ARTICLE IN PRESS

Metabolic Engineering 10 (2008) 281–292

Contents lists available at ScienceDirect

Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben

Improving production of bioactive secondary metabolites in actinomycetes

by metabolic engineering
Carlos Olano, Felipe Lombó, Carmen Méndez, José A. Salas 
Departamento de Biologı́a Funcional e Instituto Universitario de Oncologı́a del Principado de Asturias (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain

a r t i c l e in fo abstract

Article history: Production of secondary metabolites is a process influenced by several physico-chemical factors
Received 10 June 2008 including nutrient supply, oxygenation, temperature and pH. These factors have been traditionally
Received in revised form controlled and optimized in industrial fermentations in order to enhance metabolite production. In
8 July 2008
addition, traditional mutagenesis programs have been used by the pharmaceutical industry for strain
Accepted 9 July 2008
Available online 15 July 2008
and production yield improvement. In the last years, the development of recombinant DNA technology
has provided new tools for approaching yields improvement by means of genetic manipulation of
Keywords: biosynthetic pathways. These efforts are usually focused in redirecting precursor metabolic fluxes,
Streptomyces deregulation of biosynthetic pathways and overexpression of specific enzymes involved in metabolic
Metabolism
bottlenecks. In addition, efforts have been made for the heterologous expression of biosynthetic gene
Antibiotic biosynthesis
clusters in other organisms, looking not only for an increase of production levels but also to speed the
Precursor engineering
Heterologous expression process by using rapidly growing and easy to manipulate organisms compared to the producing
Regulation organism. In this review, we will focus on these genetic approaches as applied to bioactive secondary
Ribosome engineering metabolites produced by actinomycetes.
Genome shuffling & 2008 Elsevier Inc. All rights reserved.

1. Introduction
Microorganisms are the source of many drugs including
antibiotics, antitumor compounds, immunosuppressants, antivir-
al, antiparasitic agents and enzyme-inactivating compounds.
About 23,000 bioactive secondary metabolites produced by
microorganisms have been reported, and only 150 of them are
being used in pharmacology, agriculture or other fields. Over
10,000 of these compounds are produced by actinomycetes,
representing 45% of all bioactive microbial metabolites discov-
ered, 80% if we only consider those compounds in practical use.
Among actinomycetes, around 7600 compounds are produced by
Streptomyces species (Bérdy, 2005).
Drugs in commercial use are obtained at industrial scale either
by fermentative production, chemical synthesis or semisynthetic
processes. When fermentation is used, it requires microbial
strains producing high titers of compound. However, wild-type
strains isolated from nature usually produce only discrete
amounts of a particular secondary metabolite, which in terms of
its isolation implies the need for production improvement to meet
commercial requirements. This improvement will, in addition,
determine in most cases whether a new natural product goes to
market or is abandoned, since it is clear that when production
 Corresponding author. Fax: +34 985103652.
E-mail address: jasalas@uniovi.es (J.A. Salas).
yields increase then costs are reduced. The large-scale production
of drugs from microbial fermentation has been the basis of the
industry since the development of penicillin in the 1940s. The
titers of products made by industrial cultures nowadays are very
high after years of intense improvement programs using the
traditional ‘‘mutate-and-screen’’ method of strain improvement
that was early developed for the penicillin strain. Today, penicillin
producer Penicillium chrysogenum makes over 70 g/l of drug
starting from a strain capable of producing only 60 mg/l, which
represents a 1000-fold increase. Other impressive examples of
strain improvement are the production of riboflavin by Ashbya
gossypii that has been improved 40,000 times and the production
of a 100,000-fold excess of vitamin B12 by Pseudomonas
denitrificans (Demain, 2006).
Current methods to increase the productivity of industrial
microorganisms go from the classical random mutagenesis
performed in close association with optimization of large-scale
industrial fermentations, to the use of more rational methods. One
of these is metabolic engineering where, in order to maximize
product yields, primary metabolic fluxes are redirected by
the introduction of genetic modifications through recombinant
DNA technology, in a manner that supports high secondary
metabolite productivities (Adrio and Demain, 2006; Nielsen,
1998). In addition, the development of modern technologies such
as DNA sequencing, transcription profiling, genomics, proteomics,
metabolomics, transcriptomics and metabolite profiling has
created new opportunities to engineer microorganisms for the

