes enable the metabolic engineering and synthetic biology of biosynthetic pathways to produce higher amounts of desired
metabolites and/or creation of novel metabolites for drug discovery.
2 Introduction
Higher plants produce a wide range of low molecular weight specialized chemicals of over 200,000, so called secondary
metabolites; so far, >30,000 terpenoids, and about 20,000 alkaloids and 8000 phenolic substances have been identified.1,2
These metabolites are composed of structurally diversified chemicals, which are classified as phenolics, terpenoids, alkaloids and
others, mainly based on the nature of chemicals and origins of biosynthesis. Whereas their physiological roles in plants are still
under investigations, these metabolites are often produced on the response to environmental stresses, such as high irradiation,
pathogen attach, wounding, insect and cattle feeding to protect plant body, because plants have no mean to escape physically from
these damages and stresses. Thus, these chemicals have protective characteristics against oxidative, digestive, destructive stresses.1–3
Thus, these chemicals are often poisonous, but also used to promote human health as pharmaceuticals, and also as dietary
supplements and functional foods.2,4–8
Alkaloids, alkaline nitrogen-containing, and often heterocyclic compounds, are most biologically active, and used pharmaceu-
ticals among these chemicals. World Health Organization (WHO) listed many plant alkaloids as key chemicals; e.g., atropine,
caffeine, codeine, ephedrine, hyoscine (scopolamine), morphine, quinine, vinblastine, and vincristine (Fig. 1) in “Model list of
essential medicines 20th ed. 2017” (http://www.who.int/medicines/publications/essentialmedicines/en/). Whereas we can chem-
ically synthesize many pharmaceuticals (e.g.,9–11), plant-derived pharmaceuticals are still commonly harvested from natural
resources, either field growing or artificially cultivated ones, due to their complicated and stereospecific natures of chemicals.12
These plant secondary metabolites are, however, often produced at low quantities and also as a mixture of structurally similar
chemicals in plants. Thus, more considerable advances in methodology for the production of these chemicals are desired. Whereas
breeding of superior plant variety, optimization of cultivation or greenhouse cultivation of selected cultivar and also cell or tissue
cultures are investigated,12–14 recent progresses in the molecular biology in plant metabolism, especially biosynthetic enzyme genes
and engineering technology, open the new horizon of metabolic engineering and synthetic biology for secondary metabolite
production and pathway engineering in plants and also in microbes (see later section, and Ref. 8).
Morphine (IQA) Codeine (IQA) Ephedorine (IQA)
R=CH3, Vinblastine (MIA) Quinine (MIA)

R=CHO, Vincristine (MIA)
Caffeine
Atropine (TA) Hyoscine (scopolamine) (TA)

Fig. 1 Alkaloids in model list of essential medicines of WHO 20th ed. IQA, isoquinoline alkaloid; MIA, monoterpenoid indole alkaloid; TA, tropane alkaloid.
On the other hand, the alkaloid families are so diverse due to the diversified origin (precursors) of biosynthesis, in comparison
with terpenoids or phenolics, which are synthesized common precursor, such as isopentenyl pyrophosphate or phenylala-
nine.1,15,16 Therefore, investigation of alkaloid engineering is more fragmented and difficult. Then, detailed discussion on the
pathway engineering should be done based on the origin of alkaloids as follows; tyrosine-derived isoquinoline alkaloids (IQAs;
often benzylisoquinoline alkaloids, BIAs), tryptophan-derived monoterpenoid indole alkaloids (MIAs), putrescine-derived tropane
alkaloids (TAs) and nicotines, and some others, in which their molecular information are sufficiently accumulating for
engineering.8,17–20 In this review, steroidal glycoalkaloids (SGAs), such as tomatine and solanine in tomato and potato, are also
included, because their recent molecular characterization of biosynthesis based on genome sequence is useful model for the future
progresses in the pathway engineering, whereas SGAs are pseudoalkaloids and not true alkaloids derived from amino acids.
Since the progresses in the genomics and transcriptomics based on next-generation sequencing (NGS) are so rapid with the
advances in instrumental analyses of chemicals, frequent update of this field is requested based on the past good review
articles.8,17,21–23
Before go to the detailed discussion of metabolic engineering and synthetic biology, some progresses in instrumental and
analytical measurements are summarized (also see24).
2.1 Instrumental and Analytical Advances

2.1.1 Mass spectrometry (MS)
The development of high-resolution mass spectrometry (MS), especially combination with liquid chromatography (LC) such as
LC-MS and LC-MS/MS provides powerful method to characterize and identify low molecular weight compounds with similar
chromatographic properties. For example, accurate mass-time (AMT) tag approach is now available for the LC-MS based identifi-
cation of plant natural products (PNPs) in complex extracts.25 The LC-MS and MS/MS data sets were integrated into online spectral
search tools and repositories (Spektraris and MassBank; Table 1), thus researchers can interrogate their own data sets for the
potential presence of PNPs. The utility of the AMT tag library approach was used for the annotation of active principles in
27 different medicinal plant species with diverse chemical constituents.25
Table 1 Useful URL for plant alkaloid engineering.
Tools for natural products and metabolic pathways
Spektraris (a collection of tools for identifying plant natural products) http://langelabtools.wsu.edu/

spektraris/
MassBank (database of comprehensive, high-resolution mass spectra of metabolites) http://www.massbank.jp
Plant Metabolic Network (PMN) (plant metabolic pathway databases, including PlantCyc, which provides access to manually http://www.plantcyc.org/
curated or reviewed information about shared and unique metabolic pathways present in over 350 plant species)
KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway map https://www.genome.jp/kegg/
Genetic Sequence Database (an annotated collection of all publicly available DNA sequences): GenBank at NCBI, DNA DataBank of Japan (DDBJ), and the European
Nucleotide Archive (ENA) at EMBL-EBI comprise the International Nucleotide Sequence Database Collaboration
GenBank https://www.ncbi.nlm.nih.gov/genbank/
DDBJ https://www.ddbj.nig.ac.jp/index.html
ENA https://www.ebi.ac.uk/ena
Medicinal plant transcriptome database
PhytoMetaSyn Project www.phytometasyn.ca
Tools for sequence analysis
RefSeq (a comprehensive, integrated, nonredundant, well-annotated set of reference sequences including genomic, https://www.ncbi.nlm.nih.gov/refseq/
transcript and protein)
InterPro (InterPro provides functional analysis of proteins by classifying them into families and predicting domains and https://www.ebi.ac.uk/interpro/
important sites)
Protein data bank (PDB) (biomolecular structure database) https://www.rcsb.org
Gene Ontology (GO provides the tool to predict the molecular functions, cellular locations, and processes gene products) http://geneontology.orge
EsPASy (enzyme nomenclature database) https://enzyme.expasy.org
Uniprot (comprehensive, high-quality and freely accessible resource of protein sequence and functional information) https://www.uniprot.org
plantiSMASH (tool to find densely clustered biosynthetic gene clusters (BGCs)) http://plantismash.
secondarymetabolites.org
List of P450s http://drnelson.uthsc.edu/biblioD.html
2.1.2 Stable isotope labeling

With the development of high-resolution MS, identification of metabolites is much easier and informative to trace the pathway.
Especially, stable isotope labeling become easy tool for the characterization of metabolic pathway and also for flux analysis. For
example, [ring-13C6]-tyramine was fed to opium poppy (Papaver somniferum) seedlings to monitor morphinan alkaloid biosynthesis
and incorporation of the labeled tyramine into about 20 alkaloids was elucidated both by direct infusion high-resolution Electro
Spray Ionization-Fourier transform-ion cyclotron resonance (ESI-FT-ICR)-MS and LC-electrospray tandem MS.26
2.1.3 Mass spectrometry imaging (MSI)

Imaging mass analysis is new technology to determine the localization of metabolites in planta. Whereas in situ hybridization
analysis of messenger RNAs expressed show the localization of the biosynthesis of metabolites, actual abundance of metabolites is
usually not clear, since some metabolites such as nicotine and tropane alkaloids are transferred and accumulate at the different cells
from the biosynthesis site. MSI of sectioned tissues now provides useful information about the abundance of precursor and
products.8,27
2.1.4 Nuclear magnetic resonance (NMR)

Beside MS, improvement in NMR hardware and methodology considerably increased the efficacy of NMR based profiling of plant
natural products with much smaller amount (such as under 1 mg) of sample needed for identification and characterization.28 Even
1
H NMR revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content29 and useful
platform for a functional genomics.30
2.2 Outlines of Metabolic Engineering and Synthetic Biology

It was not so long time ago, when we could artificially modify the plant metabolic pathway or re-produce plant metabolites in
heterologous system. First metabolic engineering was done in Atropa belladonna with Hyoscyamus hyoscyamine 6b-hydroxylase
(H6H) over-expression,31 just after the isolation of the key gene (cDNA) of H6H from Hyoscyamus niger. Whereas this first proof-of-
concept experiment was very successful, such simple over-expression of biosynthetic enzyme gene was not so fruitful, because the
bottlenecks in biosynthesis exist at several points. So, gene mining and characterization of metabolic pathway is critical and endless
processes, whereas with the progresses in NGS, coexpression analysis of metabolite profiles (metabolomes) and transcriptomes, and
considerable progresses in molecular and cellular biology facilitated the gene mining in pathway, which enabled the large extension
of metabolic engineering and even synthetic biology of metabolic pathway.
Besides the expansion of gene collection in secondary metabolism, transformation and engineering techniques are also rapidly
developed. For example, finding of gene silencing enhanced the possibility of pathway engineering and also facilitate the
identification of gene. One of the successful application of RNA silencing is the production of blue rose, which was established
with ectopic expression of viola flavonioid 30 50 -hydroxylase, a key enzyme for blue delphinidin biosynthesis and silencing of
endogenous dihydroflavonol 4-reductase, which is more favorable for the production of native red anthocyanin.32 However, gene
silencing also brought another pitfall, because the shutdown of pathway increased the accumulation of intermediates, and increased
the new flow of metabolites over the barrier of enzyme substrate affinity.33 Recent progresses in genome editing with CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 or other editing tools further open the possibility for the
pathway engineering.34–37
Whereas the transformation and regeneration of plants for the metabolic engineering are still big obstacles to improve the
efficiency of engineering in nonmodel medicinal plants, recent progresses in genome editing also provide alternative path to
overcome the difficulty of stable transformation. Whereas microbombardment and protoplast transformation are low efficient
methods due to multigene copy integration and low stable transformation efficiency, these methods would be promising tool for
transient expression for genome editing tools to modify the target gene.38,39 Especially, swapping of gene using homologous
recombination would be most promising method for the metabolic engineering and synthetic biology in future.
When the progresses in molecular techniques in the pathway engineering are evident, the isolation of target enzyme gene is the
real obstacle.40 Therefore, the identification and characterization of biosynthetic enzyme genes is firstly discussed in next section.
3 Isolation and Identification of Molecular Tools in Pathway Engineering

3.1 General Background
The availability of engineering tools, i.e., biosynthetic enzyme genes and regulatory factor genes, is exponentially growing due to the
progresses in molecular biology including NGS. Progresses in NGS enable low cost RNA sequencing and even genome sequence
analysis for the accumulation of nucleotide sequence data in public database domain such as GenBank at NCBI, ENA at EMBL-EBI
and DDBJ (Table 1).
However, we should note that our current knowledge on the key enzyme in secondary metabolism is limited due to the low
expression of transcripts of enzymes with the low and spatially and temporally specialized activity in plant and the redundancy of
enzyme genes. That is, we still need empirical characterization to isolate and identify the desired genes in each specialized pathway,
whereas some genes involved in secondary metabolism in plants are clustered41,42; i.e., the candidate gene in cluster was estimated
in secondary metabolism of potato, tomato, Madagascar periwinkle, opium poppy and so on.43–45 But, it is also noted that the gold
mine of gene cluster is not so common.46
Since biochemical purification and characterization have been well established and used to isolate some key enzymes (e.g.,47,48),
updated methods are briefly summarized as below.
3.2 Coexpression Analysis to Isolate Candidate Genes in Alkaloid Biosynthesis

Comprehensive coexpression analyses of the transcripts (transcriptome) and metabolites (metabolome) using metabolite produc-
ing and nonproducing tissues/cells or plant varieties with different metabolite accumulation are now widely used to isolate the
candidate genes of the biosynthetic enzymes after the more focused analysis.19,49–55
3.2.1 Transcriptome analysis of metabolite producing and nonproducing tissues/cells or plant varieties
with different metabolite accumulation
High metabolite-accumulating cells such as high berberine-producing Coptis japonica cells was efficiently used to isolate several key
enzyme genes in BIA biosynthesis, such as CYP719A1,56 CYP82G257 and so on after the transcriptome analysis. Analysis of
transcriptome of epidermal cell, site of MIA biosynthesis, of C. roseus was also used to isolate MIA biosynthetic enzyme genes.19
Out of 2000 expressed sequence tags obtained from P. somniferum, a gene, exhibiting increased expression in morphinan
alkaloid containing species, high sequence similarity to reductase and similar expression pattern to previously isolated cDNAs
coding for enzymes in BIA biosynthesis, was identified to salutaridine reductase (SalR; EC 1.1.1.24858). Similarly, analysis of cDNAs
on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species
identified three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species and a putative
O-methyltransferase was identified as S-adenosyl-L-methionine: (R,S)-30 -hydroxy-N-methylcoclaurine 40 -O– methyltransferase
(4’OMT; EC 2.1.1.11654).
More integrated approach using targeted metabolite profiles and EST libraries established for BIA-producing cell cultures of
Eschscholzia californica, Papaver bracteatum and Thalictrum flavum allowed identification and functional characterization of four
N-methyltransferases (NMTs). One cDNA from T. flavum, pavine N-methyltransferase (TfPavNMT), showed a unique preference for
(+/−)-pavine and represents the involvement in pavine pathway.59
Comparison of transcript and metabolite profiles of eight opium poppy chemotypes revealed that four cytochrome P450s, i.e.,
three CYP82s and one CYP719, were tightly correlated with noscapine accumulation. The combined biochemical and physiological
data support that CYP82Y1 catalyzed 1-hydroxylation of (S)-N-methylcanadine in the noscapine pathway.60
3.2.2 Induced expression of biosynthetic enzyme genes in secondary metabolism for gene isolation
Coexpression analysis using transcriptome and metabolome data is useful when mutants or specific cells with different metabolite
accumulation (more correctly, different ability of biosynthesis) are available. But, secondary metabolism is usually expressed at low
level and sparse in plant. Therefore, another approaches to prepare metabolite producing or nonproducing cells are desired; one of
commonly used methods is treatment of secondary metabolism-inducing chemicals (e.g., elicitors such as microbial cell wall or
methyl-jasmonate; MeJA) to activate secondary metabolism.61–64 Or, the modulation of certain enzyme gene or transcription factor
gene might be used to induce the variation of biosynthetic activity and metabolite profiles among plant or cultured cells55,65: for
example, the ectopic expression of Coptis japonica scoulerine-9-O-methyltransferase (CjSMT) in E. californica enhanced the diversi-
fication of the BIA profile in transgenic cells due to the modification of metabolic flow and somaclonal variation. After the
diversification, the corelationship between product, allocryptopine and protopine 6-hydroxylae (P6H; EC 1.14.13.55) was
characterized.65,66
Physiological relevance to use elicitation for the isolation of the biosynthetic enzymes of MIAs was validated on C. roseus leaves
treated with Manduca sexta larvae. The transcriptomic and metabolic analyses showed that C. roseus respond to herbivore by both
local and systemic processes with the activation of specific gene sets and biosynthesis of distinct MIAs after the JA production.67
Whereas the JAs are critical regulator in plant defense against herbivores and induce secondary metabolite production in
response, we should be careful to use them, because JAs include several derivatives and the conjugate jasmonoyl isoleucine (JA-
Ile) induce some different physiological responses from JAs.68
Coexpression analysis is now more efficiently used on the basis of genome sequence. Identification of GLYCOALKALOID
METABOLISM 9 (GAME9), an APETALA2 (AP2)/Ethylene Response Factor (ERF) in steroidal glycoalkaloids (SGAs) biosynthesis is
excellent example to use genome information for coexpression analysis.69 Further reverse genetic approach such as GAME9
knockdown and overexpression in tomato and potato supports the involvement of GAME9 in SGAs and upstream mevalonate
pathways including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2).
Whereas coexpression analysis is powerful to predict the candidate genes involved in the biosynthesis of secondary metabolites,
it is not sufficient to identify the gene function. Thus, the biochemical characterization of biosynthetic enzymes expressed in
heterologous host cells, or the reverse genetic approaches using over-expression or down-regulation of target genes are used to
identify the gene function. Or, characterization of peptide sequence of purified enzyme as well as proteomics analysis of protein
extracts provide complementary support, when heterologous expression is not successful. Using this proteomic approach, a
representative enzyme in morphine biosynthesis was detected within the serum fraction of latex from opium poppy.70
3.3 Comparative Genomics and Gene Cluster Analysis Based on the Sequence Homology
With the progresses in molecular characterization of biosynthetic enzyme genes as mentioned in previous Sections 3.1 and 3.2,
considerable molecular information about the biosynthetic enzyme and regulatory genes in secondary metabolism is accumulating.
Therefore, prediction of the gene candidate based on the sequence homology from transcriptome data becomes much easier.
3.3.1 Homology search based on the transcriptome data

Several transcriptome data for medicinal plants are now available. The 1000 plants (1KP) project, an international multi-
disciplinary consortium, was prepared for over 1000 plant species, which cover all of the major lineages across the Viridiplantae
(green plants) clade. These data can be analyzed with the data additionally uploaded by users.71 Data for 75 nonmodel plants that
produce biotechnologically interested compounds were also available at PhytoMetaSyn Project (www.phytometasyn.ca).72 The
sequences were annotated using RefSeq, InterPro, Gene Ontology (GO), Enzyme Commission (EC) annotations and associated
with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps (Table 1). Whereas biosynthetic enzymes listed in the
Plant Metabolic Network (http://www.plantcyc.org/) or KEGG are limited, homology search is useful to predict the candidate genes,
especially in BIA biosynthesis, in which many genes were annotated as discuss below.
3.3.1.1 BIA pathway

Hagel et al.73 analyzed RNA from 20 species representing four families within the Ranunculales by NGS to generate RNA sequence
libraries with de novo assembly and subsequent full-length coding sequence (CDS) predictions. Homology-based search using BIA-
biosynthetic enzymes yielded 850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis provides
information for the selection of gene candidates. Phylogenetic analysis was performed for each of 15 different enzyme/protein
groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including
morphinan, aporphine, and phthalideisoquinoline alkaloids.
Transcriptomic analysis of a high noscapine–producing poppy variety (HN1) revealed the exclusive expression of 10 genes
encoding five distinct enzyme classes. Analysis of an F2 mapping population indicated that these genes are tightly linked in HN1,
and bacterial artificial chromosome (BAC) sequencing confirmed that they exist as a complex gene cluster, which showed sequence
similarity with known genes in BIA biosynthesis.74
Similarly, candidate genes involved in IQA biosynthesis in Dactylicapnos scandens (D. Don) Hutch (Papaveraceae), a traditional
Chinese herb used for treatment of hypertension, inflammation, bleeding and pain for centuries was reported based on the
sequence similarity.75
Phenylalkylamines, such as ephedrine and pseudoephedrine, are produced in Ephedra sinica Stapf (Ephedraceae) from phenyl-
alanine in similar manner with BIAs, whereas BIAs are produced from tyrosine. RNA sequences of Ephedra and khat were prepared
using NGS,76 and two EsAroAT1 and EsAroAT2 Kilpatrick et al.77 and phenylalkylamine N-methyltransferase (PaNMT)78 involved
in the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines in BIA biosynthesis were isolated.
3.3.1.2 MIA pathway

Transcriptome data generated by NGS was used to identify the penultimate hydroxylase and final O-methyltransferase steps in
ibogaine biosynthesis, which named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT).
Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) confirmed the predicted activities of
both enzymes.79
However, we should note that the homology-based search on gene candidate has pitfall. When highly homologous isoforms
were expressed, it is essential to remove redundant contigs during clustering and filtering steps and annotate the transcripts carefully;
e.g., redundant secologanin synthases (SLS), and tabersonine 16-hydroxylase (T16H1 and T16H2) needed such careful
inspections.80
3.3.2 Coexpression analysis and comparative genomics based on genome sequence

In bacteria, genes are often clustered as functional groups (operons), and cotranscribed by a signal. Whereas gene cluster had been
not considered in plants, now the presence of gene clusters is evident, particularly for the biosynthesis of specialized metabolites;
e.g., avenacins in oat, benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) in maize, noscapine and mor-
phinan alkaloid in P. somniferum, and SGAs in Solanaceae.41–45
After the finding of gene cluster in specialized metabolism, plantiSMASH (http://plantismash.secondarymetabolites.org) was
developed to find biosynthetic gene clusters (BGCs) (Table 1). This platform also allows integration of transcriptomic data to
prioritize candidate BGCs based on the coexpression patterns of predicted biosynthetic enzyme-coding genes, and facilitates
comparative genomic analysis to study the evolutionary conservation of each cluster. plantiSMASH identified a rich diversity of
candidate plant BGCs from 48 high-quality plant genomes.81
3.3.2.1 BIA pathway

Recent draft sequence of the opium poppy genome, with 2.72 gigabases indicated the cluster of biosynthetic genes for noscapine
and morphinan alkaloids of 15 genes.45 Recent determination of genome sequence of sacred lotus (Nelumbo nucifera Gaertn.),
which produces BIAs, also predicted biosynthetic enzyme genes for aporphine and bisbenzylisoquinoline type BIAs.82 The genome
assembly of Aquilegia coerulea 23 “Goldsmith” is also available.83
3.3.2.2 MIA pathway

Genome assembly for C. roseus facilitated high-resolution coexpression analyses and revealed three distinct clusters of coexpression
in MIA pathway. Genome sequence also revealed the localization of enzyme-rich genic regions and transporters near known
biosynthetic enzymes, tandem gene duplication and putative neofunctionalization.44
The gene cluster was also found in the genome of the oxindole MIA producer Gelsemium sempervirens (Gelsemiaceae) using a gene
cluster from C. roseus. The gene cluster contains at least six enzymatic steps downstream from the last common intermediate shared
between the two plant alkaloid types, although the corresponding enzymes act on entirely different substrates.84
3.3.3 Gene family analyses based on genome sequence

Genome information provides not only the platform for the coexpression analysis, but also molecular information about the genes
in hidden pathway. For example, Hori et al.46 determined the California poppy draft genome sequence (489 Mb of 502 Mb) and
characterized the cytochrome P450 enzyme-encoding genes. In silico analysis showed the unique abundance of CYP80, CYP82 and
CYP719 genes in this plant species (probably also in BIA producing plants) and identified novel P450 (CYP82P2 and CYP82P3),
which exhibited 10-hydroxylase activities in late step of more oxidized BIA, i.e., macarpine, biosynthesis.
3.4 Key Enzymes in Alkaloid Biosynthesis

Enzymes are key player in the biosynthesis and engineering. As mentioned above, the candidate enzyme genes in the biosynthesis
are considerably annotated based on the biochemical characterization, coexpression analysis and sequence homology search of
genes expressed in target cells. But, the exact functions of genes predicted by sequence-homology search should be characterized
biochemically or reverse genetically. In the past, biochemical purification was the only way to identify the enzymes and to isolate
candidate gene, we had commonly faced the difficulty to purify enzyme, due to the time consuming purification processes, the low
quantities, instability and also the contamination of other proteins with similar properties (e.g.,85,86). Therefore, recombinant
proteins expressed from the candidate gene in heterologous host, such as E. coli, yeast, Pichia, insect cells, or plants, which usually do
not produce target metabolites, are used to determine biological function of candidate genes.
Microbial production of recombinant protein is well established, but it is still difficult to express some membrane proteins such
as cytochrome P450s (P450s) in large amounts. Also, some enzymes, such as flavin-binding enzymes, e.g., berberine bridge enzyme
(BBE) or tetrahydroberberine oxidase (THBO), have some difficulties to express in E. coli or yeast.86 Furthermore, excess production
of recombinant protein also often makes protein insoluble. Thus, we need more information about the optimal conditions for the
recombinant protein expression and characterization. When it is not easy to express recombinant protein in microbial cells,
transient expression in plant cells with virus-vector system with Agro-infiltration might be effective alternative to evaluate the
enzyme activities,87 especially with the combination of other biosynthetic enzyme genes to reconstitute the metabolic pathway.88
Since the understanding of chemical logics of enzymes is key for the expression of recombinant protein and reconstruction of
metabolic pathways, the features of key enzymes are summarized in this section. Biosynthetic enzymes are classified to scaffold-
generating ones and tailoring reactions that convert common scaffolds to more structurally diversified products.89–91 The key
scaffold-generating enzymes are amine–aldehyde condensation promoted by Pictet-Spenglerases, such as norcoclaurine synthase
(NCS) or strictosidine synthase (STR). Additionally, key scaffolds are generated by CdC bond formation through radical coupling
such as berberine bridge enzyme (BBE) or corytuberine synthase (CYP80G2), noncanonical oxidation of amino acids (diamine
oxidase), aryl-CoA acylation, and carbocation-mediated cyclization such as iridoid synthase.
3.4.1 Amine–aldehyde condensation by Pictet-Spengler catalyzing enzymes

The condensation of amine and aldehyde discovered by Pictet and Spengler is the most important reaction to construct stereo-
chemically and structurally complex alkaloids,92 whereas several enzymes are known in this reaction.
3.4.1.1 Norcoclaurine synthase (NCS) and related enzymes

(S)-Norcoclaurine is the entry compound in BIA biosynthesis and produced by the condensation of dopamine and
4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS; EC 4.2.1.78) (Fig. 2). cDNA of NCS, which shows
substantial identity with the pathogenesis-related (PR) 10 family protein, has been isolated from Thalictrum flavum (TfNCS96),
Papaver somniferum (PsNCS1 and PsNCS297), and Coptis japonica (CjPR10A98) and their enzyme activities were confirmed with
recombinant protein in E. coli and yeast. Physiological relevance of PR10-like NCS was confirmed by the silencing of NCS in opium
poppy with large reduction of alkaloid levels and nearly complete immunoprecipitation of NCS activity with specific antibodies,97
whereas Minami et al.98 reported dioxygenease-like enzyme also catalyzed NCS reaction (see below).
3.4.1.1.1 PR-10 like NCS

Interestingly, members of the Papaveraceae family showed tandem domain repeats in predicted NCS translation products when
21 BIA-producing species of four plant families were searched. Functional analysis of six active NCS enzymes, including four
containing either two, three or four repeated catalytic domains showed that truncation of the first 25 N-terminal amino acids from
the remaining polypeptides revealed two additional enzymes and multiple catalytic domains correlated with a proportional
increase in catalytic efficiency. The metabolic conversion capacity of engineered yeast positively correlated with the number of
repeated domains.99
Analyses with holo X-ray crystal structure and on computational docking revealed the dopamine-first mechanism for TfNCS.94
Suppression of the nonenzymatic background reaction also revealed its low catalytic efficiency, indicating the ineffectiveness of
recombinant NCS in in vivo systems, and the function as a metabolic gatekeeper in planta. The amino acid substitution Leu76Ala,
situated in the proposed aldehyde-binding site, results in the alteration of the enzyme’s aldehyde activity profile.
NCS
R1=H, R2=H, R3=OH R1=H, R2=H, R3=OH

4-Hydroxyphenylaldehyde (S)-Norcoclaurine
R1=H, R2=OH, R3=OH R1=H, R2=OH, R3=OH
3,4-Dihydroxyphenylaldehyde (S)-Norlaudanosoline
R1=H, R2=H, R3=H TfNCS truncated R1=H, R2=H, R3=H
Phenylacetaldehyde R1=F, or CH3, R2=H, R3=H
R1=F, or CH3, R2=H, R3=H R1=H, R2=F or CH3, R3=H
R1=H, R2=F or CH3, R3=H Corresponding simple benzylisoquinolines
Fig. 2 Norcoclaurine synthase (PR-10 homolog); EC 4.2.1.78. and its reaction. X-ray crystal structure of NCS was confirmed by Ilari et al.93 and truncated structure
(5n8q.pdb1-500) was confirmed by Lichman et al.94 NCS was also used for chemical biosynthesis (see Refs. 95, 100–103 and also text).
Enzymatic strategies to produce (S)-norcoclaurine and its derivatives were reviewed by Ghirga et al.95 Chemical biosynthesis
using NCS is reported by several groups.100–103 N-terminal deletion involved in membrane anchoring might be useful for
heterologous expression.
3.4.1.1.2 Dioxygenase-like NCS

Beside PR10-like NCS (e.g., CjPR10A), a distinct dioxygenase-like NCS cDNA was isolated from C. japonica (CjNCS1). The
inhibition of both native NCS and recombinant CjNCS1, but not that of CjPR10A with o-phenanthroline (ferrous ion chelating
agent) indicated that CjNCS1 is the major NCS in C. japonica,94 whereas Lee and Facchini97 claimed that they could not detect NCS
activity for recombinant CjNCS1.
3.4.1.1.3 NCS homolog, which involved in other BIA biosynthesis (also see Section 3.3.1)
Norbelladine synthase (NBS) involved in Amaryllidaceae alkaloid biosynthesis was isolated from wild daffodil (Narcissus pseudo-
narcissus) database, using transcript search for NCS orthologs. Phylogenetic analysis showed that NpNBS belongs to a unique clade
of PR10 proteins and shared 41% amino acid identity to PsNCS1. Expression of NpNBS cDNA in E. coli produced a recombinant
enzyme able to condense tyramine and 3,4-DHBA into norbelladine.104
3.4.1.1.4 Other PR10s with distinct functions; Thebaine synthase and neopinone isomerase
Whereas thebaine formation was long presumed to be a spontaneous reaction to form thebaine from (7S)-salutaridinol 7-O-acetate.
Thebaine synthase (THS) activity was recently found in opium poppy latex and to be a member of PR10 superfamily.105 THS is
encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes
immediately upstream.
Very recently, an isomerization enzyme, neopinone isomerase (NISO), which convert neopinone to codeinone, was also found
to be PR10-superfamily, along with thebaine synthase,106 whereas these two enzymes did not show NCS activity.
3.4.1.2 Strictosidine synthase (STR)

Strictosidine synthase (STR) is another key Pictet-Spengler reaction in plant alkaloid biosynthesis, which stereoselectively synthe-
sizes a beta-carboline product, strictosidene, from tryptamine and secologanin in MIA biosynthesis (Fig. 3). Maresh et al.109
suggested that the enzyme efficiently catalyzed the rate-controlling acid-catalyzed step, most likely protonation of a carbinolamine
intermediate. Structural analysis suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for
productive cyclization to form the diastereoselective product.
The substrate requirements for STR have been systematically and quantitatively examined.110 STR accepts a variety of sub-
stitutions on the indole ring of tryptophan, as well as benzofuran and benzothiophene heterocycles, but not accept tryptophan,
phenylethylamine, and pyrrole derivatives. Also STR does not accept other naturally occurring iridoid aldehydes, but accept certain
semisynthetic derivatives of secologanin.
The stringent substrate specificity of STR is the key for the precursor-directed biosynthesis of unnatural alkaloids in C. roseus.
Although STR uses some analogs of tryptamine and secologanin, many substrates are not recognized by this enzyme, such as
tryptamine analogs with substituents. With a different expression system and screening method, STR variants that accept tryptamine
Fig. 3 Strictosidine synthase (STR) and its reaction. Cristal structure of strictosidine synthase isolated from Rauwolfia was determined by Ma et al.107 and then its
complex with 2-(1-methyl-1H-indol-e-yl)ethanamine (4imb.pdb1-500) determined by Zhu et al.108 was shown here.
analogs not turned over by the wild-type enzyme was identified111 and enzyme mutant (STR Val214Met) with broadened substrate
specificities for halogenated tryptamine was designed based on the crystal structure (see Sections 3.5.2 and 3.10112).
3.4.2 Berberine bridge enzyme (BBE) and flavin-binding proteins

3.4.2.1 BBE
Berberine bridge enzyme (BBE) catalyzes the conversion of the N-methyl group of (S)-reticuline into the methylene bridge moiety
of (S)-scoulerine113 and forms the scaffold in the biosynthesis of benzophenanthridines, noscapine and protoberberines, BIAs
(Fig. 4). BBE is vesicular located flavin adenine dinuclotide (FAD)-binding protein. FAD is covalently attached to enzyme as cofactor
at His and Cys residues and often difficult to produce in microbe.
BBE also oxidizes (S)-scoulerine to the deeply red protoberberine alkaloid dehydroscoulerine with four electron oxidation at a
much lower conversion rate.115 As discuss below, BBE-homologues, i.e., (S)-tetrahydroprotoberberine oxidase (STOX) and tetra-
hydroberberine oxidase/canadine oxidase (THBO), are simple oxidases.
BBE-Like protein in nicotine biosynthesis is known to be involved in the nicotine or nicotinic alkaloid formation (see
Section 4.3), whereas its biochemical characterization is not confirmed yet.
3.4.2.2 (S)-Tetrahydroprotoberberine oxidase (STOX or THBO) in the last step of berberine biosynthesis
While isolation of (S)-tetrahydroprotoberberine oxidase (STOX or THBO; EC 1.3.3.8) cDNA was once reported, its recombinant
protein expressed in E. coli showed triosephosphate isomerase (TPI) activity, and THBO activity was not detected85). Since STOX/
THBO enzyme was very labile during purification and also difficult to express recombinant protein, TPI contaminated in the
purified enzyme fraction was identified at the first report. Then, the intensive purification was conducted, and CjTHBO belongs to
the FAD-containing BBE family was isolated based on the partial amino acid sequence of purified enzyme and its function was
characterized in transgenic California poppy (Eschscholzia californica) cells.86 Transgenic California poppy cells with CjTHBO
showed modified accumulation of coptisine and dehydrocheilanthifoline.
On the other hand, STOXs isolated from Argemone mexicana and Berberis wilsoniae were expressed in Spodoptera frugiperda Sf9
insect cells,116 whereas these proteins were also very unstable. Enzyme activities were thus measured without enzyme extraction,
and rather broad substrate specificity of STOXs for canadine, tetrahydropalmatine and scoulerine were detected.116 DBOX in
benzophenanthridine and papaverine pathways, with homology to STOX, isolated from opium poppy, was however expressed in
Pichia pastoris.117
3.4.3 Cytochrome P450s (P450s)

The number of P450 genes in plant genomes is estimated up to 1% of the total gene annotations of each plant species, implying that
plants are huge sources for various P450-dependent reactions. Plant P450s catalyze a wide variety of monoxygenation/hydroxyl-
ation reactions in secondary metabolism, and some unusual reactions such as methylenedioxy-bridge formation generate increase
chemical diversity as scaffold modifier. But, P450s also catalyze phenol-coupling reactions, oxidative rearrangement of carbon
skeletons, and oxidative CdC bond cleavage and produce new chemical scaffolds118 (Fig. 5).
3.4.3.1 Scaffold generating P450s

3.4.3.1.1 CdC bond formation through radical coupling
Some P450s catalyze intramolecular CdC phenol-coupling and intermolecular CdO phenol-coupling reactions. CYP80A1
isolated from Berberis stolonifera is the first intermolecular CdO phenol-coupling P450, and involved in the biosynthesis of
berbamunine (a bisbenzylisoquinoline alkaloid).119 CYP80G2 is the first characterized a CdC phenol-coupling P450 cDNA
Fig. 4 Berberine bridge enzyme (BBE, EC 1.21.3.3) and its reaction. Crystal structure of BBE isolated from Eschscholzia californica was determined with (S)-
reticuline (3D2D.pdb1-500) by Winkler et al.114
Fig. 5 Some cytochrome P450s (see Section 3.4.3.1), which catalyze phenol-coupling reactions, oxidative rearrangement of carbon skeletons. Also unique P450
(i.e., STORR) which fused with reductase was recently identified to be involved in the (S)-reticuline to (R)-reticuline epimerization.
(CYP80G2) isolated from plants and produce (S)-corytuberine (an aporphine) from (S)-reticuline. Interestingly, CYP80G2 had
high amino acid sequence similarity to CYP80A1.57
Another CdC phenol-coupling P450 is salutaridine synthase (CYP719B1) involved in morphine biosynthesis in opium
poppy.120 In these reactions, new scaffolds are generated by radical coupling.
3.4.3.1.2 Hydroxylation enzymes also generate new scaffold

Whereas (S)-cis-N-methylstylopine 14-hydroxylase (MSH; CYP82N5121) and protopine 6-hydroxylase (P6H; CYP82N2v266) are
member of the CYP82N subfamily and introduce hydroxy-group in their reactions, such reactions generate protopine scaffold from
N-methylprotoberberine (e.g., N-methylstylopine) and benzophenanthridine scaffold (e.g., dihydrosanguinarine) from protopine
scaffold (Fig. 5). Similarly, CYP82X1 introduce hydroxy group at C8 of 1-hydroxy 13-acetoxyl-N-methycanadine to generate
phthalideisoquinoline scaffold in noscapine biosynthesis.122 Whereas these P450s catalyze hydroxylation reactions, the reaction
products formed are highly reactive to rearrange to new chemical structure, and would be classified as scaffold generating enzymes.
3.4.3.1.3 Hybrid P450; STORR (reticuline epimerase: REPI)

Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes
both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of
mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to
1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning
as a P450 redox partner. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein
STORR was the key enzyme for microbial-based morphinan production.123
Interestingly, this fusion protein, which named as reticuline epimerase (REPI), because it catalyzes the S-to-R epimerization of
reticuline via 1,2-dehydroreticuline, was isolated almost simultaneously from opium poppy and Papaver bracteatum, which
accumulates thebaine. Physiological function of REPI was confirmed using virus-induced gene silencing (VIGS) in opium poppy,
with the reduction of (R)-reticuline and morphinan alkaloids and the increase in the overall abundance of (S)-reticuline and its
O-methylated derivatives.124
3.4.3.2 Scaffold modifier

3.4.3.2.1 Hydroxylation enzymes
Many P450s have been identified to catalyze hydroxylation reaction in secondary metabolism. For example, CYP80B1 catalyzes 30 -
hydroxylation of N-methylcoclaurine in BIA pathway.125 Tabersonine 16-hydroxylase (T16H), encoded by CYP71D12 catalyzes
hydroxylation reaction at the C-16 position in tabersonine to vindoline reaction.126 In tabersonine metabolism in root, tabersonine
19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT) have been isolated.127
3.4.3.2.2 Methylene dioxy bridge enzymes

Methylene dioxy bridge is formed from a hydroxy and a methoxy group adjacently attached to benzene ring. First methylenedioxy
bridge enzyme gene was isolated from Coptis japonica, which produce canadine (tetrahyhdroberberine) from tetrahydrocolumba-
mine and named CYP719A1.56 Later on, two methylenedioxy bridge forming enzymes involved in (S)-stylopine synthesis for the
biosynthesis of benzophenanthridine alkaloids, such as sanguinarine, were identified from Eschscholzia californica. Whereas
recombinant EcCYP719A2 and EcCYP719A3 expressed in Saccharomyces cerevisiae produced stylopine from cheilanthifoline as
stylopine synthase, but not convert scoulerine to cheilanthifoline. On the other hand, similar but distinct EcCYP719A5 showed
cheilanthiforline synthase activity. Interestingly, enzymatic analysis showed that EcCYP719A2 had high substrate affinity only
toward (R, S)-cheilanthifoline, whereas EcCYP719A3 showed broader activity not only for (R,S)-cheilanthifoline, but also for (S)-
scoulerine, and (S)-tetrahydrocolumbamine.128 Among additional four kinds of EcCYP719A genes isolated from E. californica, a
CYP719A9 preferentially expressed in plant leaf showed methylenedioxy bridge-forming activity toward (R, S)-reticuline.129
AmCYP719A13 and AmCYP719A14, which showed 77% and 60% identity to EcCYP719A2 were isolated from Argemone.
AmCYP719A14 and AmCYP719A13 expressed in S. frugiperda Sf9 insect cells produced (S)-cheilanthifoline from (S)-scoulerine and
of (S)-stylopine from (S)-cheilanthifoline, respectively. Substrate specificity analysis indicated that although AmCYP719A14
participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in
A. mexicana.130 These results suggest that sequence similarity provide only limited reaction characteristics, and actual activity should
be examined experimentally.
Whereas many CYP719s show methylene dioxy ring formation, other clan of CYP family are also involved in methylene dioxy
ring formation; e.g., CYP81Q1 in lignan (sesamin) biosynthesis.131 Also, not all CYP719 shows methylene dioxy ring formation, as
CYP719B1 shows saltaridine synthase activity.120
3.4.3.3 Cytochrome P450 oxidoreductases (CPR)

P450s require a two-electron transfer from cytochrome P450 oxidoreductases (CPRs) for catalysis. CPR isoforms usually group into
two distinct classes; i.e., class I (CPR1) for primary and basal specialized metabolisms and class II (CPR2) for inducible specialized
metabolism. When the role of CPR isoforms in Catharanthus roseus was investigated, CPR2 was expressed in leaf and cotyledon
tissues hosting MIA biosynthetic steps catalyzed by P450s and silencing of CPR1 did not affect the biosynthesis of MIA, whereas
in vitro assay showed that both classes of CPR performed equally.132
3.4.4 Carbocation-mediated cyclization; iridoid synthase (IS)

Iridoid synthase (IS), an atypical reductive terpene synthase, activates its substrate 8-oxogeranial into a reactive enol intermediate to
produce the key secoiridoid secologanin in MIA pathway. IS belongs to a member of the progesterone 5b-reductase (P5bR) family
and six P5bR genes are reported in C. roseus. Characterization of recombinant CrP5bR proteins demonstrates that all but CrP5bR3
can reduce progesterone and thus can be classified as P5bRs. Recombinant P5bR proteins prepared from angiosperm plant species,
which are not known to produce iridoids, are also capable to use 8-oxogeranial as a substrate, suggesting that IS activity is intrinsic to
angiosperm P5bR proteins.133
IS catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent
cyclization into nepetalactol. A class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) was identified from
catmint (Nepeta mussinii) that catalyzes the stereoselective cyclisation into distinct nepetalactol stereoisomers.134
3.4.5 Glucosidases
Reactive aglycons are often stabilized by the glucosylation and released by specific glucosidase. In MIA biosynthesis, strictosidine
aglycon is released by strictosidine b-glucosidase (SGD) to generate variety of MIA precursor. Characterization of SGD from C. roseus
with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine
substrate. This SGD also hydrolyzes both strictosidine and its stereoisomer vincoside, indicating that this enzyme is not completely
diastereoselective, although the naturally occurring 3a(S)-epimer (strictosidine) is preferred.135
Deglucosylation of N-deacetylisoipecoside by b-D-glucosidase, IpeGlu1, spontaneously forms the highly reactive aglycon for
emetine biosynthesis in Carapichea ipecacuanha. IpeGlu1 lacked stereo-specificity for its substrates, whereas ipecoside 1b(R), a major
alkaloidal glucoside in C. ipecacuanha, was preferred to 1a(S)-epimers.136
3.4.6 Scaffold modifying methyltransferases (MTs)

3.4.6.1 O-methyltransferases (OMTs)
O-methylation of hydroxy group of secondary metabolites with S-adenosyl methionine (SAM) is critical to increase the stability of
chemicals and diversity due to the restriction of chemical reactions. This reaction is catalyzed by SAM-dependent
O-methyltransferases (OMTs), which have consensus signature for SAM binding. The manipulation of class I and V SAM-dependent
MTs including OMTs and the use of synthetic SAM analogs have been reviewed for rational redesign toward biotechnological
applications.137
OMTs are rather common enzyme in secondary metabolism and easy to annotate due to the consensus signature sequence, but
the identification of biosynthetic enzyme functions in pathway is rather difficult due to their redundancy and rather broad substrate
specificity. In fact, scoulerine 9-O-methyltransferase (SOMT, or SMT) in benzophenanthridine alkaloid biosynthesis of California
poppy showed broad OMT activity; i.e., not only for protoberberine scoulerine, but also for the simple benzylisoquinolines,
including reticuline and norreticline with dual regio-reactivities. That is, EcSOMT showed 9-O-methylation and 2-O-methylation for
scoulerine, and 7-O-methylation and 30 -O-methylation for simple benzylisoquinoline.138
In addition, OMTs isolated from Carapichea ipecacuanha showed considerable redundancy and variation of enzyme character-
istics. Whereas they had high sequence identity of 98%, IpeOMT2 catalyzed the 70 -O-methylation of 70 -O-demethycepaeline to form
cephaeline in emetine biosynthesis,139 but distinct CiOMT1 from IpeOMTs showed both 70 -O-methylation and 60 -O-methylation
activities at the last two steps of emetine biosynthesis, indicating that small differences in amino acid residues are responsible for
distinct regional methylation specificities.140
Based on the high sequence similarity, the possibility of creating a chimeric enzyme with novel substrate specificity has been
investigated using three OMT cDNAs involved in berberine biosynthesis. Chimeric OMT with an N-terminal half of 6-OMT and a
C-terminal half of 40 -OMT (640 -OMT) indicated that 640 -OMT retained the regio-specificity of 6-OMT with novel reactivity.141
As mentioned before, OMTs are easily annotated from transcriptome data with signature sequence. When cDNAs of Glaucium
flavum were searched with known OMTs involved in BIA biosynthesis, three OMTs were found to accept 1-benzylisoquinolines with
differential substrate and regio-specificity.142 GflOMT2 with the highest amino acid sequence identity with 6OMT, showed a
preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 30 and 40 hydroxyl groups of certain alkaloids.
GflOMT1 with the highest sequence identity with 40 OMT catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently
40 -O-methylated 6-O-methylnorlaudanosoline and its N-methylated 6-O-methyllaudanosoline. GflOMT1 also effectively 30 -O-
methylated both reticuline and norreticuline. GflOMT6 was most similar to Cjscoulerine 9-OMT and efficiently catalyzed both
30 - and 70 -O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes
accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine
using combinations of E. coli producing (1) GflOMT1, (2) either GflOMT2 or GflOMT6, and (3) coclaurine N-methyltransferase
from C. japonica.
3.4.6.2 N-methyltransferases (NMTs)

N-Methylation is one of the most common tailoring reactions, yielding tertiary and quaternary pathway intermediates and
products. However, NMTs show little sequence similarity, because they are evolved from distinct primary metabolic enzymes.
3.4.6.2.1 Putrescine N-methyltransferase (PMT)

Putrescine N-methyltransferase (PMT) catalyzes SAM-dependent methylation of the diamine putrescine in nicotine, tropane, and
nortropane alkaloid biosynthesis. PMT was firstly identified in nicotine biosynthesis. PMT sequences are resembled to those of plant
spermidine synthases (putrescine aminopropyltransferases) and show little similarity to other plant MTs. PMT is likely to have
evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein
structure; they bind the same substrate putrescine and similar cosubstrates, SAM and decarboxylated S-adenosylmethionine.143
3.4.6.2.2 NMTs in BIA and ephedrine biosynthesis

In BIA biosynthesis, NMTs accepting (i) early 1-benzylisoquinoline intermediates in (S)-reticuline biosynthesis (CNMT144) and
(ii) protoberberines in phthalideisoquinolines and antimicrobial benzo[c]phenanthridines (TNMT59; pavine NMT145), and (iii) 1--
benzylisoquinoline and aporphine (RNMT146), were characterized. NMTs in BIA biosynthesis showed sequence similarity to
choline NMT. NMT also show rather broad substrate specificity; CNMT showed NMT activity against simple tetrahydroisoquinoline
and corytuberine.47,57 Since CjCNMT has relatively broad substrate specificity, it can be used for chemical biosynthesis. The crystal
structure of CNMT was also determined recently with mutagenesis studies to define the enzyme active site architecture.147
The crystal structure of pavine NMT from Thalictrum flavum was determined with selenomethionine-substituted protein as well
as the native protein and binary complexes with SAM or the reaction product S-adenosylhomocysteine. Pavine NMT converted
racemic tetrahydropapaverine to laudanosine, but the enzyme showed a preference for ()-pavine and (S)-reticuline as
substrates.145
NMT from Ephedra sinica catalyzed the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines,
including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine NMT (PaNMT) shares substantial amino acid
sequence identity with the NMT family involved in BIA metabolism. PaNMT accepted a broad range of substrates with phenylalk-
ylamine, tryptamine, b-carboline, tetrahydroisoquinoline, and BIA structural scaffolds.78
3.4.6.2.3 NMT in MIA biosynthesis