1096-7176/$ - see front matter & 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymben.2008.07.001
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282 C. Olano et al. / Metabolic Engineering 10 (2008) 281–292

production of natural products in high yields (Bro and Nielsen, increase of acetyl-CoA, precursor of undecylprodigiosin and
2004). actinorhodin, increasing production of these antibiotics (Fig. 2)
In this review, we focus on the different genetic approaches (Butler et al., 2002).
used for the production improvement of secondary metabolites
produced by actinomycetes. In general, a particular metabolite
2.2. Fatty acid precursors
can be overproduced following different approaches: (i) altering
the metabolic flux distribution of its different precursors,
Engineering the availability of coenzyme A (CoA) activated
(ii) deregulating its specific biosynthetic pathway, (iii) increasing
fatty acid precursors has been reported for the enhanced
self-resistance or inducing resistance to several antibiotics,
production of several polyketides such as erythromycin, oligomy-
(iv) overexpressing structural genes coding for enzymes involved
cin, monensin B and actinorhodin in the producer organisms. In
in the biosynthesis of the metabolite, (v) using global genetic
the case of erythromycin, two of its precursors are malonyl-CoA
approaches such as genome shuffling and (vi) by expressing the
derived from the carboxylation of acetyl-CoA and methylmalonyl-
biosynthetic gene cluster in a heterologous host or an industrially
CoA, which can be synthesized through different pathways such
optimized strain. Examples of each of these approaches are
as carboxylation of propionyl-CoA and rearrangement of succinyl-
described below, summarized in Table 1 and schematized in Fig. 1.
CoA (Fig. 3). Engineering the methylmalonyl-CoA metabolic node
in erythromycin producers Saccharopolyspora erythraea and Aero-
2. Precursor engineering microbium erythreum leads to the enhanced production of the
antibiotic depending on the fermentation medium used. Over-
The availability of biosynthetic precursors is a key factor production of erythromycin was achieved by inactivating the
determining the productivity of secondary metabolites. Primary methylmalonyl-CoA mutase (MCM) gene mutB and cultivation of
metabolism is the supplier for those precursors that are generally Sac. erythraea or A. erythreum in a carbohydrate-based medium
formed through the catabolism of various carbon substrates such while in an oil-based medium erythromycin production dimin-
as fatty acids, monosaccharides or proteins. The identification and ished (Reeves et al., 2004, 2006). In a carbohydrate-based
genetic manipulation of key enzymes regulating carbon flux medium, the MCM reaction acts like a drain on the methylma-
through the metabolic network of the central carbon metabolism lonyl-CoA pool, but in an oil-based medium, the same reaction
can lead to an increase in the availability of a particular precursor acts to fill the methylmalonyl-CoA pool (Reeves et al., 2006). In
as shown in Figs. 2 and 3. addition, overproduction of erythromycin was accomplished by
duplication of the MCM operon (mutA, mutB, meaB and mutR) and
cultivation of Sac. erythraea in an oil-based fermentation medium
2.1. Carbohydrate metabolism
(Reeves et al., 2007). These experiments led to the conclusion that
the carbon flow under oil-based growth conditions was from
In glucose catabolism the Embden–Meyerhof and pentose
succinyl-CoA to methylmalonyl-CoA (Reeves et al., 2007).
phosphate (PPP) pathways are interlinked to form the metabolic
Antiparasitic avermectins and cell growth inhibitor oligomycin
network shown in Fig. 2. Key enzymes in the individual pathways
are macrocyclic lactones produced by Streptomyces avermitilis
regulate the carbon flux among them. The first intermediate in
(Omura et al., 2001). During the biosynthesis of the avermectins,
glucose catabolism, glucose-6-phosphate (G6P), is used as a
two different starter units are used: isobutyryl-CoA and
common substrate for phosphoglucose isomerase (Pgi), glucose-
2-methylbutyryl-CoA produced by the degradation of branched-
6-phosphate dehydrogenase (Zwf), and phosphoglucomutase
chain amino acids valine and isoleucine. Inactivation of bkdF
(Pgm). Zwf channels glucose to the PPP. Pgm catalyzes the
encoding a branched-chain a-keto acid dehydrogenase leads to
reversible interconversion of G6P and glucose-1-phospate (G1P),
the abolition of avermectins production and in turn to the
potentially leading to the deviation of carbon to the biosynthesis
overproduction of the macrolide oligomycin by enabling addi-
of glucose-based polymers such as glycogen (Ryu et al., 2006) or
tional extender units (malonyl-CoA, methylmalonyl-CoA and
to the biosynthesis of 6-deoxyhexoses (6DOH) characteristic, but
ethylmalonyl-CoA) to enter the biosynthetic pathway of oligomy-
not exclusive, of secondary metabolism and found in many
cin (Cropp et al., 2001; Wei et al., 2006). In Streptomyces
bioactive secondary metabolites (Salas and Méndez, 2005). Pgi
cinnamonensis, inactivation of crotonyl-CoA reductase (CCR)
leads carbohydrate catabolism through the Embden–Meyerhof
involved in the reduction of crotonyl-CoA to butyryl-CoA leads
pathway and subsequently to the tricarboxylic acid cycle, where
to the depletion of ethylmalonyl-CoA synthesized from butyryl-
other precursors for secondary metabolite formation are provided.
CoA and subsequently to the accumulation of monensin B instead
Attempts of genetic manipulation of the initial steps in the
of monensin A. Monensin B requires only methylmalonyl-CoA
Embden–Meyerhof pathway and PPP have been reported for the
while monensin A requires both methylmalonyl-CoA and ethyl-
enhanced production of clavulanic acid, actinorhodin and un-
malonyl-CoA during its biosynthesis (Cropp et al., 2001). Actinor-
decylprodigiosin. The glycolytic pathway in Streptomyces clavuli-
hodin production by Streptomyces coelicolor is another example of
gerus was genetically engineered by disruption of gap1 and gap2
enhanced production of a polyketide by modification of its
genes coding for glyceraldehyde-3-phosphate dehydrogenases
precursor supplies. In this case, the overexpression of the genes
involved in the conversion of D-glyceraldehyde-3-phosphate
accA2, accB and accE, coding for the different subunits of the
(G3P) into 1,3-diphosphoglicerate (1,3-BPG). Since G3P and
enzyme acetyl-CoA carboxylase (ACC) in S. coelicolor, was
L-arginine are precursors in the biosynthesis of clavulanic acid,
sufficient to enhance carbon flux to malonyl-CoA, which is a
the accumulation of G3P in the gap mutants correlated with a
precursor of actinorhodin together with acetyl-CoA, leading to a
significant increase in the production of the antibiotic, increase
six-fold increase in actinorhodin production (Ryu et al., 2006).
that reached 3.1-fold when L-arginine was fed to the cultures
(Li and Townsend, 2006). PPP was engineered by independently
removing two different sets of genes: zwf1 and zwf2 coding for 2.3. Cofactors
isoenzymes of glucose-6-phosphate dehydrogenase, and devB
coding for a 6-phosphoglucolactonase in Streptomyces lividans. Additional elements of the central carbon metabolism take
These deletions lead to channeling the precursors flux through the part in the biosynthesis of secondary metabolites as precursors
Embden–Meyerhof instead of PPP, which in turn lead to the and are also targets for metabolic engineering approaches. This is
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Table 1
Increase in production of secondary metabolites produced by actinomycetes achieved by metabolic engineering