A SAM-dependent NMT isolated from C. roseus in vindoline biosynthesis is most similar to the plastidic g-tocopherol
C methyltransferases of vitamin E biosynthesis.148
3.4.7 Dioxygenases
Whereas cytochromes P450 (CYPs) as often considered the most versatile oxidative enzymes, the 2-oxoglutarate/Fe(II)-dependent
dioxygenases (2ODDs) also catalyze many different reactions in plant metabolism, and their diversity and complexity of reactions
catalyzed is comparable to the CYPs. Their reactions include hydroxylations, demethylations, desaturations, ring closure, ring
cleavage, epimerization, rearrangement, halogenation, and demethylenation.149
Hyoscyamine 6b-hydroxylase isolated from Hyoscyamus niger is the first 2ODD isolated in alkaloid biosynthesis.150 H6H
catalyzes two-step epoxidation reactions from hyoscyamine to scopolamine. In BIA pathway, beside NCS reported in
C. japonica,94 two unique 2ODDs, i.e., thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), were identified
in morphine biosynthesis in opium poppy,151 whereas cytochrome P450s function in mammals.
Biochemical characterization of CODM and T6ODM and the functionally unassigned paralog DIOX2, named protopine
O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,
O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups. CODM and protopine O-dealkylase
preferred protopine alkaloids for O, O-demethylenation.152 Additionally, a 2ODD, papaverine 7-O-demethylase (P7ODM) isolated
from opium poppy, catalyzes the efficient substrate- and regio-specific 7-O-demethylation of papaverine to pacodine, tri-O-
methylated analogs of papaverine.153
3.5 Additional Molecular Option for Pathway Engineering

3.5.1 Molecular tools obtained from nonalkaloid producing organisms
In the synthetic biology, we can use any option to reconstruct and optimize the pathway. Whereas metabolite-producing cells are
commonly used as the host, even nonproducing cells can be used as the host to reconstitute the pathway with ant available and
necessary genes. Even if biosynthetic steps in secondary metabolism are not characterized at molecular level, we can recruit
molecular tools from other resources to reconstruct the pathway.
3.5.1.1 Substrate supply in BIA biosynthesis (see also Fig. 6)

Whereas late pathway has usually intensively investigated in specialized metabolism, early steps are often not so clarified yet. For
example, the formation of DOPA from tyrosine in BIA biosynthesis in plant is postulated to be catalyzed by phenolase and a CYP
has been reported as hydroxylation enzyme, actual enzyme is not clarified yet. Thus, bacterial tyrosinase enzyme is used to generate
DOPA from tyrosine to reconstruct BIA pathway. But this enzyme has additional o-diphenolase activity and produces byproduct
with the consumption of substrate. Therefore, specific bacterial enzyme from Streptomyces castaneoglebiosporus with low
o-diphenolase was firstly used, but later more specific Ralstonia solanacearum enzyme with less o-diphenolase activity was selectively
used.154
Or, mammalian tyrosine hydroxylase (TH) without o-phenolase activity was selectively used, whereas this tyrosine hydroxylase
needs tetrahydrobiopterin as cofactor. In this option, additional four mammalian tetrahydrobiopterin biosynthesis and recycling
enzymes are also included with the expression of mammalian tyrosine hydroxylase.155,156
Production of aldehyde substrate in NCS reaction is another hurdle for the reconstruction of BIA biosynthesis. Bacterial
monoamine oxidase (MAO) was successfully used to produce 3,4-dihydroxyphenylacetaldehyde for NCS reaction to skip later
hydroxylation in reticuline biosynthesis.157 Whereas MAO is powerful enzyme, excess activity of MAO also brings another problem
to produce excess aldehyde, which induces spontaneous condensation with dopamine to form racemic norlaudanosoline. MAO
also brings another pitfall, because MAO reacts with tyrosine to form 4-hydroxyphenylacetaldehyde (4HPAA), which forms
norcoclaurine with dopamine in NCS reaction. These problems would be controlled with fine-tuning of MAO expression and
also use of bacterial DOPA specific decarboxylase (DODC) from Pseudomonas putida158 instead of plant tyrosine/DOPA decarbox-
ylase in the reconstruction in E. coli.156,157 DOPA specific decarboxylase was also used to avoid the loss of substrate tyrosine in
yeast155 (also see Section 4.1.6).
3.5.1.2 Modification enzymes in BIA pathway

3.5.1.2.1 Mammalian enzymes
Whereas whole morphinan alkaloid biosynthetic enzyme genes were identified, some mammalian enzymes involved in morphinan
alkaloid biosynthesis might be useful for the morphine biosynthesis. In fact, human P450s 2D6 and 3A4 catalyzed the phenol-
coupling reaction using (R)-reticuline and NADPH to produce salutaridine, whereas CYP719B1 catalyzes this reaction in opium
poppy. Human CYP2D6 and 3A4 also produced mixture of (−)-isoboldine, (−)-corytuberine, (+)-pallidine, and salutaridine from
(R)-reticuline, and yielded (+)-isoboldine, (+)-corytuberine, (−)-pallidine, and sinoacutine from (S)-reticuline.159 Rat CYP2D2 also
generated corytuberine, pallidine, salutaridine and isoboldine from (R)-reticuline, while it showed the 3-O-demethylation with
thebaine and codeine.160
H3CO
OH
HO O NCH3
O HO
COOH N
H H
HO
NH2 HO
HO 3,4-dihydroxy
benzaldehyde Norbelladine HO
Tyrosine
NBS Galanthamine
TH or Tyramine
Tyrosinase
PO
TAT 4HPP
HO COOH
NH2 Aro10p (in yeast) Berbamunine

HO
HPDC (a bisbenzylisoquinoline)
O
TDC CYP80A1
DODC H Plant pathway
HO
4HPAA HO H3CO
HO NH 6OMT NCH3
HO
HO
H HO
H Various
NH2 CNMT
HO NCS CYP80B1
BIAs
dopamine HO H3CO (see Fig. 7)
(S)-Norcoclaurine (S)-Reticuline
MAO (in E. coli ) NCS HO
NH 6OMT
HO O HO CNMT
H
HO
H
HO
3,4DHPAA HO
(S)-Norlaudanosoline
Microbial reconstituted pathway
Fig. 6 Early pathway in IQA biosynthesis and some IQAs, Tyrosine hydroxylase (TH) or bacterial tyrosinase can bypath the early dopamine production in microbial
systems, PO, phenol oxidase; TAT, tyrosine aminotransferase; TDC, tyrosine decarboxylase; NCS, norcoclaurine synthse; CNMT, coclaurine N-methyltransferase;
6OMT, norcoclaurine 6-OMT; CYP80B1, N-methylcoclaurine 30 -hydroxylase; 4’OMT, 30 -hydroxy N-methycoclaurine 40 -OMT; NBS, norbelladine synthase; 4HPP,
4-hydroxyphenylpyruvate; 4HPAA, 4-hydroxyphenylacetaldehyde.
A search in the human genome for MTs also identified NMT (EC 2.1.1.49) that converted all four benzylisoquinolines with a
strict preference for (R)-configured morphine precursors.161
3.5.1.2.2 Bacterial enzymes for further modification

Catabolic reaction of secondary metabolites in microbes might be used to modify the chemical structure of target metabolite. The
enzyme, which forms demethyleneberberine from berberine, was isolated from berberine-utilizing bacteria. Interestingly, this
demethyleneberberine forming enzyme is tetrahydrofolate dependent and similar to a sesamin (a lignan) demethylenase (SesA),
isolated from the sesamin-metabolizing bacterium Sinomonas sp. no. 22, and distinct from CYP2D6, CYP1A2 and CYP3A4
responsible for this reaction in human liver microsome.162
Recently, Augustin et al.163 isolated morphinan N-demethylase (MND) from a Methylobacterium. This enzyme is robust and
versatile N-demethylase on structurally diverse substarates and also tolerate in some organic solvent.
3.5.1.2.3 Halogenation enzymes

Halogenation is important step to increase drug efficiency. Whereas feeding of precursor analogs with halogenation can be used for
the halogenated products, introduction of bacterial flavin-dependent halogenases, i.e., RebH or PyrH with RebF reductase,
generated chlorinated tryptophan (e.g., 5-chloro, or 7-chloro-tryptophan) and yielded chlorinated alkaloids (e.g.,
15-chlorotabersonine, or 12-chloro-19, 20-dihydroakumamicine, respectively) in C. roseus.164 Furthermore, this halogenase
RebH was engineered to incorporate chlorine preferentially onto tryptamine rather than the native substrate tryptophan, because
tryptophan decarboxylase accepts 7-chlorotryptophan at only 3% of tryptophan and transgenic cells with wild type RebH
accumulated substantial levels of 7-chlorotryptophan. The RebH Y455W mutant overcame this bottleneck and did not accumulate
7-chlorotryptophan, whereas only minor quantities of 12-chloro-19, 20-dihydroakuammicine was produced in transgenic cells
with RebH mutant.165
3.5.2 Protein engineering based on the 3D-structure of enzyme and/or with mutagenesis
As mentioned above, biosynthetic enzymes in secondary metabolism are often not so specific, not strict, or not so efficient.
Therefore, protein engineering based on the protein structure or random mutagenesis is crucial to improve the enzyme performance
as well as the search of better enzymes.
3.5.2.1 Strictosidine synthase (STR)

Strictosidine synthase (STR), the first committed step in the biosynthesis of MIAs, was rationally redesigned to accommodate
secologanin substrate analogs to the STR binding pocket to generate unnatural intermediate.166 With further characterization of a
different expression system and screening, STR variants were created to accept tryptamine analogs not used by the wild-type
enzyme.111
The crystal structure of STR was also determined to broaden substrate specificities for the production of a greater variety of
strictosidine analogs. The reengineered enzyme gene expressed in transgenic cultured C. roseus cells produced a variety of unnatural
alkaloid compounds when cocultured with simple, achiral, commercially available precursors.112
3.5.2.2 Other proteins

There are several examples for protein design to improve enzyme performance. Codeinone reductase (COR), which catalyzes the
reversible NADPH-dependent reduction of codeinone to codeine in opium poppy, also irreversibly reduces neopinone, which is
generated by spontaneous isomerization from codeinone, to neopine. In a parallel morphinan biosynthesis pathway, COR also
converts morphinone to morphine reversibly, and neomorphinone to neomorphine irreversibly. Whereas plant accumulates low
neopine and no detectable neomorphine, transgenic yeast for opiate production accumulated neopine and neomorphine as major
products by COR reaction. Based on the finding that an isoform (CORdB) showed higher catalytic activity than previously
characterized CORs, and mostly produced neopine in vitro and in engineered yeast, native COR isoforms were site-directed
mutagenized and four residues (V25, K41, F129 and W279) were identified to affect protein stability, reaction velocity, and product
selectivity to improve COR.167
Also the atomic structure of salutaridine reductase (SalR; EC 1.1.1.248) in BIA biosynthesis was determined and site-directed
mutagenized to reduce the substrate inhibition.168
In MIA biosynthesis, crystal structure of C. roseus heteroyohimbine synthases (HYSs), which catalyzes formation of a hetero-
yohimbine isomer, was determined to characterize the mechanism of reactivity and stereo-selectivity, and one loop was mutagen-
ized to modify product specificity.169
3.6 Transcription Factors

Transcription factor is most efficient tool to control gene expression in pathway engineering. Transcriptional regulation of
metabolite production; e.g., the introduction of transcriptional regulator R and C1170 or random activation of transcription factor
with activation tagging171 show the high potential of activation of transcription factor. Furthermore, a successful transcription factor
decoy strategy for targeted activation of large secondary metabolism gene clusters was reported in microbe streptomycetes.172 As in
microbes, plant genomes have many uncharacterized and un-expressed genes in secondary metabolism. Thus, activation and
modification of transcriptional regulation would be powerful strategy in pathway engineering, especially novel drug discovery.
However, this transcriptional activation strategy needs more information about TFs in secondary metabolism.173,174
3.6.1 APETALA2 (AP2)/ethylene response factors (ERFs)

AP2/ERFs are plant specific TFs involved in ethylene and JA signal transduction and/or flower development. ERFs, which belong to
group IX subfamily, interact with target genes via GCC-box cis-elements in ethylene and JA signaling. Involvement of ERFs in
alkaloid biosynthesis is reported in MIA and nicotine biosynthesis.174
ORCA3, a transcription factor with a jasmonate (JA)-responsive AP2/ERF domain, is the first TF identified in alkaloid pathway.
ORCA3 was isolated in MIA pathway from Catharanthus roseum using activation tagging.175 Whereas the ectopic expression of
ORCA3 in cultured C. roseus cells increases the expression of the MIA biosynthetic genes and the accumulation of more tryptophan
and tryptamine, it did not increase the production of MIAs. This result and other evidences indicate that several transcription factors
would be involved in the regulation of alkaloid biosynthesis, especially for MIAs and BIAs as discuss below.174
ORCA3 in MIA biosynthesis forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Interestingly, the ORCA
gene cluster is differentially regulated. ORCA4, functionally overlapping with ORCA3, modulates an additional set of MIA genes.
Unlike ORCA3, ORCA4 overexpression dramatically increased MIA accumulation in C. roseus hairy roots. In addition, CrMYC2, a
bHLH TF, is capable to activate ORCA3 and coregulate MIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and
CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1.
Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated MIA pathways genes and increased MIA accumulation.176
Unique gene clusters of NtERFs are also identified in nicotine alkaloid biosynthesis (see also Refs. 177–179 and Section 4.3.2).
GLYCOALKALOID METABOLISM 9 (GAME9) in steroidal glycoalkaloids (SGAs) biosynthesis is also an AP2/ERF related to
those in the biosynthesis of nicotine in tobacco and MIAs in C. roseus. GAME9 controls gene expression in SGAs biosynthesis and
also upstream mevalonate pathways including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2) in
potato and tomato.69 Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooper-
atively with the SlMYC2 transcription factor as in the case of the D(7)-sterol-C5(6)-desaturase (C5-SD) gene promoter.
3.6.2 Basic helix-loop-helix (bHLH) family; MYC2 and non-MYC2 type

Besides ERFs, several other TFs are isolated in alkaloid biosynthesis. The basic helix-loop-helix (bHLH) superfamily functions in
diverse biological processes as TFs both in nonplant eukaryotes and plants. In Arabidopsis, 147 bHLH genes are annotated using
signature bHLH motif.180 The N-terminal region of bHLH motifs (about 60 amino acids) consists of approximately 15 mainly basic
amino acids, and is responsible for the recognition of E-box (50 -CANNTG-30 ), such as G-box (CACGTG) cis-elements in promoter
sequence. Approximately 45 amino acids located at the C-terminus form two amphipathic a-helices, that are connected by a
variable loop region, also form homo- or heterodimer complexes.
Among bHLH superfamily, Arabidopsis MYC2 is most characterized TF involved in the jasmonate (JA) signaling. The MYC2
activity is blocked by JAZ proteins in the absence of JA, whereas JAZ proteins are degraded and MYC2 activity is released in the
presence of JA. Arabidopsis MYC2 homologues were isolated in the biosynthesis of MIAs in C. roseus or in the biosynthesis of
nicotine in Nicotiana.181,182
On the other hand, a non-MYC2-type bHLH transcription factor (CjbHLH1), which lacks JID (JAZ-interacting domain)
sequence, was also isolated from C. japonica in BIA biosynthesis. Transient RNAi and over-expression experiments showed
comprehensive regulation of BIA biosynthesis in C. japonica by CjbHLH1. Interestingly, CjbHLH1 homologous genes were found
in many BIA-producing plant species; e.g., suppression of California poppy homologues, EcbHLH1, caused a decrease in BIA
accumulation, indicating that non-MYC2-type bHLH1s regulate BIA biosynthesis both in California poppy and C. japonica.183–185
bHLH Iridoid Synthesis 1 and 2 (BIS1/2), which belong to another clade IVa of the bHLH family, were also isolated in MIA
biosynthesis in C. roseus. CrBIS1/2 do not contain a known JID as CjbHLH1, and different from MYC2-type. BIS1/2 trans-activated
the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid
precursor geranyl diphosphate to the iridoid loganic acid.186 Transcript profiling indicated that CrBIS2 expression is part of an
amplification loop, and silencing of CrBIS2 in C. roseus cells completely abolished the JA-induced upregulation of the iridoid
pathway genes, despite the presence of induced BIS1, indicating that BIS2 is essential for MIA production in C. roseus.187
3.6.3 WRKY
WRKY is plant specific TFs involved in defense response. A group-II WRKY TF, which interacts with W-box cis-elements via WRKY
DNA-binding domain, was isolated in BIA biosynthesis from C. japonica using a transient RNAi and overexpression of the candidate
gene. Ectopic expression of CjWRKY1 activated the BIA biosynthesis in C. japonica specifically and comprehensively.188
CrWRKY1 isolated from C. roseus is TF preferentially expressed in roots and induced by jasmonate (JA), gibberellic acid, and
ethylene. Whereas the overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key MIA pathway genes, especially
tryptophan decarboxylase (TDC) gene, it also up-regulated the expression of ZCT1 (zinc-finger C. roseus TF1), ZCT2, and ZCT3,
which act as repressors of MIA biosynthesis in C. roseus.189 In fact, CrWRKY1 overexpression repressed the transcriptional activators
ORCA2, ORCA3, and CrMYC2, whereas CrWRKY1-overexpressed hairy roots accumulated up to threefold higher levels of
serpentine in comparison with control roots.190
3.6.4 Posttranslational regulation of TFs in secondary metabolism

Whereas transcriptional activation by the over-expression of TFs is useful strategy, some TFs function as repressor. Furthermore, the
TF activity was not only regulated by transcriptional level, but also regulated posttranscriptionally. For example, the phosphoryla-
tion of CjWRKY1 lost its nuclear localization, DNA-binding activity, and transactivation activity compared to nonphosphorylated
one.191 Furthermore, CjWRKY1 protein showed the rapid degradation via 26S proteasome and vacuolar proteases, suggesting TFs in
secondary metabolism would be fine-tuned to optimize the production of metabolites to avoid excess production of toxic
compounds and loss of the cell viability. Auto-regulatory feedback of MYC2-MTB (MYC2-TARGETED bHLH) was also reported
to shut down the deleterious effects of a sustained response.192
3.7 Genes for Transport, Accumulation and Storage

Since secondary metabolites are often produced in specialized plant cells and accumulate in cells/organs apart from the site of
biosynthesis, transport and accumulation/storage or secretion have physiological importance.193,194 This is partially due to the toxic
nature of produced specialized metabolites. Therefore, the metabolites are often stored in vacuole as inactive form such as glucose
conjugates and activated after the destruction of cellular organization and cleavage with digesting enzymes such as glucosidases, or
released to extracellular space after the outer signals. The importance of transports and accumulation processes were also indicated
by the ectopic expression of yeast PDR5 (pleiotropic drug resistance-type ATP-binding cassette transporter) genes in transgenic
tobacco, which stimulated the secretion of metabolites.195 Whereas our knowledge is still limited, some typical transporters are
briefly summarized below. Also some recent progresses are discussed in the section on nicotine, tropane and BIAs.
3.7.1 ABC transporters

Berberine in C. japonica is produced in root and transferred to rhizome across several membranes. CjMDR (a MultiDrug Resistance
gene; now called CjABCB1) was identified in rhizome for the accumulation and detoxification of these biologically active
metabolites.196
Whereas plant cells generally accumulate these toxic secondary metabolites in the vacuole, some environmental signals induce
the secretion; for example, benzyladenine (BA) induces the production of a BIA, berberine, in cultured Thalictrum minus cells and the
release into the medium. Isolation of cDNAs of two novel B-type ABC transporter genes from T. minus, TmABCB1 and TmABCB2,
and their increased expression by BA suggest that TmABCB1 and TmABCB2 participate in berberine transport in T. minus cells.197
Characterization of MIA biosynthesis in C. roseus indicated extensive movement of metabolites such as movement of cathar-
anthine from leaf epidermis to the leaf surface and vindoline to internal leaf cells. A unique catharanthine transporter (CrTPT2)
expressed predominantly in the epidermis of young leaves was identified. CrTPT2 gene expression is activated by treatment with
catharanthine, and its silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the
leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that
may be unique to MIA-producing plant species.198
3.7.2 Antiporter and multidrug and toxic compound extrusion protein (MATE)
The characterization of vacuolar transport of berberine by H+/berberine antiporter indicates that this transporter was also involved
in subcellular and intercellular transport of berberine in C. japonica cells.199
Nicotine is translocated via xylem transport from the root tissues, where it is biosynthesized, to the accumulation sites, i.e., the
vacuoles of leaves. A tobacco jasmonate (JA)-inducible alkaloid transporter 1 (NtJAT1), belonging to the family of multidrug and
toxic compound extrusion transporters, was isolated. NtJAT1 expressed in yeast localized mainly in the plasma membrane and
showed nicotine efflux activity. Biochemical reconstitution experiment showed that NtJAT1 functioned as a proton antiporter and
recognized endogenous tobacco alkaloids, such as nicotine and anabasine, and other alkaloids, such as hyoscyamine and berberine,
but not flavonoids, suggesting that NtJAT1 plays an important role in the nicotine translocation by acting as a secondary transporter
responsible for unloading of alkaloids in the aerial parts and deposition in the vacuoles.200 Multidrug and toxic compound
extrusion protein 1 (MATE1) also reported to act as a berberine transporter in cultured C. japonica cells.201
3.7.3 Nitrate/peptide family transporter (NPF)

MIAs are synthesized in several different cellular locations. For example, the MIA precursor, secologanin, is biosynthesized between
internal phloem-associated parenchyma and epidermis cells. Based on the identification of an Arabidopsis thaliana nitrate/peptide
family (NPF) transporter, AtNPF2.9, as a putative iridoid glucoside importer in Xenopus oocytes, orthologs CrNPF2.4, CrNPF2.5
and CrNPF2.6, isolated from C. roseus were identified to transport the iridoid glucosides 7-deoxyloganic acid, loganic acid, loganin
and secologanin in oocytes.202 On the other hand, tonoplast localized NPF transporter, CrNPF2.9, was identified to export
strictosidine, into the cytosol from the vacuole in C. roseus.203
3.7.4 Purine permease (PUP)

The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-
coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A tobacco PUP-family
transporter, nicotine uptake permease 1 (NtNUP1), was identified to transport tobacco alkaloids and to affect both nicotine
biosynthesis and root growth in tobacco plants. Because NtNUP1 could transport various compounds containing a pyridine ring
such as vitamin B6 derivatives (pyridoxamine and pyridoxine) beside anatabine and nicotine, NtNUP1 might affect on physiology
due to such broad transport activity, whereas the naturally occurring (S)-isomer of nicotine was preferentially transported over the
(R)-isomer.204
3.8 Progresses in Engineering Methods

Expression of genes in target cells is critical factors for pathway engineering with the mining of genes in target pathway.
Transformation, especially that of nonmodel medicinal plants, is critical barrier for pathway engineering. Agrobacterium mediated
stable transformation is commonly used for gene expression in plants, but the toxicities of natural products hinder the transfor-
mation via reduced viability of Agrobacterium and also Agrobacterium-infected plant cells. Therefore, whole pathway reconstruc-
tion in heterologous host, especially microbial hosts, might be promising alternative, when molecular tools for whole pathway is
available. In most cases, however, molecular characterization of pathway is limited and the partial modification of pathway in target
plants would be more strategic choice. In this section, firstly engineering of pathway via stable transformation, then, reconstitution
of pathway in microbes with synthetic biology are discussed.
3.8.1 General strategies for metabolic engineering; over-expression or silencing of gene using stable transformation
3.8.1.1 Condition of transformation
Agrobacterium system is gold standard for stable transformation for plants, whereas direct gene transfer using microbombardment
or electroinjection with protoplasts would be useful alternatives. Since Agrobacterium based transformation is dependent on the
viability of bacterium and host cells, antimicrobial activity of many plant natural products would be inhibitory for the transfor-
mation. Thus, it is practical to select good viable materials such as immature embryo or young shoot with low secondary metabolite
content, or to choose some conditions, which reduce the secondary metabolite content. For example, new A. tumefaciens strain with
g-aminobutyric acid (GABA) transaminase to degrade GABA was established and successfully used to increase the frequency of
stable transformation in plant, because the accumulation of GABA is a negative factor in T-DNA transfer in Agrobacterium.205
3.8.1.2 Strategy of engineering

To improve and/or modify the metabolite production, overexpression of rate-limiting step enzyme is simple and effective, whereas
the identification of rate-limiting step is not easy. The improved scopolamine production in transgenic Atropa with over-expression
of hyoscyamine 6-b-hydroxylase isolated from Hyoscyamus was first success in alkaloid engineering31 as mentioned before.
However, simple over-expression of the enzyme in secondary metabolism is usually not sufficient to increase the biosynthesis of
the desired end product, due to a lack of sufficient flux for the pathway in upstream or downstream bottleneck in the pathway. For
example, over-expression of tryptophan decarboxylase in C. roseus cell cultures increased the product tryptamine, but did not
increase the target MIAs due to the downstream bottleneck. On the other hand, the over-expression of strictosidine synthase in
downstream of pathway increased the MIAs.206
3.8.1.3 Quantity improvement with overexpression of rate-limiting enzyme gene via stable transformation
Whereas Cauliflower Mosaic Virus 35S is commonly used promoter sequence to over-express desired gene constitutively in host
plant cells, we need more careful selection of promoter and terminator as well as enhancer sequences to express gene in more
optimized developmental stage and specific cells for metabolic engineering. For alkaloid engineering, we need more detailed
understandings about the regulation of metabolic pathway and gene expression profiles. For example, when two enzyme genes of
norcoclaurine 6-O-methyltransferase (Cj6OMT) and 30 -hydroxy-N-methylcoclaurine 40 -O-methyltransferase (Cj40 OMT) isolated
from C. japonica were over-expressed in California poppy, two genes showed different effects; i.e., the overexpression of Cj6OMT
increased alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of Cj40 OMT had only a marginal
effect.207
Similarly, whereas constitutively expression of codeinone reductase (PsCor1.1) in opium poppy showed increases of 15% and
30% in the morphinan alkaloid contents on a dry weight basis than those in control high-yielding genotypes,208 over-expression of
(S)-N-methylcoclaurine 30 -hydroxylase (CYP80B3) resulted in an up to 450% increase in the amount of total alkaloid in latex.209
These results clearly suggest the importance of the understanding of biosynthetic pathway and metabolic flux.
However, we should also note that metabolism is dynamic; i.e., multiple rate-limiting processes exist, and the regulation of
metabolic pathways is different among plant species. For example, the production capacity might be limited by the substrate supply.
In such case, enhancement of primary metabolism is needed. Or, storage or transport capacity might be modified to increase the
accumulation of metabolites. In fact, the pyramiding of metabolic engineering is sometimes required to overcome multiple rate-
limitation; e.g., the coordinated expression of two cytochrome P450 genes with a glucosyltransferase improved the production of
dhurrin in Arabidopsis.210
On the other hand, we should also be careful for the limitations in over-expression experiments; i.e., the cosuppression (or RNA
silencing, RNA interference; i.e., RNAi) of homologous gene due to the production of double stranded RNA derived from excess
transcription of gene,211,212 as discussed later. Therefore, expression of heterologous gene to endogenous gene with less homoge-
neity is recommended to use in the pathway engineering.
3.8.1.4 Quality control via stable transformation

Whereas quantity improvement of metabolite production with over-expression of rate-limiting enzyme gene is useful strategy in
metabolic engineering, quality modification is another target of pathway engineering.
3.8.1.4.1 Quality control via the introduction of branch pathway

Introduction of branch pathway is one of promising approaches for the modification of quality of metabolites.213 A crucial point to
create a new branch in pathway is the substrate-affinity and/or reaction specificity to endogenous enzyme(s) and also affinity of
downstream enzyme(s) for newly produced metabolite. For example, introduction of scoulerine 9-O-methyltransferase (CjSMT)
gene of C. japonica in berberine biosynthesis into benzophenanthridine biosynthesis in California poppy shifted the metabolites
from the sanguinarine type with two methylene-dioxy rings to chelerythrine type with one methylene-dioxy and one methoxy
groups.65 Whereas both introduced CjSMT and endogenous cheilanthiforine synthase (EcCYP719A2/A3) accept scoulerine as
substrate, high reactivity of CjSMT would dominate the pathway than the endogenous EcCYP719A2/A3.
Similarly, when (S)-tetrahydroberberine oxidase (CjTHBO) isolated from C. japonica cells was introduced to California poppy
cells, CjTHBO hijacked the intermediates in benzophenanthridine biosynthesis to protoberberine type products.86 However, it is
clear that no channeling of metabolic flow occurs, if endogenous enzyme is more reactive.
3.8.1.4.2 Knockdown the undesired endogenous pathway with RNA silencing

A more promising approach to modify the metabolite quality is the down-regulation of gene expression for undesired pathway(s) in
metabolism. Whereas down-regulation using antisense RNA and cosuppression has been shown to be effective to control the
metabolite production, the RNA-interference (RNAi) method using double-stranded (ds)RNA-induced posttranscriptional and/or
transcriptional silencing is more efficient to shut down the gene expression and to modify the pathway.214,215
For example, RNAi of berberine bridge enzyme (BBE) gene in California poppy cells induced the accumulation of the key
intermediate reticuline, a substrate of BBE, in BIA biosynthesis. In addition, gene-silenced cells secreted significant amounts of
reticuline into the medium. Such reticuline accumulation was not detected with antisense RNA method,216 suggesting the higher
efficiency of RNAi than antisense RNA. However, these cells also produced a methylated derivative of reticuline, laudanine, which
was produced from accumulating reticuline by endogenous reticuline 7-OMT. Activity of reticuline 7-OMT would be not evident in
control cells, but increased metabolite, i.e., reticuline, in transgenic cells enabled the 7-O-methylation reaction and changed the
pathway, indicating the dynamism of metabolism.33
Similar metabolic modification was also found in RNAi poppy of salutaridinol 7-O-acetyltransferase (SalAT ), which accumu-
lated the substrate (salutaridinol) of SalAT and also induced accumulation of salutaridine, the substrate for salutaridine reductase
(SalR), one-step earlier enzyme than SalAT.217 Whereas salutaridine accumulation might be induced by the inhibition of SalR via
the accumulation of salutaridinol, yeast two-hybrid and coimmuno-precipitation analyses suggested an interaction between SalR
and SalAT and the loss of enzyme complex (metabolon) by RNAi.218
Whereas RNAi is powerful tool to down-regulate the gene expression, single RNAi is still not sufficient to induce the
accumulation of intermediate; e.g., low-caffeine coffee produced by the RNAi of caffeine synthase did not accumulate any
intermediates, such as theobromine,219 suggesting that intermediates produced might be further metabolized by endogenous
enzymes.
Additionally, it should be noted that selection of the target sequence is crucial for RNAi, since only a 22 nucleotide-long perfect
match was sufficient to silence homologous genes with an identical sequence,220 and longer sequence might down-regulate
homologous genes via off-target effect as RNAi of COR affected the expression of STORR via reductase domain.221
3.8.2 Transient system to evaluate the possibility of metabolic engineering

Stable transformation is prominent and preferred method in pathway engineering. But, it is also time consuming and labor-
intensive process to establish. In this sense, transient assay is easy and rapid. Especially, transient assay would be useful to
characterize the gene functions to predict the pathway engineering in advance. Whereas protoplasts are suitable host for transient
assay to evaluate metabolic engineering, preparation of protoplast is sometimes not easy. Therefore, another system, such as virus
based expression using benthamiana tobacco as an excellent host for transient over-expression of enzyme genes, or virus-induced
gene silencing (VIGS) in target plants would be useful to evaluate the gene function (see following section).
3.8.2.1 Transient assay with protoplasts

Protoplasts are prepared from plant cells after the digestion of cell wall with cellulase, pectinase and other cell wall digesting
enzymes. Protoplasts uptake DNA, RNA, or proteins easily, by the treatment with polyethylene glycol (PEG), or electric stimulus.
For example, double-stranded (ds) RNAs prepared against candidate TF genes or over-expression plasmids for TF genes were
introduced into C. japonica protoplasts by PEG-mediated transformation, and their suppression effects of TFs on biosynthetic
enzyme genes were monitored by quantitative reverse transcription (RT)-polymerase chain reaction (PCR).188,222 This transient
assay using protoplasts was very useful for the functional characterization of TFs, but the down-regulation or overexpression of
biosynthetic enzyme genes were less effective on the metabolism due to preexisting enzyme proteins, in comparison with the large
changes of transcripts by the regulation of TFs. For the functional characterization of biosynthetic enzyme genes, prolonged
modulation of gene expression with VIGS or virus-based expression is more recommended method.
3.8.2.2 Virus-based over-expression of gene and virus-induced gene silencing (VIGS) in intact plant
3.8.2.2.1 Virus vectors
Virus-based modification of gene expression including gene silencing (VIGS) is useful tool for a functional genomics to investigate
the gene functions as well as the regulation of biosynthesis via a systematic reduction in enzyme levels.223
Commonly used virus vectors are derived from RNA viruses such as Potato virus X (PVX), Tobacco mosaic virus (TMV), and Tobacco
rattle virus (TRV) or DNA viruses such as Tomato yellow leaf curl China virus (TYLCCV). So far, the bipartite pTRV vector system, pTRV1
and pTRV2, derived from Tobacco Rattle Virus is successfully used to assay gene function in diverse plant systems, including
C. roseus,224 California poppy plants,225 meadow rue (Thalictrum spp.226), and opium poppy.97,151,227,228
3.8.2.2.2 Transient expression in benthamiana tobacco or other plants

Virus-based transient over-expression of enzyme genes in plant cells is rapid and efficient method to characterize the gene function.
Now, several transient expression vectors such as MagniCon or gemini virus-based system are available.87,229,230
Interestingly, multiple gene transfer is successfully applied to reconstitute reactions for MIA biosynthesis. Sarpagan bridge
enzyme (SBE) is the entry enzyme to convert the central MIA intermediate strictosidine into sarpagan, ajmalan and alstophyllan
alkaloid classes. Based on the hypothesis that SBE is a cytochrome P450 that acts on geissoschizine (the product of a soluble
reductase and strictosidine aglycone), SBE candidates were expressed along with enzymes responsible for production of chemically
stable vinorine from unstable SBE product, i.e., polyneuridine aldehyde (PNA). Nicotiana benthamiana (benthamiana tobacco), best
host for transient gene expression,231 was infiltrated with strictosidine and A. tumefaciens strains harboring strictosidine glucosidase
(SGD), geissoschizine synthase (CrGS, identified from C. roseus), PNAE esterase (PNAE), vinorine synthase (VS) and SBE candidates
for transient expression. Up to three SBE candidates from Rauwolfia serpentina, Gelsemium sempervirens and C. roseus were introduced
simultaneously into N. benthamiana, and SBE activity was confirmed with the formation of vinorine.232
Whereas benthamina tobacco is good host plant to express foreign genes using virus vectors,231 its adaptability in alkaloid
engineering is limited due to the restriction of substrate supply. Therefore, more universal vector system to express gene in divergent
host plant is needed to characterize the gene function in various metabolism. Recent development of gemini virus-derived
expression system, which can express in various plant species, would be more promising for functional genomics in pathway
engineering.87
3.8.2.2.3 VIGS
VIGS is widely used to characterize the gene function. In opium poppy, the final six step enzyme genes in the morphine
biosynthesis, i.e., salutaridine synthase (SalSyn or SalS), salutaridine reductase (SalR), salutaridine 7-O-acetyltransferase (SalAT),
thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), and codeine O-demethylase (CODM), were successfully down-
regulated with VIGS using pTRV vector system. Reduced SalSyn, SalR, T6ODM and CODM correlated with lower morphine levels
and a substantial increase in the accumulation of reticuline, salutaridine, thebaine and codeine, respectively. In contrast, the
silencing of genes encoding SalAT and COR resulted in the accumulation of salutaridine and reticuline, respectively, which are not
the corresponding enzymatic substrates.228 The reason of the accumulation of salutaridine and reticuline by RNAi of SalAT and
COR is discussed elsewhere.
The biosynthesis of vindoline and catharanthine in C. roseus, was also effectively studied with the bipartite pTRV vector
system.224
3.8.3 Genome editing

One of disadvantages in RNA silencing including VIGS is cross-silencing of target gene, i.e., off-target effects of silencing as shown for
VIGS of COR in opium poppy, due to the sequence homology, and also relatively low efficiency of silencing. Therefore, more
accurate silencing technique is needed. Development of new genome editing tools, especially CRISPR-Cas9, after the first and
second generation editing tools (i.e., Zinc-finger nuclease and Transcription Activator-Like Effector Nuclease; TALEN) open the new
field in breeding in living organisms including plants.35,39 Whereas the application of genome editing is still limited in medicinal
plants,34 gene knockout of one of key enzyme in solanine biosynthesis suggests the high potential of genome editing.37 Further-
more, complete knockout of the gene will reveal the hidden pathway of the biosynthesis, which will be apparent only when the
main stream was blocked.
Additionally, homologous recombination using genome-editing tool enables the incorporation or exchange of endogenous
genes with more preferable enzyme genes or promoter-elements for gene regulation. Such gain of function modification with
genome editing will open new field in pathway engineering.
3.8.4 Heterologous expression of biosynthetic enzyme genes in microbe

For the successful industrial application of secondary metabolite production in plant cells, both the quantity and quality of
metabolites must be improved, i.e., control of the metabolite profile and yields are important. Especially, simple composition of
metabolites is desirable for industrial production with limited purification process. The simplest solution to produce desired
compounds is to reconstruct entire biosynthetic processes in vitro. So far, a considerable number of genes involved in alkaloid
biosynthesis have been cloned and expressed in E. coli, yeast or insect cells. Whereas microbial enzymes have been shown to be
useful for the biotransformation of chemicals, plant enzymes with high substrate specificity, e.g., P450s118 would be useful
biocatalysts to diversify the chemical structure of products. The combination of microbial and plant enzymes as well as the use
of artificially synthesized chemicals would increase the production of more divergent chemicals.233–239 Thus, heterologous
microbial systems could be useful for the production of plant-derived metabolites, while microbial cells have less capacity to
store metabolites.
3.8.4.1 Escherichia coli (E. coli)

Expression of recombinant proteins in Escherichia coli is both efficient and cost-effective with high expression efficiency and rapid
growth for protein production. Soluble proteins are commonly expressed under the T7 promoter of the pET expression system in
E. coli, but more options are available with variety of expression systems to optimize production for labile and unstable proteins.
One of disadvantage is lack of endomembrane systems for compartmentation of proteins and metabolites, whereas some
membrane-bound proteins such as cytochrome P450 can be expressed in E. coli as active form with N-terminal modification.240
Now, large reconstruction of biosynthetic pathway is available with increase in molecular tools and also chromosomal
integration of introduced genes, whereas plasmid based transformation is more rapid and useful to evaluate certain number of
genes than genome integration. Especially, the rapid growth and easy transformation are advantages for E. coli. The molecular
knowledge of primary metabolism also makes E. coli as more advanced host for the supply of primary metabolites for specialized
metabolites; e.g., high production of tyrosine such as 9.7 g/L has been achieved in E. coli,154 whereas tyrosine-overproducing yeast
platform with accumulation of up to 192 mM tyrosine in cytosol with supplement of methionine and complete elimination of
feedback inhibition and degradation pathway was reported.241
3.8.4.2 Saccharomyces cerevisiae, yeast

Yeast, a simple eukaryotic cell, has several advantages in enzyme expression such as, copy number, transcriptional regulation,
posttranscriptional and posttranslational regulation, and spatial regulation.235 Whereas a number of constructs exist for modifying
the copy number of heterologous genes based on expression from plasmids for yeast, artificial or native chromosomes for
integration of larger gene fragments would be one of advantages in synthetic biology. Also, a variety of well-characterized promoters
and the posttranscriptional processing of transcripts via RNA-based control elements would be used for the optimization of
expression.
The largest benefit of yeast cell is endomembrane systems to increase the local concentration of pathway enzymes and
intermediates. High local concentrations of biosynthetic enzymes reduce the diffusion of intermediates and increase the metabolic
flux. In addition, such localization can prevent the loss of intermediates by competing pathways, degradation of unstable
intermediates, and the negative effects of toxic intermediates and feedback inhibition. Endomembrane systems also supply
important cofactors and separate toxic compounds in pathway engineering, whereas yeast grows rather slowly than E. coli.
3.8.4.3 Other host cells

Pichia pastoris, a methylotrophic yeast, is another choice to express proteins, because its protein production using methylotrophic
protein expression system is highly efficient than yeast expression system. For example, opium poppy DBOX, a homolog to (S)-
tetrahydroprotoberberine oxidase (STOX/THBO) was successfully expressed in P. pastoris,117 whereas no expression of THBO
activity in E. coli or yeast cells was reported.86
Spodoptera frugiperda Sf9 insect expression system is alternative choice for high expression of difficult proteins, such as berberine
bridge enzyme (BBE)113 and STOX of Argemone mexicana.116
3.9 Synthetic Biology

In previous sections, homologous or heterologous expressions of biosynthetic enzyme genes are discussed to characterize their
biochemical functions. These successes enable the reconstruction of some parts or even whole of pathway. Microbial production of
plant secondary metabolites through the heterologous expression of plant biosynthetic genes now provides new solution for the
production of these chemicals. So far, several strategies were proposed as summarized below.23,239,242–247
3.9.1 Strategy for synthetic biology; pathway identification, metabolic modeling, pathway engineering, metabolic control
network, and drug discovery
Synthetic biology provides opportunity to understand and reconstruct the biosynthetic pathways that generate the diversity, and
also to produce novel products with improved efficiency. The algorithms and databases that presently support the design and
manipulation of metabolic pathways in plants, starting from metabolic models of native biosynthetic pathways, progressing to
novel combinations of known reactions, and finally proposing new reactions that may be carried out by existing enzymes, were
reviewed recently.239 These tools are also useful to propose new pathways and identify side reactions that may affect the pathway
engineering. Methods for pathway discovery, DNA synthesis and assembly, and expression of engineered pathways in heterologous
hosts were also reviewed.238
Importance of de novo pathway identification, tunable enzyme expression, rapid pathway construction, optimization of single
enzymes and entire genomes through diversity generation and screening, whole cell analytics, and synthetic metabolic control
networks were emphasized both in yeast and E. coli.242 With the successful history of natural products in drug discovery, the targeted
evolution of secondary metabolite pathway in plants or microbes as factories would be valuable for drug production, and directed
evolution strategies have been reviewed.244
Interestingly, novel strategy to limit the enrichment of nonproducing cell populations in bioproduction was proposed by
Rugbjerg et al.247; i.e., the genes for key growth intermediates was designed to be placed under the control of a promoter responsive
to the bioproduct being made and enrich high producing cells.
3.9.2 Metabolon and design of gene fusion protein

The concept of a metabolon was reported in 1980s as a supramolecular assembly of enzymatic and structural components to
sequester and channel metabolic pathway intermediates. Whereas metabolons were initially proposed for the primary metabolism
such as citric acid cycle, glycolysis, and nucleotide and amino acid biosynthesis, more pathways are expected to form metabolon
based on the protein-protein interactions. Proteins and membrane interaction are also expected to form dynamic metabolons and
increase metabolic flux.248,249 As shown in SalAT and SalR interaction in BIA biosynthesis,218 metabolon formation might be the
key factor for the optimum production of product in microbe.
When metabolon formation is predicted, gene fusions, which are common in prokaryotic enzymes, might be attractive choice in
engineering. The occurrence of a gene fusion, in which originally separated gene products are combined into a single polypeptide,
was shown in STORR in morphinan biosynthesis.123,124 Partial or complete domain repeats and extensive domain shuffling are also
evident for norcoclaurine synthase in BIA biosynthesis.99 Whereas artificial gene fusions would be beneficial for pathway engineer-
ing, we should note that the functional impact of fused gene products is not straightforward. For example, whereas enzyme fusions
might facilitate the metabolic channeling of unstable intermediates, this channeling can also occur between tightly associated
independent enzymes. The frequent occurrence of both fused and un-fused enzymes in plant and microbial metabolism adds
additional complexity, in terms of both pathway functionality and evolution.250
3.10 Feeding of Substrate and Combination with Chemical Strategy

Feeding substrate is not new, especially in the bioconversion of metabolites. But, one of advantages in substrate feeding is possibility
to short cut the pathway and enlarge the chemical diversity, especially in MIA biosynthetic machinery.251 Feeding of secologanin
substrate analog produced nonnatural MIA in crude extracts of plant tissue.252 Synthetic seco-iridoid and strictosidine were also
used in the heteroyohimbine branch of MIA biosynthesis in C. roseus with bypassing of the stereoselectivity of early steps.253
Whereas native enzyme was applicable for such engineering, modification of substrate specificity broadens the conversion
efficiency. So, variants of strictosidine synthase (STR), that accept tryptamine analog, was generated and used in stereoselective
synthesis of beta-carboline analogs.111 Furthermore, the crystal structure of STR was used to design enzyme mutants with broadened
substrate specificities. When an alkaloid biosynthetic gene with reengineered substrate specificity was introduced into C. roseus, the
resulting transgenic cells produced a variety of unnatural MIAs when cocultured with simple, achiral, commercially available
precursors.112
Whereas feeding is effective to produce novel compounds, the contamination of endogenous metabolites hampers the
purification of product. One of the solutions is the suppression of early step enzyme as shown in suppression of tryptamine
biosynthesis in C. roseus hairy root culture, which eliminated all production of MIAs, but did not affect the expression of
downstream biosynthetic enzymes. Under the chemically silent background, an unnatural tryptamine analog was successfully
converted to a variety of novel MIAs.254
Further diversification of alkaloids can be achieved via a combination of biosynthetic and chemical strategies. Specifically,
introduction of a chlorine or a bromine into the target molecule by mutasynthesis, followed by post biosynthetic chemical
derivatization using Pd-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of MIAs.255
4 Engineering for Alkaloid Production

4.1 Engineering in Isoquinoline Alkaloid (IQA) Biosynthesis
Isoquinoline alkaloids (IQAs) are a large and diverse group of alkaloids with 2500 defined structures. They include the analgesic
morphine from Papaver somniferum (opium poppy); the antigout colchicine from Colchicum autumnale; the emetic and antiamoebic
emetine from Cephaelis ipecacuanha (Ipecac); the skeletal muscle relaxant tubocurarine from Chondodendron spp.; and the antimi-
crobial compounds berberine and sanguinarine from divergent plant species including Berberis spp., Sanguinaria spp., and
Coptis spp.
Among IQAs, benzylisoquinoline alkaloids (BIAs) derived from norcoclaurine include major IQAs such as morphine (a
morphinan), berberine (a protoberberine), sanguinarine (a benzophenanthridine), noscapine (a phthalideisoquinoline), tubocu-
rarine (a bisbenzylisoquinoline), magnoflorine (an aporphine), californidine (a pavine) and their biosynthesis pathways are most
intensively investigated. So far, norcocluarine synthase (NCS) as “Pictet-Spenglerase,” O-methyl-, and N-methyltransferases (MTs),
cytochrome P450s (CYPs), FAD-dependent oxidases, O-acetyltransferases (ATs), nonheme dioxygenases and NADPH-dependent
reductases are characterized using opium poppy (Papaver somniferum), California poppy (Eschscholzia californica) and other members
of the Ranunculales such as golden thread (Coptis japonica), Thalictrum as model systems in BIA biosynthesis. BIA metabolism has
been characterized with radioactive tracer experiments, enzyme characterization and molecular cloning, and reverse genetics
analysis such as virus-induced gene silencing (VIGS), transcriptome and metabolome analysis and reviewed several times.256–258
Opium poppy (Papaver somniferum) is the dominant commercial source for the narcotic analgesics morphine, codeine and semi-
synthetic derivatives such as oxycodone. The plant also produces several other BIAs with potent pharmacological properties
including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial
agent sanguinarine. Opium poppy is a model system to investigate the biosynthesis of BIAs, especially pharmaceutically important
morphinan and noscapine alkaloids. The characterization of genomic organization of biosynthetic genes and the cellular and sub-
cellular localization of biosynthetic enzymes provide unique characteristics of this plant species.259
California poppy (Eschscholzia californica), a member of the Papaveraceae family, also produces various types of BIAs, i.e.,
aporphine-, pavine-, protoberberine-, protopine-, and benzophenanthridine-type alkaloids. The major alkaloid biosynthesis
pathways and biosynthetic enzymes of BIAs have been characterized at the molecular level.20,258 Furthermore, its draft genome
sequence of 489 Mb, which covered 97% of the whole genome (502 Mb) was determined recently.46
The rhizome of golden thread (Coptis japonica and C. chinensis) is used in Japanese Kampo Medicine and Traditional Chinese
Medicine for intestinal disorders, heat clearing, dampness drying, fire draining, and detoxification by their major bioactive
components, BIAs. After the intensive study on biosynthetic pathway of BIAs in C. japonica,20 comprehensive transcriptome analysis
of C. chinensis has been reported.260
4.1.1 Biosynthetic enzymes and their genes in the pathway