Compound Strain Engineering approach Increase (fold)

Actinomycin S. antibioticus Ribosome engineering 5.25

Actinorhodin S. coelicolor Fatty acid precursors 6

Up-regulation 2.6–40
Ribosome engineering 1.6–180

S. lividans Carbohydrate metabolism 4–5

Cofactors 4
Up-regulation 470
Down-regulation 3.5–5

C-1027 S. globisporus Biosynthetic structural genes 2–4

Cephamycin C S. clavuligerus Biosynthetic structural genes 2–4
Up-regulation 2–3
Chromomycin S. griseus Down-regulation 3
Clavulanic acid S. clavuligerus Carbohydrate metabolism 2.1–3.1
Biosynthetic structural genes 1.6–5
Up-regulation 1.5–23.8
Clorobiocin S. coelicolor Heterologous expression 1
Daunorubicin S. peucetius Biosynthetic structural genes 8–9
Up-regulation 2.4–10
6-dEB S. coelicolor Heterologous expression and fatty acid precursors 4
E. coli Heterologous expression and fatty acid precursors 1.8
Desosaminyl tylactone S. venezuelae Heterologous expression, PKS deletion and up-regulation 17.1
Doramectin S. avermitilis Biosynthetic structural genes 4–23
Doxorubicin S. peucetius Biosynthetic structural genes 3–74
Up-regulation 4
Erythromycin A. erythreum Fatty acid precursors 2–4
Sac. erythraea Fatty acid precursors 1.25–1.5
Expression of heterologous genes 2–2.5
Plasmid integration 2–5
Expression in industrial strains 50
Fredericamycin S. chattanoogensis Ribosome engineering 26
Formycin S. lavendulae Ribosome engineering 5.2
GE2270 P. rosea Ribosome engineering 1.8
Hydroxycitric acid Streptomyces U121 Genome shuffling 5
Kanamycin S. kanamyceticus Self-resistance 3.5
Megalomycin Sac. erythraea Heterologous expression and 6DOH metabolism 3.4
15-Methyl-6-dEB S. coelicolor Heterologous expression and plasmid co-integration 4–25
Mithramycin S. argillaceus Up-regulation 2–16
Monensin B S. cinnamonensis Fatty acid precursors 1.76
Nanchangmycin S. nanchangensis Biosynthetic structural genes 3
Neomycin S. fradiae Self-resistance 6