BIA biosynthesis begins with the conversion of tyrosine to both dopamine and 4-hydroxyphenylacetaldehyde (4HPAA) by
decarboxylation, ortho-hydroxylation, and deamination.20,258
4.1.1.1 Early pathway from tyrosine to norcoclaurine biosynthesis (Fig. 6)

Tyrosine/DOPA decarboxylase (an aromatic L-amino acid decarboxylase; TYDC) converts tyrosine and DOPA to their corresponding
amines. TYDC genes (15 genes) isolated from opium poppy have distinct developmental and inducible expression patterns.261
Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and a-ketoglutarate, and produces
4-hydroxyphenylpyruvic acid and L-glutamate. 4-Hydroxyphenylacetaldehyde (4HPAA), a substrate for norcoclaurine synthase
(NCS), is produced by the decarboxylation of 4-hydroxyphenylpyruvic acid. TyrAT isolated from opium poppy showed its
transcripts were most abundant in roots and stems of mature opium poppy plants. VIGS of TyrAT in opium poppy showed a
moderate reduction in total alkaloid content, and suggests the occurrence of other sources for 4HPAA.262
For the tyrosine hydroxylation to DOPA, plants and animals use tyrosine 3-monooxygenases (EC 1.14.16.2), which require a
cofactor (tetrahydrobiopterin), or phenol oxidase. Copper-containing tyrosinases (EC 1.14.18.1) of microbial cells also show
tyrosine hydroxylase activities, but also DOPA oxidase activities to produce L-dopaquinone from L-tyrosine and form melanin.
Unexpectedly, tyrosine hydroxylation activity of CYP76AD1 from the sugar beet (Beta vulgaris) in the betanidin biosynthesis was
identified using enzyme-coupled L-DOPA biosensor and its improved tyrosine hydroxylase by error-prone PCR was used for the
production of reticuline in yeast.263
Dopamine and 4HPAA are condensed by NCS and yield (S)-norcoclaurine, the central precursor of BIAs. NCS cDNA belonging
to the PR10 family was isolated from Thalictrum flavum,96 P. somniferum (PsNCS1 and PsNCS297), and C. japonica (CjPR10A98),
whereas a novel dioxygenase-like protein (CjNCS) from C. japonica was also reported (see Section 3.4).
4.1.1.2 Reticuline biosynthesis from norcoclaurine (Fig. 6)

(S)-Norcoclaurine is sequentially converted to coclaurine by SAM-dependent norcoclaurine 6-O-methyltransferase (6OMT;
EC.2.1.1.12848), to N-methylcoclaurine by coclaurine N-methyltransferase (CNMT; EC.2.1.1.140144), to 30 -hydroxy-N-methyl
coclaurine by P450 hydroxylase (CYP80B1, EC1.14.14.102125), and then to (S)-reticuline by 30 -hydroxy N-methylcoclaurine 40 -
O-methyltransferase (40 OMT, EC2.1.1.11648). cDNAs for these reactions have been isolated from C. japonica, opium poppy,
California poppy and so on.
Detailed biochemical studies using recombinant enzymes as well as reconstitution of pathway in microbe have shown their strict
reaction specificities, and the sequential biosynthesis by enzymes in a coordinated manner.157 In fact, CjCNMT prefers coclaurine to
6-O-methylnorlaudanosoline and Cj40 OMT prefers an N-methylated substrate, suggesting the sequential reactions of N-methyla-
tion, hydroxylation and 40 -O-methylation. Interestingly, different OMTs showed some differences in enzymological properties; i.e.,
CjOMTs formed homodimer and showed specific substrate specificity,48 but Thalictrum OMTs formed heterodimers and exhibited
broad substrate specificity.264
4.1.1.3 Bisbenzylisoquinoline biosynthesis (Fig. 6)

Further diversification of BIAs is produced from the key intermediate, (S)-reticuline, while dimeric bisbenzylisoquinoline
alkaloids, e.g., berbamunine and tubocurarine, are produced from the intermediates of the (S)-reticuline pathway, by the action
of a phenol coupling P450-dependent oxidase (berbamunine synthase, CYP80A1; EC1.14.19.66119).
4.1.1.4 BIA diversification from reticuline (Fig. 7)

(S)-Reticuline is converted to scoulerine by berberine bridge enzyme (BBE, EC.1.21.3.3), then benzophenanthridine alkaloids (e.g.,
sanguinarine and marcarpine), protoberberine alkaloids (e.g., berberine and palmatine), or a phthalideisoquinoline (e.g., nosca-
pine). Or, (S)-reticuline is converted by Papaver STORR (EC.1.14.19.154/ EC.1.5.1.27) to (R)-reticuline, then to morphinan
alkaloids (e.g., morphine and codeine). (S)-reticuline is converted to corytuberine by CjCYP80G2 (EC.1.14.19.51), then
N-methylated to magnoflorine (an aporphine alkaloid).
(S)-scoulerine can be converted to (S)-stylopine in benzophenanthridine alkaloid biosynthesis, by two P450s, (S)-
cheilanthifoline synthase (EcCYP719A5, AmCYP719A14; EC.1.14.19.65) and (S)-stylopine synthase (EcCYP719A2/A3,
AmCY719A13; EC.1.14.19.65), which form two methylenedioxy groups in products.128–130
On the other hand, (S)-scoulerine is converted to (S)-tetrahydrocolumbamine in protoberberine biosynthesis by the SAM-
dependent scoulerine 9-O-methyltransferase (CjSMT; EC.2.1.1.117265), and then to tetrahydroberberine (canadine) by a P450
canadine synthase (CDS or CjCYP719A1; EC.1.14.19.6856). The characterization of these enzymes has confirmed that berberine
biosynthesis proceeds via canadine and not via columbamine in C. japonica. Again, the enzyme substrate specificity shows a clear
preference for the pathway.
Scoulerine is also 9-O-methylated in benzophenanthridine biosynthesis to produce chelerythrine via allocryptopine in Califor-
nia poppy. Functional analysis of recombinant California poppy OMTs in reconstituted pathway indicated an enzyme with broad
substrate specificities and also multiple regio-specificity, i.e., scoulerine 9-O-/2-O-MT with reticuline 7-O-/30 -O-MT activity func-
tions in chelerythrine biosynthesis in California poppy and can be used for multiple biosynthesis in microbial system.138
4.1.1.5 Papaverine pathway

Papaverine is a simple BIA, but its biosynthetic pathway is controversial and still under debate. Direct examination of the metabolic
fate of the stable-isotope-labeled precursors in opium poppy suggested the central and earliest BIA, norcoclaurine, is converted into
(S)-reticuline and then sequentially methylated to (S)-laudanine, laudanosine, then N-demethylate to the known precursor of
Fig. 7 Late BIA biosynthesis derived from (S)-reticuline produce divergent chemicals. Enzyme reactions shown by dotted lines are not characterized yet at
molecular level. Enzyme names are shown in abbreviations.
papaverine, i.e., tetrahydropapaverine.266 On the other hand, gene-suppression and comparative transcriptomics studies suggested
the N-desmethyl pathway as the major route to papaverine.267,268
4.1.1.6 Morphinan alkaloid biosynthesis (Fig. 8)

(S)-Reticuline is converted to its (R)-enantiomer by STORR (also called S-reticuline epimerase,123–124) in morphinan alkaloid
biosynthesis. Subsequent intramolecular carbon-carbon phenol coupling of (R)-reticuline by a P450, salutaridine synthase (SalS/
CYP719B1; EC.1.14.19.67) forms salutaridine.120
Salutaridine: NADPH 7-oxidoreductase (SalR; EC. 1.1.1.253) then reduces salutaridine to (7S)-salutaridinol. Salutaridinol is
acetylated by acetylcoenzyme A: salutaridinol-7-O-acetyltransferase (SalAT/SAT; EC. 2.3.1.150269), then converted into thebaine via
the closure of an oxide bridge between C-4 and C-5. Whereas this cyclization step was thought to occur spontaneously, the
involvement of thebaine synthase (THS), a PR-10 homolog, was recently identified.105 Thebaine is then converted to codeinone by
thebaine demethylase (TODM; EC1.14.11.31), and then reduced to codeine by cytosolic NADPH-dependent codeinone reductase
(COR; EC1.1.1.247270) or to oripavine by codeine demethylase, then morphinone by COR. Recent study also revealed that COR
converts codeinone to neopinone and additional neiopinone isomerase (NISO) forms codeine from neopinone. Finally, codeine or
morphinone is demethylated by codeine demethylase (CODM; EC.1.14.11.32) or reduced by COR to give morphine.
4.1.1.7 Noscapine biosynthesis (Fig. 7)

Noscapine, a phthalideisoquinoline alkaloid in the latex of opium poppy, is a potential anticancer drug. The complete pathway of
noscapine has been characterized based on molecular genetics, functional genomics, and metabolic biochemistry and its microbial
production was established.271,272
Noscapine biosynthesis starts from the 9-O-methylation of (S)-scoulerine by scoulerine 9-O-methyltransferase (PsSOMT;
EC.2.1.1.117). O-Methylated product, (S)-tetrahydrocolumbamine, is converted to (S)-canadine by PsCYP719A21
(EC.1.14.19.68) with methylenedioxy bridge formation, and then N-methylated to N-methyl-canadine by a N-methyltransferase
H3CO H3CO H3CO
HO
NCH3 SalS HO SalR/SalATHO
H
HO
NCH3 NCH3
H H
H3CO H3CO H3CO
O AcO H
(R)-Reticuline
Salutaridine Salutaridinol-7-O-acetate
THS
HO H3CO
CODM
O
HO TODM NCH3
O
NCH3
H H
H3CO H3CO Thebaine
O Oripavine
H
NCH3 TODM
H3CO
O
COR Morphinone
O
HO
NCH3
H
O Neopinone
O
Plant Alkaloid Engineering

NCH3
H NISO
HO
H3CO H3CO
Morphine
O
CODM NCH3
COR O
H NCH3
H
HO
Codeine O Codeinone
29
103 104
Fig. 8 Biosyntheis of morphine from (R)-reticuline. Thebaine synthase (THS) and neopinone isomerase (NISO) were recently identified (see Section 3.4.1.1.4).
(EC.2.1.1.122). Recombinant CYP719A21 displayed strict substrate specificity and high affinity for (S)-tetrahydrocolumbamine.
VIGS confirmed the function of CYP719A21 in noscapine biosynthesis in opium poppy. N-methylcanadine is 1-hydroxylated by a
P450, CYP82Y1, which accepts (R,S)-N-methylcanadine as a substrate with strict specificity and high affinity, to 1-hydroxy-N-
methylcanadine. VIGS of CYP82Y1 significantly reduced noscapine, narcotoline, and a putative downstream secoberbine interme-
diate and also increased the upstream intermediates scoulerine, tetrahydrocolumbamine, canadine, and N-methylcanadine.60
1-Hydroxy-N-methylcanadine is further converted to narcotoline hemiacetal; Two P450s (CYP82X2, CYP82X1) introduce
hydroxylation at C13 and C8 on the protoberberine scaffold, in which the latter step induces ring opening and forms an aldehyde
moiety. Acetylation at C13 before C8 hydroxylation introduces a protective group subsequently hydrolyzed by a carboxylesterase,
which triggers rearrangement to a cyclic hemiacetal.122
4.1.1.8 Novel IQAs; amaryllidaceae alkaloids biosynthesis (Fig. 6)

Amaryllidaceae alkaloids with over 300 known structures include haemanthamine, haemanthidine, galanthamine, lycorine, and
maritidine attract high research interests,273 because galanthamine isolated from daffodil (Narcissus spp.), snowdrop (Galanthus
spp.), and summer snowflake (Leucojum aestivum), is a medicine for the symptoms of Alzheimer’s disease. Amaryllidaceae alkaloids
are synthesized from norbelladine, which is produced by the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-
DHBA) by norbelladine synthase (NBS). NBS, PR10 family protein, was isolated from wild daffodil (N. pseudonarcissus) transcrip-
tome, and enzyme activity was confirmed in E. coli.104
Norbelladine 40 -O-methyltransferase (NpN40 OMT) gene274 was identified from daffodil transcriptome using in silico search of
plant OMT candidates and biochemical characterization of recombinant protein expressed in E. coli. Next CdC phenol-coupling
reaction that can have para-para’, para-ortho’, or ortho-para’ regiospecificity, was also investigated through comparative transcrip-
tomics of Narcissus sp. aff. Pseudonarcissus, Galanthus sp., and G. elwesii. Then, para-para’ CdC phenol coupling P450, CYP96T1,
was identified to form (10bR, 4aS)-noroxomaritidine and (10bS,4aR)-noroxomaritidine from 40 -O-methylnorbelladine,275
whereas CYP96T1 also formed para-ortho’ phenol coupled product, N-demethylnarwedine, as <1% of the total product.
NpCYP96T1 and NpN40 OMT showed similar expression profile.
To identify the reductase involved in the reduction of norcraugsodine to norbelladine, a short chain alcohol dehydrogenase/
reductase was searched based on the coexpression analyses with N40 OMT from Narcissus sp. and Galanthus spp. Identified enzyme
(named noroxomaritidine reductase; NR) reduced norcraugsodine to norbelladine with a 400-fold lower specific activity than the
formation of oxomaritinamine from noroxomaritidine.276 This NR was suitable for abroad range of imine reduction as an enone
reductase and was active with all four tested imine compounds (up to 99% conversion, up to 92% ee).277 NR also reduced two keto
compounds as well.
4.1.1.9 Ipecac alkaloids

Ipecac alkaloids, such as emetine and cephaeline produced in Carapichea ipecacuanha (Ipecac, also named Psychotria ipecacuanha, or
Cephaelis ipecacuanha, Rubiaceae) is monoterpenoid-tetrahydroisoquinoline formed by condensation of dopamine and secologa-
nin. The condensed products N-deacetylisoipecoside (1a(S)-epimer) is deglycosylated for emetine biosynthesis, whereas its 1b(R)-
epimer N-deacetylipecoside is converted to ipecoside in C. ipecacuanha.278
Firstly, Ipecac alkaloid b-D-glucosidase (named IpeGlu1) gene was isolated from C. ipecacuanha. Recombinant IpeGlu1 prefer-
entially hydrolyzed glucosidic Ipecac alkaloids except for their lactams, but showed poor or no activity toward other substrates,
including terpenoid-indole alkaloid glucosides. Deglucosylated products of N-deacetylisoipecoside revealed spontaneous forma-
tion of the highly reactive aglycons, for emetine biosynthesis. Because IpeGlu1 activity was extremely poor toward 7-O-methyl and
6,7-O, O-dimethyl derivatives, and, 6-O-methyl derivatives were hydrolyzed as efficiently as nonmethylated substrates, 6-O-
methylation was suggested prior to deglucosylation by IpeGlu1. Because ipecoside (1b(R)) is a major alkaloidal glucoside in
C. ipecacuanha, the compartmentalization of IpeGlu1 from ipecoside is suggested.136
Three OMTs, i.e., IpeOMT1-IpeOMT3, coordinately transcribed with the IpeGlu1, were identified to encode ipecac alkaloid
OMTs of C. ipecacuanha. Biochemical characterization showed that N-deacetylisoipecoside was 6-O-methyled by IpeOMT1, with a
minor contribution by IpeOMT2, and deglucosylated by IpeGlu1. IpeOMT3 methylated 7-hydroxy group of the aglycon to form
protoemetine. Protoemetine is condensed with a second dopamine molecule, then sequentially O-methylated by IpeOMT2 and
IpeOMT1 to form cephaeline and emetine, respectively.139 On the other hand, another OMT, CiOMT1, with subtle sequence
difference from IpeOMTs was identified to catalyze both OMT reactions against 70 -O-demethylcephaeline to cepaheline and
emetine, suggesting high redundancy of OMTs in Ipecac alkaloid biosynthesis.140
4.1.2 Cell specific expression

Biosynthesis of certain alkaloids, such as morphinan alkaloids in opium poppy, needs cell-type-specific localization of biosynthetic
enzymes. In situ localization of gene transcripts indicated that seven biosynthetic enzymes (6OMT, CNMT, CYP80B, 40 OMT and
BBE in reticuline biosynthesis, and SAT and COR in morphine pathway) were localized in sieve elements, whereas corresponding
gene transcripts were localized in the supporting companion cells.279,280 Further analysis with polyclonal antibodies for each of six
enzymes involved in converting (R)-reticuline to morphine detected corresponding enzymes in sieve elements of the phloem, as for
all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Shotgun proteomics revealed that SalS was detected in the
whole stem, but not in the latex. The final three enzymes converting thebaine to morphine were most abundant in latex and a
limited occurrence in laticifers. Multiple isoforms of two key O-demethylases were detected in the latex by two-dimensional
immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to
morphine is predominant in adjacent laticifers, which contain morphine-rich latex.281
Tissue-type-specific expression was also reported in protoberberine alkaloid biosynthesis in C. japonica56 and Thalictrum flavum
spp.282 Whereas gene transcripts for biosynthetic enzyme were most abundant in rhizomes and lower level in roots and other organ
in T. flavum, higher expression was detected in roots and lower expression in rhizome and other organ in C. japonica. In situ RNA
hybridization analysis in T. flavum revealed that all transcripts were mainly localized in the immature endodermis and pericycle in
roots, while they were localized in the protoderm of leaf primordia in rhizomes. These data and an analysis of alkaloid
accumulation clearly indicated that distinct and different tissue types are involved in the biosynthesis and accumulation of BIAs
in C. japonica, T. flavum and P. somniferum. These and other results suggest diverse strategies for the biosynthesis and accumulation of
alkaloids as defensive compounds. Recruitment of cell types involved in BIA metabolism, both within and external to the phloem,
was likely driven by selection pressures unique to each species.283
4.1.3 Transport and accumulation

As mentioned before (see Section 3.7), plant alkaloids are often translocated from the biosynthetic site to storage tissue.193
Berberine in C. japonica is transported from root tissue to rhizome across several membranes. CjMDR (a MultiDrug Resistance
gene) was identified for the uptake and accumulation in rhizome.196 The characterization of the vacuolar transport of berberine by
H+/berberine antiporter indicates that multiple transporters are also involved in subcellular and intercellular transport.199 Differ-
ences in the inhibition of berberine transport by its analogs suggest that transporter activity might be critical for the biosynthesis.
Multidrug and toxic compound extrusion protein 1 (CjMATE1) is also suggested to act as a berberine transporter in cultured
C. japonica cells.201
Whereas the transporter activity of C. japonica vacuoles suggests the high affinity for reticuline, transgenic California poppy cells
with RNAi of BBE showed the secretion of large amounts of reticuline into the culture medium.33 When we consider the intercellular
network in BIA biosynthesis, especially of morphinan alkaloids, these transporter activities would be important determinants of BIA
biosynthesis.
4.1.4 Metabolic engineering

Since all biosynthetic genes from norcoclaurine to berberine, sanguinarine, noscapine and morphine have been isolated and their
recombinant enzyme activities have been demonstrated in microbial systems, the complete reconstruction of the biosynthetic
pathway of these BIAs is now accomplished in heterologous system in microbes (see Refs. 221,236,272,284 and also Section 4.1.5).
But, metabolic engineering in plant cells is still effective to produce specialized BIAs and understand the pathway.20 Whereas one-
step bioconversion of novel chemicals by biosynthetic enzyme is practical to produce stereo-specific and site-specific conversion,
this topic is discussed at the end of this section (also see Sections 3.4 and 3.10; e.g., NCS, NMT and other enzymes have high
potentials in chemical biosynthesis).
4.1.4.1 Over-expression of biosynthetic enzyme genes; pros and con

Whereas biochemical conversion is practical, the supply of substrates is often limited. Therefore, metabolic engineering using
introduction of biosynthetic enzyme genes to enhance the production or down-regulate the un-desired pathway in target plants is
more realistic. The first metabolic engineering in IQA biosynthesis was performed in California poppy with the overexpression of
C. japonica scoulerine O-methyltransferase (CjSMT) cDNA. The alkaloid profile was modified with the introduction of CjSMT; i.e.,
the alkaloid profile changed from sanguinarine (benzophenanthridine-type) to columbamine (protoberberine-type).213 However,
when alkaloid profiles of transformants were investigated more in details, 9-O-methylated scoulerine (tetrahydrocolumbamine)
was further metabolized in benzophenanthridine pathway to produce chelerythirine type alkaloids (one methylenedioxy bridge
and one set of methoxy and hydroxy group) instead of sanguinarine type (two methylenedioxy bridges). Since the main alkaloid in
California poppy is sanguinarine, overexpression of CjSMT competes the endogenous enzyme, i.e., chelerythrine synthase, which
also reacts with scoulerine, and shifted the flow from sanguinarine production to chelerythrine production.65,213 This result
indicates that our understanding of enzyme characteristics is limited and metabolic engineering provides more information
about the metabolic pathway.
Above experiment also provides that metabolic flow is dynamic and a newly introduced pathway can provide the substrate for
further enzymatic conversion and produce novel compounds, which are usually marginal in wild-type cells. This type of dynamic
metabolic flow is often observed in the down-regulation of certain metabolic step, whereas the effect of over-expression of
biosynthetic enzyme is more quantitative and not qualitative for the production; The overexpression of Cj6OMT in California
poppy cells, or overexpression of (S)-N-methylcoclaurine 30 -hydroxylase (CYP80B3) in opium poppy induced the large increase in
the amount of total alkaloid. This increase occurred either without changing the ratio of the individual alkaloids.207,209
It would be noted that over-expression of biosynthetic enzyme for target metabolites might induce the excess accumulation of
products, which would be toxic for the growth. Whereas C. japonica is a slow-growing plant to grow for >5 years for harvest,
overexpression of Cj40 OMT in Coptis plants with increase of berberine 1.5 fold inhibited the growth.285 On the other hand, opium
poppy transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1) showed significant increases in capsule
alkaloid content in glasshouse and field trials over 4 years without growth penalty. The morphinan alkaloid contents on a dry
weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control nontransgenic
segregant.208
4.1.4.2 Down-regulation of gene expression; pros and con

In the morphinan alkaloid biosynthesis, the top1 mutant of opium poppy overproducing thebaine286 has great economic interests.
Thus, the last step for morphine biosynthesis catalyzed by COR was knocked down with RNAi techniques.287 Unexpectedly, RNAi
with full-length cDNA of COR not only down-regulated COR but also stopped other biosynthetic activities and induced the
accumulation of reticuline. Whereas some enzymes form the large complex with other biosynthetic partner (see metabolon,
Section 3.9), the inhibition of biosynthesis with COR RNAi was the off-target effects on STORR, a fusion of P450 and reductase,
due to the sequence similarity of reductase part. This means that the results of RNAi silencing including VIGS should be examined
with specific cautions.
On the other hand, RNAi of BBE in California poppy induced the accumulation of reticuline without further interference in the
biosynthesis, whereas produced reticuline was mainly found in the medium.33 This secretion/leakage of metabolite might make the
product to escape from further catabolism, but accumulation of reticuline still formed 7-O-methylreticuline in California poppy
cells with BBE RNAi as opium poppy with reticuline accumulation induced by COR RNAi.282 This result also provides another
evidence that the accumulation of chemical(s) would induce the activation of a silence pathway or the creation of a new pathway
from an intermediate.
4.1.5 Metabolic engineering with transcription factor

Metabolic engineering using transcription factor in BIA biosynthesis was firstly done by Apuya et al.288 using Arabidopsis WRKY1 in
California poppy and opium poppy. Successful enhancement of BIA biosynthesis suggested the usefulness of TF activation.
However, metabolic engineering of BIA biosynthesis using native TF is not so easy.191
Even heterologous expression of CjWRKY1 in cultured California poppy cells induced limited increases in transcripts of limited
genes encoding BIA biosynthetic enzymes. These data indicated that each TF would have some specificity to activate down-stream
genes. In fact, several TFs, i.e., WRKY, bHLH and probably ERF are involved in BIA biosynthesis. They would be interacted and also
regulated posttranscriptionally191,289 as discussed in previous section (see Section 3.6).
4.1.6 Synthetic biology

Recent progresses in molecular biology in BIA pathway and synthetic biology have enabled the introduction of complete plant
pathways into microbes for the production of plant alkaloids. Microbial production of modified alkaloids has the potential to
accelerate the semi-synthesis of alkaloid-derived drugs by providing advanced intermediates that are structurally closer to the final
pharmaceuticals and used for the synthesis of novel drug.11,236,290,291 Some highlights of synthetic biology in BIA pathway are
summarized here.
4.1.6.1 Production of BIA from intermediates

4.1.6.1.1 Reticuline and related compounds from dopamine or norlaudanosoline ( Fig. 6)
So far microbial system was thought to be not suitable to produce plant alkaloid, because of their anti-microbial nature. However,
Minami et al.157 combined microbial monoamine oxidase (MAO), which produces 3,4-dihydroxyphenylacetaldehyde (3,4-
DHPAA) from dopamine, and four plant enzymes (i.e., NCS, 6OMT, CNMT and 40 OMT), which produce a key intermediate of
benzylisoquinoline alkaloids, i.e., (S)-reticuline, from dopamine, in transgenic E. coli with the yield of 11 mg/L reticuline.
Furthermore, an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, was synthesized from dopamine via
reticuline using cocultures of transgenic reticuline-producing E. coli and yeast cells with CYP80G2 or BBE, respectively, whereas the
final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/L culture medium.
These results indicate that microbial systems with incorporation of plant genes can produce plant BIAs, whereas hydroxylation
step catalyzed by CYP80B1 was skipped in this E. coli system via the production of norlaudanosoline from dopamine and its derived
3,4-DHPAA. On the other hand, Hawkins and Smolke290 used norlaudanosoline as starting material to skip entry NCS reaction in
BIA pathway and produced reticuline at 150 mg/L. Either results indicated the potentials of microbial production of plant BIAs.
4.1.6.1.2 More complex BIAs, i.e., protoberberine, benzophenanthridine or noscapine production from
norlaudanosoline (Figs. 6 and 7)
Production of protoberberine alkaloid from norlaudanosoline was conducted using seven enzymes Ps6OMT, Ps40 OMT, PsCNMT,
three membrane-bound enzymes (i.e., PsBBE, the canadine synthase (TfCYP719A1), and a cytochrome P450 reductase (AtCPR)),
and TfSMT. Enzyme variant screening, genetic copy number variation, and culture optimization increased the production of
canadine over 70-fold, whereas the yield was around 1.8 mg/L from 1 mM norlaudanosoline.292
With the characterization of sanguinarine pathway, the yeast cells with expression of 10 genes in three blocks [block1, Ps6OMT,
PsCNMT, Ps40 OMT; block2, PsBBE, Cheilanthifoline synthase (PsCFS; CYP719A25), Stylopine synthase (PsSPS; CYP719A20);
block3, PsTNMT, PsMSH, EcP6H] were constructed to produce dihydrosanguinarine in 1.5% conversion from 10 mM norlaudanoso-
line, whereas TNMT N-methylated scoulerine and cheilanthifoline as side-products.293 The production of side-product, such as
N-methylcheilanthifoline, in yeast was reduced by the screening of NMT and CYP719A; i.e., the conversion efficiency of norlau-
danosoline to dihydrosanguinarine was improved from 1.5% to 10%.294
Protoberberine producing platform developed by Galanie and Smolke292 was further modified with EcCFS, EcSTS, PsTNMT,
PsMSH, EcP6H, and AtCPR to produce 676 mg/L stylopine, 548 mg/L cis-N-methylstylopine, 252 mg/L protopine, and 80 mg/L
sanguinarine from 2 mM norlaudanosoline. Modification of PsBBE expression from a high-copy plasmid to the chromosome or
low-copy plasmid was effective to optimize the production; high copy plasmid of BBE to the chromosome improved the production
of scoulerine from 419 to 717 mg/L and conversion efficiency from 23% to 67%. Also, EcCFS was most active, when several CFSs
(i.e., EcCFS, AmCFS and PsCFS) were tested.295
Since noscapine biosynthesis enzyme genes were discovered in the cluster on the genome of P. somniferum, a 14-step biosynthetic
pathway of noscapine from the simple alkaloid norlaudanosoline was reconstructed in canadine-production yeast strain of seven
heterologous plant enzymes with additional expression of nine heterologous plant enzymes from the genes in gene cluster and
PsTNMT (in total 16 genes). Whereas genes in cluster contain four P450s (PsCYP719A21 as canadine synthase, CYP82Y1 as
1-hydroxy-N-methylcanadine synthase, CYP82X1 and CYP82X2), three methyltransferases (PsMT1 as scoulerine 9-OMT, PsMT2
and PsMT3), one carboxylesterase (PsCXE1), one short-chain dehydrogenase/reductase (PsSDR1), and one acetyl transferase
(PsAT1), PsCYP719A21 and PsMT1 were not used because previous construct contain necessary genes (CjCAS and PsSOMT) for
the production of canadine. Reconstruction of pathway in yeasts not only produced many pathway intermediates and a novel
derivative. This reconstitution experiment also revealed that MT2 and MT3 would be functional as a heterodimer in noscapine
biosynthesis.296
4.1.6.1.3 Protoberberine production from reticuline in Pichia using coculture system

Reticuline is the key intermediate in BIA biosynthesis and its de novo fermentation became available as mentioned below. Thus,
some BIAs production from reticuline was also investigated. For the production of protoberberine, benzophenanthridine or
noscapine, BBE expression is the bottom-neck reaction for the pathway reconstruction in microbe. So, stylopine biosynthesis
from reticuline was examined using EcBBE, EcCHS (CYP719A5), and EcSTS (CYP719A2), in a methanol-utilizing yeast (Pichia
pastoris) either in consolidated or coculture system. Although both systems efficiently converted reticuline to stylopine, the
consolidated system was more rapid and efficient than the coculture system. However, substrate-feeding experiments revealed a
decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the
coculture system remained constant, probably due to the compartmentation of enzyme in successive reactions. The final amount of
stylopine produced after successive feedings in the coculture system was >150 nmoles from 375 nmoles of (R,S)-reticuline.297
Interestingly, BIAs produced were mainly released into medium, then further converted except stylopine, which accumulated
in cells.
A OMT with broad substrate specificities, i.e., scoulerine 9-O/2-O-methyltransferase and also reticuline 7-O-/30 -O-methyltrans-
ferase, was isolated from California poppy.138 When this OMT was coincubated with BBE, CYP719A5, CYP719A2, CYP719A3,
TNMT and MSH in Pichia coculture system,297 cocultured Pichia cells produced N-methylstylopine and N-methylcanadine as most
abundant products. This result also suggested that BBE was more active than scoulerine/reticuline OMT against reticuline and only
low level of O-methylated reticuline was produced. On the other hand, this multifunctional OMT actively produced canadine with
CYP719A3, indicating that this coculture system would be useful to analyze the possible physiological role of enzymes in reactions.
Also, this coculture system has advantage to reconstruct the pathway easily by the mixing Pichia cell with expression of desired
enzyme.
4.1.6.1.4 Morphine biosynthesis from BIA intermediates ( Fig. 8)

Reconstruction of morphine biosynthesis in microbe was first tried from thebaine in yeast cells, which expressed T6ODM, CODM,
and COR1.3 with high affinity for codeinone genes from P. somniferum. Also, NADP-dependent morphine dehydrogenase (an aldo-
keto reductase; MorA) and NADH-dependent morphinone reductase (an a/b-barrel flavoprotein oxidoreductase; MorB) isolated
from Pseudomonas putida M10 were used to convert thebaine to semisynthetic opiates, hydromorphone, hydrocodone and
oxycodone. Optimization of strain titer, specificity by titrating gene copy number, and supply of cosubstrate (2-oxoglutarate for
T6ODM and CODM reaction) with high-density fermentation resulted in total opioid production up to 131 mg/L from 1 mM
thebaine, whereas the initial yield was 0.2 mg/L.298
Since pathway reconstruction for morphinan alkaloids from the precursor (R,S)-norlaudanosoline in recombinant yeast was
difficult due to the strict enantioselectivity of OMTs in reticuline production for (S)-reticuline from (R,S)-norlaudanosoline, the
pathway for codeine and morphine from (R)-reticuline was reconstructed with seven genes including salutaridine synthase (PsSalS),
salutaridine reductase (PsSalR) and salutaridinol acetyltransferase (PsSalAT) at the yield of ca. 0.3 mg/L. In this reconstruction, the
pH conditions had to be optimized to allow for productive spontaneous rearrangement of salutaridinol-7-O-acetate,299 because the
involvement of thebaine synthase for the conversion of salutaridinol-7-O-acetate to thebaine was only found recently.105
In transgenic yeast engineered to produce morphine from thebaine, the production of neopine and neomorphine hindered the
production efficiency of morphine. Recent finding of neopinone isomerase (NISO) and its expression in yeast dramatically
enhanced the formation of thebaine or semisynthetic opiates with the reduction of neopine and neomorphine accumulation.106
4.1.6.2 De novo synthesis of BIA intermediate

4.1.6.2.1 Reticuline production in E. coli
First de novo fermentative plant alkaloid production system from simple carbon sources was established in E. coli, using selected
enzymes to construct a tailor-made biosynthetic pathway through norlaudanosoline. Substrate supply pathway for the production
of reticuline was firstly designed; i.e., enhancement of aromatic amino acid production with the disruption of repressor tyrR gene
and introduction of two feedback inhibition resistant 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (fbr-
DAHPS: aroGfbr) and fbr-chorismate mutase/prephenate dehydrogenase (fbr-CM/PDH: tyrAfbr), and overexpression of key enzymes
in tyrosine biosynthesis, i.e., phosphoenolpyruvate synthetase (PEPS:ppsA) and transketolase (TKT: tktA) were integrated. Further-
more, tyrosinase (TYR) from Ralstonia solanacearum was selected instead of TYR from Streptomyces castaneoglobisporus to reduce the
o-diphenolase side-reaction for DOPA consumption. Transgenic E. coli with this improved dopamine production pathway and the
BIA biosynthetic enzymes, i.e., MAO, CjNCS, Cj6OMT, CjCNMT and Cj40 OMT, enabled the production of (S)-reticuline at the yield
of 46.0 mg/L culture medium without additional BIA substrates.154
To reduce the intermediate degradation by tyrosinase, more specific tyrosine hydroxylase reaction was also integrated; i.e.,
Drosophila melanogaster tyrosine hydroxylase and cofactor (tetrahydrobiopterin; BH4) regeneration system [rat (Rattus norvegicus)
sepiapterin reductase (SepR), rat 6-pyruvoyl-tetrahydropterin synthase (PTPS) and Bacillus subtilis GTP cyclohydrolase I] were
introduced into the pathway instead of tyrosinase. This modification improved the production of reticuline to 160 mg/L with
reduced byproduct formation.156
4.1.6.2.2 Reticuline production in yeast via norcoclaurine

The production platform of the key BIA intermediate (S)-reticuline from glucose was also constructed in yeast using a sugar beet
DOPA oxidase (CYP76AD1), which has tyrosine hydroxylase activity. Improvement of tyrosine hydroxylase activity of CYP76AD1
(W13L, F309L) with reduced DOPA oxidase activity by PCR mutagenesis increased its L-DOPA yields to about 3.6 mg/L from
1.3 mg/L. Production of dopamine from DOPA with coexpression of DOPA decarboxylase and production of 4HPAA by endog-
enous yeast pathway enabled the production of (S)-reticuline of 80 mg/L with expression of PsNCS (module B), and Ps6OMT,
PsCNMT, Ps40 OMT and EcCYP80B1 (module C).263
Yeast platform was also constructed with a rat tyrosine hydroxylase system, including four rat tetrahydrobiopterin biosynthesis
and recycling enzymes (sepiapterin reductase (SepR), 6-pyruvoyl-tetrahydropterin synthase (PTPS), quinonoid dihydropteridine
reductase (QDHPR) and pterin carbinolamine dehydratase (PCD)) to supply cofactor (BH4) for tyrosine hydroxylase. Coexpression
of a P. putida DOPA decarboxylase enabled norcoclaurine production with endogenous 4HPAA supplied via enhanced tyrosine
biosynthesis in yeast. Mutant tyrosine hydroxylase enzymes with reduced substrate and product inhibition, an optimized plant NCS
variant (i.e., CjNCS), and optimizing culture conditions with expression of five additional plant enzymes (three MTs; Ps6OMT,
PsCNMT and Ps40 OMT, and a P450; EcCYP80B1, and its reductase; PsCPR) produced reticuline at the yield of 19.2 mg/L from
glucose.155
4.1.6.2.3 De novo synthesis of morphinan alkaloids in yeast

Fermentative production of BIAs in microbes has gone beyond intermediates to desired products both in yeast and E. coli.
Engineered yeast to produce opioid compounds, such as thebaine and hydrocodone, was finally generated based on the de novo
reticuline-production platform and morphine-production platform from norlaudanosoline. Discovery of key enzyme, i.e., STORR,
enzyme engineering, and optimization of pathway and strain with the expression of 21 (thebaine) and 23 (two hydrocodone)
enzyme from plants, mammals, bacteria, and yeast achieved the opiate biosynthesis in single yeast cell, whereas the production was
far below the industrial production level.221
For the fermentative production, tyrosine overproduction module was designed in yeast with the overexpression of three or four
yeast proteins, which include mutants of DAHP synthase and chorismate mutase (Aro4p Q166K, Aro7p T226I) with less inhibition
by tyrosine, and transketolase (TKT1p) only or with additional phenylpyruvate decarboxylase (Aro10p). Cofactor BH4 module was
designed for the synthesis and recycle of BH4 using rat SepR, PTPS, QDHPR and PCD. Norcoclaurine module was designed with a
mutant tyrosine hydroxylase (TyrHWR with R37E, R38E, W166Y mutations), rat BH4 salvage enzyme dihydrofolate reductase
(DHFR), DOPA decarboxylase from P. putida, and CjNCS. Reticuline module consisted of Ps6OMT, PsCNMT, Ps40 OMT, PsCPR
and EcCYP80B1. Thebaine module consisted of newly identified PsDRS-DRR (STORR), expression optimized hybrid SalSyn
(yEcCFS1-83-yPbSalS92-504), PbSalR, and PsSalAT. Transgenic yeast with all modules produced 6.5 mg/L thebaine. Further expression
of PsT6ODM and morphine reductase (morB) from P. putida M10 in Yeast Artificial Chromosome produced hydrodcodone of
0.3 mg/L with addition of 50 mM 2-oxoglutarate for T6ODM activity.
Whereas the yield in the first attempt to produce thebaine in microbe was low, recent identification of thebaine synthase suggests
that the considerable improvement of thebaine yield is expected in engineered microbes as shown in transgenic yeast with thebaine
synthase; i.e., the conversion to thebaine with thebaine synthase expression was improved from 29 to 479 mg/L when 2.5 mM (R, S)-
norlaudanosoline was used as precursor.105
4.1.6.2.4 De novo synthesis of morphinan alkaloids in E. coli

After the first de novo production of opiate in yeast, fermentative production of opioid was also achieved in E. coli using stepwise
culture of four engineered strains. In E. coli system, (R)-reticuline was produced using CjCNMT and Cj40 OMT from racemic
norlaudanosoline, which was formed by spontaneous condensation of dopamine and 3,4-DHPAA without NCS reaction, because
expression of STORR in E. coli was not so successful, probably due to the membrane-bound nature of STORR. Racemic reticuline was
further converted with N-truncated PsSalS with Arabidopsis CPR (ATR2), PsSalR, PsSalAT to thebaine. Whereas racemic reticuline
was used, this E. coli system yielded thebaine at 2.1 mg/L, which is 300-fold higher than yeast production system, probably due to
strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli.279 In the production of thebaine, glucose was
also added to support the production of acetyl CoA for SalAT reaction. Mammalian CYP2D6 and CYP3A4 with SalS activity were
not used due to the undesirable by-product formation.
4.1.6.2.5 De novo synthesis of noscapine

After the establishment of norlaudanosoline to noscapine pathway in yeast,291 the de novo production of noscapine was
accomplished based on the de novo reticuline producing yeast platform with engineering of over 30 enzymes from plants, bacteria,
mammals, and yeast itself, including seven plant endoplasmic reticulum (ER)-localized enzymes. Improvement of (i) reticuline
production with codon-optimized TyrHWR, N-truncated NCS, and additional copy of 40 OMT, as well as (ii) enhanced noscapine
pathway with additional copy of SOMT and CYP82X2, (iii) enhanced NADPH generation system and (iv) enriched fermentation
medium (yeast extract with peptone) with 10% glycerol conditions improved the yield over 18,000-fold to 2.2 mg/L.272 These
integrated engineering suggests the more possibility to produce novel plant alkaloids in microbial system.
4.1.7 Enzymatic production; single or stepwise conversion

4.1.7.1 Enzymatic bioconversion
Simplest engineering in secondary metabolism is the bioconversion of metabolite using recombinant enzymes produced in
transgenic microbes. Norcoclaurine synthase (NCS) (Section 3.4.1.1), which catalyzes the stereo-selective Pictet-Spengler reaction
between dopamine and 4-hydroxyphenylacetaldehyde as the first step of BIA synthesis, was used alone or in combination for the
chemical expansion of drug discovery in BIAs. Relatively relaxed substrate specificity of NCS toward aldehydes enabled to synthesize
unnatural, optically active tetrahydroisoquinolines.100,101 Especially, N-terminal truncation of NCS i.e., NCS-D 29, showed 40-fold
higher activity than intact CjNCS protein, probably due to the deletion of vesicular localization signal sequence. NCS-D 29 produced
various 1-substituted-1,2,3,4-tetrahydroisoquinolines stereospecifically from dopamine and aromatic aldehydes or n-alkyl alde-
hydes as substrates; e.g., 6,7-dihydroxy-1-phenethyl-1,2,3,4-tetrahydroisoquinoline (19.7 g/L) was produced with hydrocinnamal-
dehyde, and 6,7-dihydroxy-1-propyl-1,2,3,4-tetrahydroisoquinoline (13.5 g/L) was produced with 1-butylaldehyde in molar yields
of 86.0% and 99.6% and in enantiomer excess of 95.3% and 98.0%, respectively.101 Production of 6,7-dihydroxy-1-phe-
nethyl-1,2,3,4-tetrahydroisoquinoline was especially significant because its derivatives are in high demand as pharmaceutical
intermediates. These chemicals would be useful substrate to produce phenethyl isoquinoline alkaloids such as dysoxyline and
colchicine.
4.1.7.2 Stepwise production

Bacteria lack organelles and the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently
inhibited or disrupted by undesirable reactions. Indeed, the yield to synthesize norlaudanosoline using a single strain of engineered
E. coli154 was low (<5 mM), mainly because of the oxidation of norlaudanosoline by tyrosinase, an essential enzyme in this system.
Thus, the stepwise production of norlaudanosoline to separate dopamine-production and the subsequent norlaudanosoline-
production greatly improved the yield of norlaudanosoline to 1.0 mM (287 mg/L).300
Stepwise enzymatic production of (S)-benzylisoquinoline was also achieved in the combination of Chromobacterium violaceum
transaminase (TAm) and N-truncated TfNCS. TAms were used instead of MAO to control the conversion of dopamine to aldehyde
with the limitation of cosubstrate (pyruvate) quantity. (S)-tetrahydroprotoberberines were further produced by the chemical
cyclization with formaldehyde in a one-pot, one-substrate “triangular” cascade.102
More radically, norlaudanosoline (3.4 g/L) containing supernatant produced by conversion of 100 mM dopamine using
transgenic E. coli expressing MAO and NCS was reacted with crude enzyme mixture of Ps6OMT, PsCNMT and Cj40 OMT expressed
in E. coli to produce (S)-reticuline at the yield of 593 mg of pure (S)-reticuline from 1 L of the reaction mixture.301
Stepwise strategy is also useful even in eukaryotic cells, such as yeast. As mentioned before (Section 4.1.6.1.3), the consolidated
system in P. pastoris revealed a decrease in the conversion efficiency from reticuline to stylopine during successive cultures, but the
conversion efficiency in the coculture of each independent enzyme expressing yeast remained constant, suggesting the product
inhibition of catalytic enzyme and efficiency of separation of pathway.297
Ephedrine is not IQA but similar strategy using enzymatic bioconversion can be applicable for the production from phenyla-
cetylcarbinol. PaNMT enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and
pseudoephedrine, respectively, by the coexpression of recombinant PaNMT and an o-transaminase (PP2799) from Pseudomonas
putida in E. coli.78
4.1.7.3 Novel nonnatural metabolite productions using feeding chemicals and nonplant enzymes
Obvious advantage in enzymatic production is to produce nonnatural metabolites artificially. Not only novel reaction intermedi-
ates can be produced artificially with the modified combination of enzymes (e.g., 138), but also feeding of unnatural substrates,272
or introduction of modification enzymes such as human sulphotransferases can be used to produce novel chemicals for drug
discovery.156 For example, expression of hSULT1E1op in reticuline fermentative E. coli produced (S)-reticuline 30 -O-sulphate from
glucose in a one-pot culture at titers reaching 90.9 mg/L. Or, feeding modified tyrosine derivatives assisted the production of
halogenated reticulines in noscapine-producing strain,272 indicating that BBE is critical for further modification in BIA pathway.
4.2 Engineering in Terpenoid Indole Alkaloid Biosynthesis

Terpenoid indole alkaloids (TIAs) or monoterpenoid indole alkaloids (MIAs) consist of about 3000 compounds, including the
antineoplastic vinblastine from Madagascar periwinkle (Catharanthus roseus), camptothecin from Camptotheca acuminata, and the
antimalarial quinine from Cinchona spp.302
Antineoplastic vinblastine and vincristine are essential medicine listed in WHO list and extracted from Madagascar periwinkle
(Catharanthus roseus). Because C. roseus is a rich source of MIAs, including aspidosperma, corynanthe, iboga, and bisindole types, but
the contents of vinblastine and vincristine in plant is rather low, biotechnological production of these MIAs in C. roseus is intensively
investigated and made this plant as a model plant in molecular studies on MIA biosynthesis.
Camptothecin (CPT) is a water-insoluble quinoline alkaloid with a unique pentacyclic structure, and used as the substrate to
form water-soluble anti-cancer drugs such as topotecan and irinotecan. CPT, which lacks the basic indole structure, is synthesized
via the MIA pathway from tryptophan and terpenoid precursors in Camptotheca acuminata and Nothapodytes foetida, whereas at least
other 14 CPT producing plants including Ophiorrhiza filistipula (Rubiaceae), and O. pumila are known.303 The biosynthesis of CPT is
unique among MIAs, because strictosidine is not immediately deglycosylated. Instead, a lactam is formed between the amine of
strictosidine and the methyl ester derived from the secologanin moiety to yield the intermediate strictosamide. Computer-aided
atomic reconstruction of metabolism and tracer experiments with [1-13C] glucose indicated that CPT is formed by the combined
activities of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway.304
Cinchona species (C. robusta, C. officinalis, C. ledgeriana) produce variety of quinoline alkaloids including quinine, a highly
effective anti-malarial agent. Feeding studies with radiolabeled tryptophan, monoterpenes, and strictosidine indicate that the
Cinchona quinoline alkaloids are derived from strictosidine.302
R. serpentina, along with other Rauwolfia species, produces numerous MIAs such as ajmaline, yohimbine and ajmalicine. Since
Rauwolfia is slow-growing woody plant and difficult to cultivate, the biosynthetic pathway for ajmaline in R. serpentina is intensively
characterized with in vitro cell culture system.305
4.2.1 Outline of biosynthesis

The central intermediate in the biosynthesis of MIAs is strictosidine, which is produced from tryptamine and the iridoid glucoside
secologanin by strictosidine synthase (STR, EC4.3.3.2, Fig. 3). Tryptamine is produced by tryptophan decarboxylase (TDC,
EC4.1.1.105). Whereas a single gene in C. roseus responds in both developmental and inducible expression,306 two genes in
C. acuminata showed different expression profiles, suggesting that one is involved in developmentally controlled camptothecin
production in shoot apex and bark, while the other is involved in an inducible defense mechanism.307
4.2.1.1 Biosynthetic genes involved in the iridoid pathway19

The first step to iridoid biosynthesis is generation of 10-hydroxygeraniol from geraniol by a P450 (geraniol 10-hydroxylase,
CYP76B6), which was cloned and functionally characterized from C. roseus.308 10-Hydroxygeraniol is further oxidized into the
dialdehyde 10-oxogeranial by an oxidoreductase, then converted by NADPH-dependent cyclase, iridodial synthase (IRS).309 The
cyclized product is further oxidized by a P450 to form deoxyloganetic acid, glucosylated to produce deoxyloganic acid, and then
hydroxylated by deoxyloganic acid hydroxylase (DLH) to form loganic acid. Loganic acid was methylated by loganic acid
methyltransferase (LAMT) to form loganin.310 Finally, the cyclopentane ring of loganin is cleaved by secologanin synthase (SLS,
EC1.3.3.9), an unusual P450 (CYP72A1) with epidermis-specific expression in immature leaves of C. roseus.311
The supply of terpenoid precursors should be rate limiting in MIA biosynthesis. The addition of secologanin or loganin to
C. roseus cell culture increases alkaloid accumulation and the level of G10H activity is positively correlated with the accumulation of
alkaloids.312 However, secologanin is inefficiently used in STR when added to the medium, since exogenous secologanin appears to
be compartmentalized differently from endogenous secologanin. These isoprenoid precursors are derived from a nonmevalonate
pathway in plastids.
4.2.1.2 Strictosidine synthesis and downstream pathway

Strictosidine is produced from tryptamine and the secologanin by strictosidine synthase (STR), a key enzyme in MIA biosynthesis.
cDNAs of STR have been isolated from Rauvolfia serpentina313 and C. roseus.314 Strictosidine is deglucosylated by strictosidine
glucosidase (SGD, EC 3.2.1.105), and then converted via several unstable intermediates.
Chemical diversity is generated from the intermediate strictosidine. The concerted action of two enzymes commonly involved in
natural product metabolism, i.e., an alcohol dehydrogenase and a P450, produces diversified rearrangements in strictosidine when
assayed simultaneously. The tetrahydro-b-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid.
Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-
cancer agents, vinblastine and vincristine, as well as other biologically active compounds.315
Reduction of strictosidine aglycone with NaBH4 or NaCNBH3 yielded the heteroyohimbines, ajmalicine (raubasine), tetrahy-
droalstonine, and 19-epi-ajmalicine, which differ only in the stereochemical configuration at carbons 15, 19, and 20, in various
ratios. These three diastereomers were again observed, also in varying relative amounts, when crude C. roseus protein extracts were
incubated with strictosidine aglycone and NADPH, but not in the absence of NADPH. Based on the observations, cDNA of an
alcohol dehydrogenase homolog, which converts strictosidine aglycone to the heteroyhimbine-type alkaloid tetrahydroalstonine
was isolated.316
O N
N
HO
N Ajimaline N
OH
N O (Rauwolfia) N OCOCH3
H H
O CO2CH3
OH O O N HO CO2CH3
N
Campthothecin H3CO
CO2CH3 R
(quinoline type,
Quinine N
Camptotheca) R=CH3, Vinblastine
(quinoline type,
N R=CHO, Vincristine
Cinchona) H Polyneuridine
aldehyde
NH N
N
H
N H
(sarpagan type) Leaf
H OGlc H H
O OH N
H3CO O SGD O
O Strictosidine aglycon N
H
O O
Strictosidine
N PAS/DPAS
CS Catharanthine
N
OH (Catharanthus)
H
O O
Stemmadenine TS
Root O N
N H TEX1/TEX2 N
OCOCH3
T19H(CYP71BJ1)
OCOCH3
H3CO N HO CO2CH3
N CO2CH3 TAT Tabersonine N CO2CH3 C
H3
H H
(aspidosperma type) CYP71D12/351 Vindoline
(T16H)/16OMT/ (Catharanthus)
19-O-Acethylhoerhammericine
NMT/D4H/DAT
Fig. 9 Many MIAs are synthesized from strictosidine. Strictosidine glucosidase (SGD), reticuline oxidase homolog (PAS), alchol dehydrogenase (DPAS), tabersonine
synthase (TS), catharanthine synthase (CS), tabersonine 16-hydroxylase (T16H), 16-hydroxytabersonine 16-O-methyltransferase(16OMT), 16-methoxytabersonine
N-methyltransferase (NMT), deacetylindoline O-acetyltransferase (DAT), desacetoxyvindoline 4-hydroxylase (D4H).
A short-chain alcohol dehydrogenase (SDR) isolated from C. roseus and R. serpentina reduced strictosidine aglycone and
produced an alkaloid that does not correspond to any previously reported compound. Structural characterization of this product,
named vitrosamine, as well as the crystal structure of the SDR highlighted the structural versatility of the strictosidine aglycone
biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyze.317
Sarpagan bridge enzyme (SBE) catalyzes the key cyclization reaction for the central MIA intermediate strictosidine and produces
sarpagan, ajmalan and alstophyllan alkaloid classes. Based on the hypothesis that SBE is a cytochrome P450 that acts on
geissoschizine, the product of a soluble reductase and strictosidine aglycone, P450s were identified from three monoterpene indole
alkaloid-producing plants (R. serpentina, Gelsemium sempervirens and C. roseus) that provide entry into two distinct alkaloid classes,
the sarpagans and the b-carbolines using Agrobacterium-mediated transient expression in Nicotiana benthamiana as discussed in
previous Section 3.8.2.2.2.232
Two missing enzymes necessary for vinblastine biosynthesis in C. roseus: an oxidase and a reductase that isomerize stemmade-
nine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or taberso-
nine via two hydrolases characterized herein, were isolated using sophisticated biochemistry and transient expression in
benthamiana tobacco.88
4.2.1.3 From tabersonine to vindoline

The first of six steps in the conversion of tabersonine to vindoline consists of hydroxylation at the C-16 position by tabersonine
16-hydroxylase (T16H, EC 1.14.14.103), a P450 encoded by CYP71D12.126 Interestingly, C. roseus has at least two T16H genes,
whereas it has a single copy of the TDC, STR and cytochrome P450 reductase (CPR) genes. The 16-hydroxylation of tabersonine is
followed by 16-O-methylation by a cytosolic SAM: 16-hydroxyltabersonine OMT (16OMT, EC 2.1.1.94318), hydration of the 2,3-
double bond by an as yet uncharacterized enzyme, and N-methylation of the indole-ring nitrogen by a thylakoid-associated SAM:
2,3-dihydro-3-hydroxytabersonine-NMT. Recombinant NMT isolated from C. roseus was highly substrate specific, and showed a
strict requirement for a 2,3-dihydro bond in the aspidosperma skeleton.148
CYP71D12 was firstly isolated as T16H from undifferentiated C. roseus, but CYP71D351 with T16H activity was also isolated.
Whereas both CYP71D12 and CYP71D351 exhibit high affinity for tabersonine and narrow substrate specificity, CYP71D12
(T16H1) expression is restricted to flowers and undifferentiated cells, and the CYP71D351 (T16H2) expression profile is similar
to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization and in situ
hybridization analyses demonstrated the specific localization of CYP71D351 mRNAs in leaf epidermis, as 16OMT. Comparison of
high- and low-vindoline-accumulating C. roseus cultivars also showed the direct correlation between CYP71D351 transcript and
vindoline levels. In addition, VIGS confirmed the involvement of CYP71D351 in vindoline accumulation in leaves.319
The penultimate step in vindoline biosynthesis is catalyzed by a cytosolic 2-oxoglutarate-dependent dioxygenase(2ODD) that
hydroxylates the C-4 position of desacetoxyvindoline (desacetoxyvindoline-4-hydroxylase; D4H320), and the final step is catalyzed
by the cytosolic acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase (DAT321).
A P450 (CYP71D1, named 16-methoxytabersonine 3-oxygenase, 16T3O), which forms an epoxide that can undergo rearrange-
ment to yield the vincamine–eburnamine backbone, was identified for the biosynthesis in the aspidosperma- and eburnamine-type
alkaloids.322 C2–C3 epoxidation by tabersonine 3-oxidase (T3O) yielding to 16-methoxytabersonine epoxide/imine alcohol, when
this reaction is performed on 16-methoxytabersonine or to tabersonine epoxide.
4.2.1.4 Root MIA pathway

Lochnericine is a major MIA in the roots of C. roseus. Lochnericine is derived from the stereoselective C6, C7-epoxidation of
tabersonine and can be metabolized further to generate other MIAs. By combining gene correlation studies, functional assays, and
transient gene inactivation, two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-
epoxidase isoforms 1 and 2 (TEX1 and TEX2) were isolated. Both P450s are quite divergent from the previously characterized
tabersonine 2,3-epoxidase and more closely related to T16H in vindoline biosynthesis in leaves. Biochemical characterization of
TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating
that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary
expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. TEX1 and TEX2 were estimated to be originated
from a gene duplication and acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis.323
In tabersonine metabolism in root, tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT) have
been isolated. T19H, CYP71BJ1, which stereoselectively hydroxylates tabersonine and lochnericine at the 19-position of the
aspidosperma-type alkaloid scaffold, is a branch point enzyme to either vindoline or 19-O-acetylhörhammericine.127 On the
other hand, the recombinant MAT isolated catalyzes MIA acetylation at low efficiency in vitro, but MAT was inactive when expressed
in yeast and in planta. Therefore, native enzyme for tabersonine derivative 19-O-acetyltransferase (TAT), which acetylates the
19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, was searched. Kinetic studies showed
that the recombinant TAT newly identified was specific for root MIAs and showed up to 200-fold higher catalytic efficiency than
MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in N. benthamiana
supported the prominent role of TAT in acetylation of root-specific MIAs.324
4.2.1.5 Gelsemium alkaloids

The gene cluster was also found in the genome of the oxindole MIA producer Gelsemium sempervirens (Gelsemiaceae) using a gene
cluster from C. roseus, although the corresponding enzymes act on entirely different substrates.84
4.2.2 Cell specific expression and transcription factors

4.2.2.1 Localization of biosynthesis of MIAs
A series of experiments with leaves of C. roseus showed that at least three cell types are involved in vindoline biosynthesis. The
nonmevalonate pathway genes (1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and
2C-methyl-D-erythriotol 2,4-cyclodiphosphate synthase) as well as G10H were expressed in internal phloem parenchyma of the
young aerial organs. Other early-stage enzymes in the biosynthesis of strictosidine, such as TDC, SLS and STR, were expressed
specifically in the upper and lower epidermis of young leaves, stem, and flower buds. Late-stage enzymes in vindoline biosynthesis,
such as D4H and DAT, were localized in laticifer and idioblast cells, which show greater yellow autofluorescence with few
chloroplasts, compared to the surrounding red-autofluorescent mesophyll cells. Light is required for activation of the localized
expression of the late stages of vindoline biosynthesis, whereas it is not required for the formation of laticifer and idioblast cells.325
Vindoline biosynthesis is restricted to the aboveground organs, and the pathway beyond tabersonine is not expressed in tissue
cultures, whereas catharanthine accumulates in cultured cells as well as etiolated seedlings. These results, along with the recovery of
vindoline biosynthesis in regenerated shoots, suggest that the biosynthesis of catharanthine and vindoline is differentially regulated
and that vindoline biosynthesis is under more rigid tissue-, developmental- and environmental-specific control than that of
catharanthine.326
4.2.2.2 Transcription factors

The transcription factors in MIA biosynthesis was intensively investigated but still remains fragmentary.327 First TF in MIA
biosynthesis, Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) with a jasmonate (JA)-responsive
ERF/AP2 domain, was isolated from C. roseus using activation tagging.175 However, no MIAs were induced by the ectopic expression
of ORCA3 in cultured C. roseus cells, although this increased the expression of the MIA biosynthetic genes and the accumulation of
tryptophan and tryptamine.
ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. The ORCA gene cluster was differentially
regulated and ORCA4 modulated an additional set of MIA genes. Unlike ORCA3, ORCA4 overexpression dramatically increased
MIA accumulation in C. roseus hairy roots. In addition, CrMYC2 was capable to activate ORCA3 and coregulated MIA pathway genes
with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously unchar-
acterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated MIA pathways genes
and increased MIA accumulation.176
Beside ERF type TF (ORCAs), a JA-regulated bHLH, iridoid synthesis 1 (BIS1), was isolated in C. roseus. BISI transactivated the
expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor
geranyl diphosphate to the iridoid loganic acid and also acted in a complementary manner to ORCA3. In contrast to ORCA3,
overexpression of BIS1/2 was sufficient to produce high value iridoids and MIAs in C. roseus suspension cell cultures. Thus, BIS1/2
would be an effective tool to produce MIAs in C. roseus plants or cultures (186,187; also see Section 4.2.3).

Metabolic engineering in MIA has been reviewed.328 Therefore, some highlights are shown here.
RNA mediated suppression of tryptamine biosynthesis in C. roseus hairy root culture eliminated all production of MIA.
Suppression of tryptamine biosynthesis, however, did not affect the expression of downstream biosynthetic enzymes and growth
or development in C. roseus root culture. Thus, an unnatural tryptamine analog was added to the culture media and successfully
converted to a variety of novel products derived.254
Further alternation was done with the replacement of the indole with one of four aza-indole isomers. Two aza-tryptamine
substrates were successfully incorporated into the products of MIA pathway in C. roseus.329
Interestingly, some combinatorial effects of overexpression of biosynthetic enzyme gene and transcription factor or that of
multiple transcription factors were examined in MIA biosynthesis. Pan et al.330 reported that cooverexpression of G10H with
ORCA3 significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine in comparison with single
ORCA3 overexpression, whereas this coexpression still showed limited effects on anhydrovinblastine and vinblastine levels.
Furthermore, Schweizer et al.331 examined combinatorial overexpression of the transcriptional activators BIS1, ORCA3 and
MYC2a, and revealed the transcriptional control of MIA biosynthesis. More interestingly, using the expression of an engineered
de-repressed MYC2a, they succeeded to increased accumulation of MIAs considerably. This success would provide the model of
future metabolic engineering with transcription factors.
4.2.4 Synthetic biology

De novo production of strictosidine was successfully reconstructed in yeast with 14 known MIA pathway genes, along with an
additional seven genes and three gene deletions that enhance secondary metabolism.332 Yeast was used as a host, because functional
expression of microsomal plant P450s. To enhance genetic stability, the necessary biosynthetic genes was integrated by homologous
recombination to the genome under the control of strong constitutive promoters (TDH3, ADH1, TEF1, PGK1, TPI1). To increase flux
through the isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) pathway in the yeast, a truncated HMG CoA
reductase gene (tHMGR), which encodes a non–feedback regulated rate-limiting enzyme in the mevalonate pathway; a second copy
of IDI1, the gene encoding IPP isomerase to increase DMAPP formation; (iii) a second copy of MAF1, a negative regulator of tRNA
synthesis, to direct IPP away from tRNA synthesis and into GPP production were integrated. Since yeast ERG20 with dual geranyl
pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) synthase activity does not release GPP in reaction, a GPP-specific synthase
from grand fir (Abies grandis; AgGPPS2) was integrated in yeast, and the native ERG20 gene was replaced to increase the production
of GPP, with mFPS144 (N144W mutation) from Gallus gallus, which has a much greater GPP synthase activity relative to FPP
synthase activity. Finally, all required biosynthetic genes for strictosidine production (geraniol synthase (GES), geraniol
8-hydroxylase (G8H), 8-hydroxygeraniol oxidoreductase (GOR), iridoid synthase (IS), 7-deoxyloganetic acid synthase/iridoid
oxidase (IO), 7-deoxyloganetic acid glucosyl transferase (7-DLGT), 7-deoxyloganic acid hydroxylase (7-DLH), loganic acid
O-methyltransferase (LAMT), SLS, STR, TDC) characterized in the MIA pathway of C. roseus were stably incorporated into the
transgenic yeast. Transgenic yeast strain showed improved production of 0.5 mg/L strictosidine and 0.8 mg/L loganin in the media
when codon optimized G8H plasmid was introduced. Accumulation of loganin also suggested that SLS may be another rate limiting
step beside G8H.
Noble MIA biosynthetic pathway was also established by mixing spatially separated enzyme expression in the plant. That is,
root-specific T19H and TAT were expressed with leaf specific T16H2 and 16OMT in yeast to produce tailor-made MIA such as
16-methoxy 19-acetyltabersonine from tabersonine.325
4.3 Nicotine and Tropane Alkaloids

Nicotiana, a member of the Solanaceae family, is important model plants, and of high agricultural and economic value. Several
important genetic maps, including a high-density map of N. tabacum consisting of 2000 markers were published in 2012 and four
whole genome sequences are determined in allotetraploid species, including N. benthamiana in 2012, and three N. tabacum cultivars
in 2014. Whole genome sequences of diploids, including progenitors N. sylvestris and N. tomentosiformis were also determined in
2013. These and additional studies on genes involved in the nicotine biosynthetic pathway derived from putrescine and the
regulation of nicotine production were summarized and compared with tomato and potato.333,334
Tropane alkaloids (TAs), which contain tropane ring, are also produced from putrescine. Whereas cocaine produced by
Erythroxylum coca is one of well-known TAs, atropine (racemic hyoscyamine) and scopolamine are other important TAs as essential
medicine listed in WHO and their biotechnological production has been investigated intensively.256 Several solanaceous plants
including thorn apple (Datura stramonium), mandrake (Mandragora officinarum), henbane (Hyoscyamus niger), belladonna (Atropa
belladonna) and corkwood tree (Duboisia spp.) produce hyoscyamine and/or scopolamine.335 Apparently, nicotine and tropane
alkaloids share the same evolutionary origin during diversification of the Solanaceae. Other classes of alkaloids derived from
putrescine include nortropane alkaloids, calystegines, and pyrrolizidine alkaloids, such as retronecine. Interestingly, Duboisia species
synthesize high levels of both nicotine and tropane alkaloids.
4.3.1 Enzymes, transcription factors and other components involved in biosynthesis ( Fig. 10)
4.3.1.1 Early biosynthetic pathway common for nicotine and tropane alkaloids
Both nicotine and tropane alkaloids are synthesized from a symmetrical diamine, putrescine, whereas putrescine is formed from
basic amino acids, ornithine and/or arginine, and is metabolized to higher polyamines in all organisms and to particular alkaloids
in a few plant species.193
L-Ornithine is converted to putrescine by ornithine decarboxylase (ODC; EC 4.1.1.17), whereas L-arginine is first decarboxylated
to agmatine by arginine decarboxylase (ADC; EC 4.1.1.19), then to putrescine via N-carbamoylputrescine. RNAi of ODC in
N. tabacum showed a marked reduction of nicotine and increase in anatabine levels. Treatment of ODC RNAi hairy roots with
methyl jasmonate, or wounding of transgenic plants did not restore normal nicotine synthesis in transgenic tissue, but markedly
increased anatabine. These results indicated that putrescine production via ODC is important for the nicotine biosynthesis.336
Putrescine N-methyltransferase (PMT; EC 2.1.1.53) catalyzes SAM-dependent methylation of putrescine.337 The
N-methylputrescine is the first specific metabolite for the biosynthesis of nicotine, tropane, and nortropane alkaloids. PMT genes
with sequence similarity with spermidine synthase (SPDS) were cloned from tobacco and other Solanaceae and characterized in
N
O CH3
AO2/QS/QPT2 N
OH
Aspartate N
Nicotine
Ornithine
Nicotinic acid BBL/A622
ODC2
PMT NH NH2
MPO1
NH CHO N N-methylpyrolium cation
NH2
H2N CH3 CH3 CH3
Putrescine 4-methyaminobutanal
N-methylputrescine + malonyl CoA
PYKS
Methylecogonone
O
O
OH
4-(1-methyl-2-pyrrolidinyo) MecgoR
N
-3-oxobutanoic acid
Phenylalanine CH3
N CH3
CYP82M3
Phenyllactoyl-CoA Methylecogonine
Tropinone
O
N CH3 N CH3
N CH3 N CH3 TRI N CH3 O
O
H OH H OH H Tropine OCH3
OH
O H6H O
CYP80F1 O OH O
O O O
O
Scopolamine Hyoscyamine Littorine Cocaine
Fig. 10 Nicotine and tropane alkaloids (TAs) are synthesized from putrescine via N-methylputrescine. ODC, ornithine decarboxylase; PMT, putrescine
N-methyltransferase; MPO, N-methylputrescine oxidase; TR, tropinone reductase; CYP80F1, littorine mutase.
E. coli. Although tobacco PMT differs from SPDS by the presence of tandem repeats of 11 amino acid residues at the N-terminus, the
repeat element is not required for enzymatic activity. Five PMT genes of tobacco have variable repeat numbers, whereas the tandem
repeats are absent in PMTs from Solanaceae plants that produce tropane alkaloids and calystegines.338–340 PMT and SPDS bind the
same substrate putrescine and similar cosubstrates, SAM and decarboxylated S-adenosylmethionine.143
Diamine oxidase (DAO; EC 1.4.3.6) catalyzes the deamination of N-methylputrescine to give 4-methylaminobutanal, which is
spontaneously cyclized to N-methylpyrrolinum cation. The DAO, specifically called N-methylputrescine oxidase (MPO) in nicotine
and tropane alkaloid biosynthesis was first characterized from N. tabacum and the corresponding gene has been isolated.341,342
MPO belongs to a class of copper dependent diamine oxidases.
The DAOs involved in nicotine and tropane alkaloid biosynthesis have higher affinity for N-methylputrescine than for
putrescine and other symmetrical diamines,343 and thus are often referred to as N-methylputrescine oxidase. In contrast, pea and
pig DAOs bind N-methylputrescine with low affinity.
4.3.1.2 Nicotine biosynthesis

In tobacco, N-methylpyrrolinum cation condenses with nicotinic acid, or its derivative, to give nicotine. Nicotinic acid is a
metabolite in the salvage pathway of NAD. In Arabidopsis thaliana, and probably in other dicotyledonous species, NAD is
synthesized from L-aspartic acid.344 The early NAD biosynthetic pathway consists of aspartate oxidase (AO; EC 1.4.3.16), quino-
linate synthase (QS), and quinolinic acid phosphoribosyl transferase (QPT; EC 2.4.2.19), all of which are localized in the plastid
and are coordinately regulated with nicotine biosynthesis.344,345
Whereas the mechanism for final condensation reaction to produce nicotine is not confirmed, pinoresinol-lariciresinol
reductase/isoflavone reductase/phenylcoumaran benzylic ether reductase (PIP) family oxidoreductase, A622, is required for the
biosynthesis of tobacco alkaloids, possibly in a step to produce a nicotinic acid-derived precursor, but the exact enzymatic reaction
catalyzed by A622 is not known.346 Suppression of the BBE-like (BBL) genes in tobacco hairy roots or in tobacco plants also highly
reduced nicotine production with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. Suppression of
BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids in the JA-elicited cultured tobacco cells,
suggesting that BBLs are involved in a late oxidation step subsequent to the pyridine-ring condensation reaction in the biosynthesis
of tobacco alkaloids.347
Conversion of nicotine to nornicotine is catalyzed by a cytochrome P450 (CYP82E4), nicotine demethylase. RNAi of CYP82E
expression suppressed nornicotine formation in “converter” tobacco plants.348
4.3.1.3 Tropane alkaloids pathway

N-Methylpyrrolinum cation is also metabolized to tropinone in tropane alkaloid-producing plants. N-methylputrescine is oxidized
by MPO and produces N-methylpyrrolinum cation. A root-expressed type III polyketide synthase from A. belladonna (AbPYKS) was
recently identified to form 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid from an unconjugated N-methyl-D-pyrrolinium cation
as the substrate with two rounds of malonyl-Coenzyme A-mediated decarboxylative condensation through a noncanonical
mechanism. Subsequent formation of tropinone is achieved by AbCYP82M3. Silencing of AbPYKS and AbCYP82M3 reduced
tropane levels in A. belladonna.349
Tropinone is then reduced either to tropine or to pseudo-tropine, by two closely related tropinone reductases with distinct
stereospecificity.350 The reductases (tropinone reductase I; TRI, and tropinone reductase II; TRII) are NADPH-dependent short-chain
dehydrogenases (SDR) with a conserved NADPH-binding domain and a somewhat divergent tropinone-binding domain.351
Several amino acid residues in the substrate-binding domain have been identified to position tropinone for stereospecific hydride
transfer from NADPH.352,353
Polyphyletic origin for TA biosynthesis in plants was indicated by the discovery of an alternate reductase enzyme for the
reduction of methylecgonone in E. coca. A homology-based approach using TR sequences from the Solanaceae failed to exhibit
methylecgonone-reducing activity of TR homolog heterologously expressed in E. coli. On the other hand, methylecgonone reductase
(MecgoR) was purified from coca leaves354 and found to belong to the aldo-keto reductase (AKR) superfamily, distinct from SDR-
type TRs.
Tropine is coupled to phenyllactate to give littorine.355 The rearrangement of the hydroxyl group of the phenyllactic acid moiety
of littorine was predicted to form atropine and scopolamine formation by a cytochrome p450 coupled with an alcohol dehydro-
genase. The reduction of hyoscyamine and the accumulation of littorine by VIGS of CYP80F1 indicated the function of CYP80F1 as
littorine mutase.356 The enzyme activity was also confirmed with synthetic deutero and arylfluoro analogs of littorine.357
Two-step epoxidation reactions from hyoscyamine to scopolamine via 6b-hydroxyhyoscyamine are catalyzed by a bifunctional
enzyme, hyoscyamine 6b-hydroxylase (H6H; EC 1.14.11.11).150 H6H belongs to the 2-oxoglutarate-dependent dioxygenase
(2ODD) family, and shows strong hydroxylase activity toward hyoscyamine and comparatively weak epoxidase activity toward
6b-hydroxyhyoscyamine. It has been demonstrated that H6H alone is sufficient for the efficient conversion of hyoscyamine to its
final product scopolamine in a transformed E. coli strain, transgenic tobacco plants, and transgenic A. belladonna plants that
ectopically expressed H6H.31,150,358
The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their
core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may
share common ancestry in some order. The diversity of their biological roles as well as the structural genes and enzymes responsible
for their biosynthesis and metabolic engineering to produce some pharmaceutically important tropanes has been reviewed.359
4.3.1.4 Transcription factors

In tobacco (N. tabacum), nicotine is produced in the roots and transferred and accumulates mainly in the leaves. Jasmonates (JAs)
are central signal for damage-induced nicotine formation. The genome sequence of tobacco provides us almost complete informa-
tion of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of
structural genes of the nicotine pathway, forming a regulon, under the control of JA-responsive ETHYLENE RESPONSE FACTOR
(ERF) TFs.333,334
Transcriptional regulation by ERF and cooperatively acting MYC2 TFs are corroborated by the frequent occurrence of cognate cis-
regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has
homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include
either nicotine-controlling ERF189 or ERF199. A large chromosomal deletion was found within one allele of the nicotine-
controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars
with a low-nicotine content.177–179
Genomes of two wild tobaccos, N. attenuata (2.5 Gb) and N. obtusifolia (1.5 Gb) were determined. In contrast to the duplication
of the polyamine pathway that is shared among several solanaceous genera producing TAs, lineage-specific duplications within the
NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ERFs that activate the expression of
all nicotine biosynthetic genes was found to result in the efficient production of nicotine in the genus Nicotiana.360
4.3.1.5 Cellular localization of synthesis, transport and storage of alkaloids

Both nicotine and tropane alkaloids are synthesized in the root, but the tissue types involved in their synthesis are different.
Immunohistochemistry and promoter:: GUS fusion reporters showed that tobacco PMT and A622 oxidoreductase gene in nicotine
biosynthesis are expressed strongly in epidermis and cortex cells of the tobacco root tip, and moderately in the outermost layer of
the cortex, and in parenchyma cells surrounding the xylem of the differentiated region of the root.361,362 On the other hand, the
PMT genes of A. belladonna are specifically expressed in the pericycle at the differentiating region of the root.363 H6H is also localized
in the root pericycle, mainly in the pericycle cells facing the xylem, of scopolamine-producing plants, whereas tropinone reductase-I
is localized in the endodermis and cortex, but not in the pericycle, of the roots of H. niger.364 The difference in the localization of
some enzymes in different cell types indicates that pathway intermediates for scopolamine biosynthesis are transported between
different root cell layers.
The localization of H6H in pericycle cells next to the xylem is a reasonable for the efficient transport of scopolamine into the
xylem. The difference in the localization patterns of PMT and A622 at the tip and the differentiated region of the tobacco roots can
also be interpreted in terms of the transport of nicotine into the xylem. In this region, nicotine synthesis in the parenchyma cells
surrounding the xylem may facilitate nicotine loading into the xylem. Once in the xylem, nicotine and scopolamine will be
transported to the aerial parts with the flow of xylem sap.
Nicotine and TAs, which are unloaded to the leaf and other aerial tissues, accumulate in the vacuole.365 The purine permeases
(PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine.
A tobacco PUP-family transporter, nicotine uptake permease 1 (NtNUP1), transports tobacco alkaloids and affects both nicotine
biosynthesis and root growth in tobacco plants. As Arabidopsis PUP1, NtNUP1 transports pyridoxineand its derivatives (vitamin
B6). Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1,
transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences.
Therefore, multiple effects of NtNUP1 on tobacco physiology would involve bioactive metabolites other than tobacco
alkaloids,204,366 and its use in synthetic biology would need special cautions.

Because of our limited knowledge about the structural genes, small number of genes such as ODC, PMT, TR and H6H has been used
for the metabolic engineering in TA pathway.31,367–369
The most successful metabolic engineering of plant alkaloids is the generation of scopolamine-type plants using hyoscyamine-
rich A. belladonna.31 Although this species has its own H6H genes in the genome, endogenous H6H genes are rather poorly
expressed, resulting in low scopolamine accumulation. Constitutive expression of the H. niger H6H gene under the CaMV35S
promoter significantly enhanced the conversion efficiency in transgenic Atropa plants, which contained scopolamine almost
exclusively in the leaf. However, the root tissues of the transgenic plants31 and Atropa root cultures over-expressing H6H150 showed
insufficient conversion of hyoscyamine to scopolamine, and accumulated the intermediate, 6b-hydroxyhyoscyamine. The improve-
ment of conversion efficiency from the root to the leaf suggests that ectopically expressed H6H in the aerial parts is involved in the
conversion during the transport process.
The several structural genes, encoding ODC, lysine decarboxylase, PMT, tropinone reductase, H6H, and P450s from plant,
mammalian, or bacterial sources, were introduced into transgenic plants, root cultures, or cultured cells to improve TA
production.367,370–376 An expressed enzyme usually catalyzes the expected reaction in the plant host cells, but the impact in the
alkaloid pathway depends on the subsequent metabolic flow to the target product and on the availability of the substrate. The
simultaneous expression of multiple enzymes in the pathway may be necessary. Transformation of a multigene construct for the
overproduction of TAs377 is repeated in several solanaceous root cultures. Combining the overexpression of both PMT and H6H in
A. belladonna has resulted in approximately a 2.5-fold increase of scopolamine above wild-type levels.378 Additional treatment of
hairy root cultures of H. niger with MeJA resulted in a fivefold increase in scopolamine levels.379
On the other hand, the inhibition of gene expression in pathway often reduces the metabolites downstream of the pathway. For
example, cosuppression of PMT gene to 16% of the wild-type tobacco reduced the accumulation of nicotine level to only 2% of that
in the wild type.213 This low-nicotine tobacco showed several morphological abnormalities, probably due to the increased
accumulation of polyamines.
Lowering gene expression to moderate levels may not significantly affect the accumulation of final products of the pathway. For
example, constitutive down-regulation of A622 expression in tobacco only partially suppressed A622 expression, and the mild
suppression phenotype was lost in the self-fertilization. When inducible suppression of A622 was tested in hairy roots and cultured
cells, this conditional RNAi line showed the inhibited cell growth, and severe decrease in the formation of all tobacco alkaloids.
Concomitantly, nicotinic acid b-N-glucoside, a probable detoxification metabolite of nicotinic acid, accumulated in both hairy
roots and methyl JA-elicited cultured cells. N-methylpyrrolinium cation, a precursor of the pyrrolidine moiety of nicotine, also
accumulated in the A622-knockdown hairy roots. These results suggest that A622 would be involved in the formation of not only
nicotine but also other pyridine alkaloids in tobacco.346
4.4 Purine Derived Alkaloids (Fig. 11)

Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methyl-
xanthines, play a significant role in pharmacology and food chemistry. A limited number of plant species accumulate purine
alkaloids, such as caffeine and theobromine, which are synthesized from xanthosine, a catabolite of purine nucleotides. The most
widely distributed methylxanthine in the plant kingdom is caffeine (1,3,7-trimethylxanthine), which accumulates in leaves and
seeds of tea (Camellia sinensis), coffee (Coffea arabica) and a limited number of other species. Considerable amounts of theobromine
(3,7-dimethylxanthine) are stored in the seeds of cacao (Theobroma cacao). The biosynthetic pathway of caffeine from xanthosine is
now well understood in coffee and tea plants.18
After the characterization of enzyme genes involved in caffeine biosynthesis, transgenic plants with enhanced or suppressed gene
expression were produced for the generation of decaffeinated coffee and tea or as natural pesticides in agriculturally important
crops. Tea tree genome was also determined recently to characterize the evolution of caffeine biosynthesis as well as the flavor.380
4.4.1 Biosynthetic enzymes and genes

Xanthosine, the initial precursor of purine alkaloid synthesis, is produced from inosine-50 -monophosphate (IMP) by the reactions
catalyzed by IMP dehydrogenase (EC 1.1.1.205) and nucleotidase (EC 3.1.3.5). 7-Methylxanthosine synthase (SAM: xanthosine N7-
methyltransferase, EC 2.1.1.158) converts xanthosine to 7-methylxanthosine in caffeine biosynthesis and its gene was isolated from
C. arabica. N-Methylnucleosidase (EC 3.2.2.25) discovered in tea leaves catalyzes the hydrolysis of 7-methylxanthosine and forms
7-methylxanthine.
SAM-dependent N-methyltransferases, distinct from the 7-methylxanthosine synthase, catalyze second methylation step in the
caffeine biosynthesis. The dual-functional caffeine synthase (EC 2.1.1.160) in C. arabica and C. sinensis catalyzes the last two steps of
caffeine biosynthesis, that is, 7-methylxanthine ! theobromine ! caffeine. In addition to caffeine synthase, genes encoding
theobromine synthase (EC 2.1.1.159) have been cloned. Four motif A, motif B0 , motif C and the YFFF are highly conserved in the
de novo synthesis
O O
H
H N H N
N XPT N
Purine catabolism
O N N O N N
H ribose-P
XMP H
Xanthine
NS O O O CH3 O
O CH3 CH3 CH3
H H H N H3C
H N N N N N
N N N N
N N O N N O N N
O N N XMT O N NS O N
ribose H ribose H TS/N3MT CH3 CS/N1MT CH3
H
Xanthosine 7-Methylxanthosine 7-Methylxanthine Theobromine Caffeine

(XR)
Fig. 11 Caffeine biosynthesis. Xanthosine monophosphate (XMP), 50 -nucleotidase (50 -NT), Xanthosine N-methyltransferase (XMT), nucleosidase (NS),
theobromine synthase (TS), N3-methytransferase (N3MT), caffeine synthase (CS), N1-methyltransferase (N1MT).
caffeine synthase amino acid sequence. Three conserved motifs are common to the binding site for SAM (motifs A, B and C) among
plant SAM-dependent OMTs. In tea and coffee seedlings, caffeine is synthesized exclusively in the green chlorophyll containing
tissues. In addition, theobromine and caffeine biosynthesis occurs in cotyledons of developing cacao fruits and in young
coffee seeds.
4.4.2 Metabolic engineering and synthetic biology

Antisense or RNAi constructs for the caffeine synthase gene were introduced into coffee or tea to produce decaffeinated plants (e.g.,
219
). Also, a multigene transfer, including all three N-methyltransferases of coffee plants, was introduced to noncaffeine producing
plant to synthesize caffeine in leaves (e.g., 381). Interestingly, transgenic caffeine-producing tobacco plants were resistant against
tobacco cutworms (Spodoptera litura) and pathogenic microbes including Pseudomonas syringe and tobacco mosaic virus.382
Microbial production of purine alkaloids including caffeine is also challenged in yeast. First, a xanthine-to-xanthosine conver-
sion pathway in native yeast metabolism was modified to increase endogenous purine flux for the production of 7-methylxanthine.
Yeast strains were further engineered by expressing combinations of different N-methyltransferases(NMTs), redirection of flux to
support the production of 270, 61, and 3700 mg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-
scale batch fermentations.383
4.5 Steroidal Glycoalkaloids (Fig. 12)

Among solanaceous plants, potato (Solanum tuberosum) and tomato (Solanum lycopersicum) produce the steroidal glycoalkaloids
(SGAs), such as a-solanine and a-chaconine, or a-tomatine, respectively. These SGAs are not real alkaloids derived from amino
acids, such as tropane and nicotine alkaloids derived from putrescine.
SGAs, also known as solanum alkaloids, are common constituents of numerous plants belonging to the Solanaceae family.
In general, the aglycones of SGAs, namely alkamine steroidal skeletons, are synthesized from cholesterol and modified with further
glycosylation at C-3b of the steroidal skeleton. Whereas the most abundant tomatidines, a-tomatine, is toxic to a variety of fungi,
Acetyl-CoA
GAME9 ox
HMGR
SQS
2,3-Oxidosqualene
CAS
SMT1
+ Campesterol
HO HO
Cycloartenol -Sitosterol
SSR2
C5-SD
N
N
*
Glu
GalO
HO
GAME1/2 Rha
-Solanine
HO Solanidine (SGT1/3)
GAME11/6/4/12
Cholesterol + -Chaconine
*PGA2/1 (CYP72A208/188): 22,26-hydroxylation, 16DOX; 16 -hydroxylation

Fig. 12 SGA biosynthesis. GLYCOALKALOID METABOLISM 9 (GAME9), a ERF-typeTF, overexpression induced the expression of the genes in red such as
CYCLOARTENOL SYNTHASE (CAS), STEROL SIDE CHAIN REDUCTASE 2 (SSR2), delta(7)-STEROL-C5(6)-DESATURASE (C5-SD), GAME11, 6, 4, 12, 1, 2 and also
increased the accumulation of cholesterol, a-solanine and a-chaconine in red.
insects and animals, tomatines were converted to esculeosides via hydroxylation and glycosylation reactions during ripening.
LC-MS metabolome analysis identified over 100 SAGs in various tomato tissues. In a recent study, comparative coexpression
analysis between tomato and potato revealed the presence of operon-like cluster of the SGA biosynthetic genes (GLYCOALKALOID
METABOLISM; GAME) in chromosomes 7 and 12. Silencing GAME4 in transgenic tomato reduced the level of SGAs in potato
tubers and tomato fruit.43,384
4.5.1 Biosynthetic enzymes and metabolic engineering

Cholesterol biosynthesis is the key in SGA biosynthesis. The reduction of desmosterol and cycloartenol to cholesterol and
cycloartanol, respectively, by biosterol side chain reductase 2 (SSR2), is branch point in the biosynthetic pathways between C-24
alkylsterols and cholesterol in both potato and tomato. StSSR2-silenced potatoes or StSSR2-disrupted potato generated by genome
editing significantly lowered the level of cholesterol and SGAs without affecting plant growth, and confirmed the importance in SGA
pathway.385
The 22- and 26-hydroxylation of cholesterol is catalyzed by P450s (POTATO GLYCOALKALOID BIOSYNTHESIS1, PGA1 and
PGA2; CYP72A208 and CYP72A188). The knockdown of either PGA1 or PGA2 in potato also significantly reduced the level of SGAs,
whereas vegetative growth and tuber production were not affected.386
The 16a-hydroxylation of hydroxycholesterols and that of (22S)-22,26-dihydroxycholesterol were catalyzed by a
2-oxoglutarate-dependent dioxygenase (16DOX). St16DOX-silenced potato significantly reduced the levels of SGAs, and accumu-
lated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same
results. St16DOX silencing also did not affect potato tuber yield.387 The knockout of St16DOX using CRISPR/Cas9 completely
abolished the accumulation of SGAs in potato hairy roots.37
4.5.2 Steroidal glycoalkaloids from Liliaceae

Members of the families Liliaceae, Apocynaceae, and Buxaceae, except Solanaceae are a rich source of steroid alkaloids. Members of
Liliaceae contain three subclasses of steroidal alkaloids: the jerveratrum-type, the cerveratrum-type, and the solanidine-type.
Cyclopamine, also known as 11-deoxojervine, is a jerveratrum-type alkaloid that exhibits potent pharmacological properties.
Augustin et al.388 discovered four Veratrum californicum enzymes that catalyze the first six steps from cholesterol to verazine, a
predicted precursor to the steroid alkaloid cyclopamine, using correlation analysis of metabolite accumulation with
transcriptome data.
Cholesterol is first hydroxylated at position C-22 in the R orientation by CYP90B27 (cholesterol 22-hydroxylase), followed by
hydroxylation/oxidation at position C-26 by CYP94N1 (22-hydroxycholesterol 26-hydroxylase/oxidase). Next, a transamination
reaction by GABAT1 (22-hydroxycholesterol-26-al transaminase, or c-aminobutyric acid transaminase 1) transfers an amino group
from c-aminobutyric acid to the C-26-aldehyde, forming 22-hydroxy-26-aminocholesterol. The C-22-hydroxy group is then
oxidized to a ketone by CYP90G1 (22-hydroxy-26-aminocholesterol 22-oxidase) to form 22-keto-26-aminocholesterol, a reactive
intermediate that cyclizes to verazine. By stepwise reconstruction of the pathway in Spodoptera frugiperda Sf9 cells, biosynthetic
intermediates were synthesized to validate the enzyme activity in pathway.
5 Future Perspectives
With the considerable technological developments, especially DNA sequencing technology for molecular information, instrumental
analysis of metabolites such as LC-MS/MS, and reverse genetic tools including genome editing, characterization of secondary
metabolic pathway can be done much easily than before. Furthermore, reconstruction of metabolic pathway with isolated genes
using either metabolic engineering or synthetic biology approaches enables the validation of gene functions in pathway and also
provides chemicals desired for analysis or even produces novel nonnatural metabolites. Now is the golden age for natural product
scientists, who study the pathway and also biological function of metabolites.
Based on the identification of genes involved in secondary metabolism and reconstruction of pathway, major problems for the
commercial application of plant secondary metabolites, such as low productivity and the high cost of purification due to the
production of structurally related compounds in biosynthesis, would be resolved in near future. Risk of fluctuation in productivity
of field grown plants due to climate change, pest infection and contamination with undesired wild plants will also be overcame by
these metabolic engineering or synthetic biology.
However, we should notice that our knowledge on biological activity of natural products are still very limited, because biological
activity of many minor metabolites are not characterized in details. Current metabolic engineering and synthetic biology provide
novel way to produce such minor components for drug discovery, because low molecular weight compounds are still major
resources for drug discovery.389
When the biological activity of analogs of a BIA, beberine, as lipid metabolism modulation for metabolic syndrome was
examined using 3T3-L1 adipocytes, 13-methylberberine, a minor component in BIAs, was found as the most active for their anti-
adipogenic effects among 11 protoberberine and 2 benzophenanthridine alkaloids.390 This result suggests that the size of chemical
library is main limiting factor for the screening. Metabolic engineering and synthetic biology have high potentials to provide more
metabolites for drug discovery.
6 Summary
Plant secondary metabolites, especially plant alkaloids, are diversified chemicals and their biosynthesis pathways have been difficult
to characterize. Recent progresses in the metabolite profiles using instrumental analysis and molecular characterization based on the
next-generation sequencing with reverse genetic approaches enable the identification and characterization of pathway and enzyme
genes involved. Using the genes identified, metabolic engineering and synthetic biological approaches are developed to further
characterize the pathway, dynamics in metabolism and production of desired and novel chemicals. Whereas our knowledge of the
basic mechanism of biosynthesis is still limited, continued progresses in molecular engineering of secondary metabolism would
lead to a new era for the production of plant alkaloids in transgenic plants as well as microbes. Production of novel chemicals using
metabolic engineering and synthetic biology would provide useful resource for the drug discovery.
Acknowledgments
The critical reading and fruitful comments by Dr. Yasuyuki Yamada of Kobe Phamaceutical University were highly appreciated. This
research was supported in part by a Grand-in-Aid for Scientific Research from the Ministry of Education, Sports, and Culture of
Japan (to F.S.). This manuscript is dedicated to the 88th birthday of Prof. Emeritus Yasuyuki Yamada of Kyoto University for his
great contributions on alkaloid biosynthesis studies.
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Plant Alkaloid Engineering

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Plant Alkaloid Engineering

Comprehensive Natural Products III: Chemistry and Biology https://doi.org/10.1016/B978-0-12-409547-2.14696-7 1

4.1.3 Transport and accumulation 31

CM/PDH Chorismate mutase/prephenate dehydrogenase

MND Morphinan N-demethylase

T16H Tabersonine 16-hydroxylase

Morphine (IQA) Codeine (IQA) Ephedorine (IQA)

R=CH3, Vinblastine (MIA) Quinine (MIA)

Atropine (TA) Hyoscine (scopolamine) (TA)

2.1 Instrumental and Analytical Advances

Table 1 Useful URL for plant alkaloid engineering.

Tools for natural products and metabolic pathways

Spektraris (a collection of tools for identifying plant natural products) http://langelabtools.wsu.edu/

Medicinal plant transcriptome database

PhytoMetaSyn Project www.phytometasyn.ca

Tools for sequence analysis

2.1.2 Stable isotope labeling

2.1.3 Mass spectrometry imaging (MSI)

2.1.4 Nuclear magnetic resonance (NMR)

2.2 Outlines of Metabolic Engineering and Synthetic Biology

3 Isolation and Identification of Molecular Tools in Pathway Engineering

3.2 Coexpression Analysis to Isolate Candidate Genes in Alkaloid Biosynthesis

3.3.1 Homology search based on the transcriptome data

3.3.1.1 BIA pathway

3.3.1.2 MIA pathway

3.3.2 Coexpression analysis and comparative genomics based on genome sequence

3.3.2.1 BIA pathway

3.3.2.2 MIA pathway

3.3.3 Gene family analyses based on genome sequence

3.4 Key Enzymes in Alkaloid Biosynthesis

3.4.1 Amine–aldehyde condensation by Pictet-Spengler catalyzing enzymes

3.4.1.1 Norcoclaurine synthase (NCS) and related enzymes

3.4.1.1.1 PR-10 like NCS

R1=H, R2=H, R3=OH R1=H, R2=H, R3=OH

3.4.1.1.2 Dioxygenase-like NCS

3.4.1.2 Strictosidine synthase (STR)

3.4.2 Berberine bridge enzyme (BBE) and flavin-binding proteins

3.4.3 Cytochrome P450s (P450s)

3.4.3.1 Scaffold generating P450s

3.4.3.1.2 Hydroxylation enzymes also generate new scaffold

3.4.3.1.3 Hybrid P450; STORR (reticuline epimerase: REPI)

3.4.3.2 Scaffold modifier

3.4.3.2.2 Methylene dioxy bridge enzymes

3.4.3.3 Cytochrome P450 oxidoreductases (CPR)

3.4.4 Carbocation-mediated cyclization; iridoid synthase (IS)

3.4.6 Scaffold modifying methyltransferases (MTs)

3.4.6.2 N-methyltransferases (NMTs)

3.4.6.2.1 Putrescine N-methyltransferase (PMT)

3.4.6.2.2 NMTs in BIA and ephedrine biosynthesis

3.4.6.2.3 NMT in MIA biosynthesis

3.5 Additional Molecular Option for Pathway Engineering

3.5.1.1 Substrate supply in BIA biosynthesis (see also Fig. 6)

3.5.1.2 Modification enzymes in BIA pathway

NH2 Aro10p (in yeast) Berbamunine

3.5.1.2.2 Bacterial enzymes for further modification

3.5.1.2.3 Halogenation enzymes

3.5.2.1 Strictosidine synthase (STR)

3.5.2.2 Other proteins

3.6 Transcription Factors

3.6.1 APETALA2 (AP2)/ethylene response factors (ERFs)

3.6.2 Basic helix-loop-helix (bHLH) family; MYC2 and non-MYC2 type

3.6.4 Posttranslational regulation of TFs in secondary metabolism

3.7 Genes for Transport, Accumulation and Storage