Nikkomycin X S. ansochromogenes Biosynthetic structural genes 1.8–2

Up-regulation 2

Novclobiocin 122 S. coelicolor Heterologous expression and 6DOH metabolism 8–26

Novobiocin S. coelicolor Heterologous expression 1

S. coelicolor Heterologous expression and up-regulation 3

Nystatin S. noursei Up-regulation 3.25

Biosynthetic structural genes 1.6

Oligomycin A S. avermitilis Fatty acid precursors 23

Pikromycin S. venezuelae Up-regulation 1.6–2.6

Pimaricin S. nataliensis Up-regulation 2.4

Down-regulation 1.8

Pristinamycin IIA S. pristinaespiralis Biosynthetic structural genes 1.25

Rapamycin S. hygroscopicus Up-regulation 1.2–1.4
e-Rhodomycinone S. peucetius Up-regulation 7–100
Salinomycin S. albus Ribosome engineering 1.5–2.3
Shengjimycin S. spiramyceticus Biosynthetic structural genes 2
Spinosyn Sac. spinosa Carbohydrate metabolism 3
Tetracenomycin D3 S. glauscescens Biosynthetic structural genes 20–30
Tylactone S. venezuelae Heterologous expression, PKS deletion and up-regulation 2.7
Tylosin S. fradiae Up-regulation 1.2–4.9
Down-regulation 1.5
Genome shuffling 6–8
Undecylprodigiosin S. coelicolor Up-regulation 31
S. lividans Carbohydrate metabolism 4
Down-regulation 11–12
Ribosome engineering 1.9–2.9
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the case of the cofactor S-adenosyl-methionine (SAM). Actinorho-

din production was reported to be enhanced both in S. lividans and
in S. coelicolor by overexpression of Streptomyces spectabilis metK
gene coding for S-adenosyl-L-methionine synthetase that cata-
lyzes the biosynthesis of SAM from ATP and L-methionine. The
same effect was obtained by addition of SAM to the culture
medium. The increase in actinorhodin production is the conse-
quence of inducing the expression of pathway-specific transcrip-
tional activator actII-orf4 (Kim et al., 2003; Okamoto et al., 2003).
A similar effect on the production of pikromycin in Streptomyces
venezuelae was reported by expression of metK1-sp gene from
Streptomyces peucetius. In this case an increase in transcripts of the
pathway-specific transcriptional activator pikD and a keto-
synthase gene were observed (Maharjan et al., 2008).

3. Engineering regulatory networks

Genes for the biosynthesis of secondary metabolism pathways

Fig. 1. Scheme representing the different approaches used for improvement of
secondary metabolite production.
are commonly grouped together in clusters on the chromosome
including their pathway-specific regulatory genes. Pathway-speci-
fic regulators can have either positive (activators) or negative
(repressors) effects on the expression of gene cluster elements.
There are clusters containing different number of pathway-specific

Fig. 2. Glucose catabolism and engineered steps involved in the biosynthesis of several secondary metabolites produced by actinomycetes. 1,3-BPG, 1,3-
diphosphoglycerate; Cas2, clavaminate synthase; Cvm1, enzyme involved in the biosynthesis of antipodal clavams; DevB, phosphoglucolactonase; 6DOH, 6-deoxyhexose
pathways; F6P, fructose-6-phosphate; Gap1 and Gap2, glyceraldehyde-3-phosphate dehydrogenases; Gdh, NDP-glucose dehydratase; G1P, glucose-1-phosphate; G6P,
glucose-6-phosphate; G3P, glyceraldehyde-3-phosphate; Gtt, NDP-glucose synthase; LAT, lysine e-aminotransferase; MetK, S-adenosyl-L-methionine synthetase; Pgi,
phosphoglucose isomerase; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucolactone; Pgm, phosphoglucomutase; Pah, proclavaminate amidino hydrolase; PPP, pentose
phosphate pathway; SAM, S-adenosyl-L-methionine; SanO, non-ribosomal peptide synthetase; SanU and SanV, glutamate mutase; Zwf1 and Zwf2, glucose-6-phosphate
dehydrogenases.
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Fig. 3. Fatty acid precursors and engineered steps involved in the biosynthesis of several secondary metabolites produced by actinomycetes. ACC, acetyl-CoA carboxylase;
BkdF, branched-chain a-keto acid dehydrogenase; ICM, isobutyryl-CoA mutase; IST, 400 -O-acyltransferase; MMT, methylmalonyl-CoA transcarboxylase; MutB,
methylmalonyl-CoA mutase; PPC, propionyl-CoA carboxylase.
positive regulatory genes ranging from one in actinorhodin path-
way (Fernández-Moreno et al., 1991) to three in daunorubicin
pathway (Stutzman-Engwall et al., 1992; Otten et al., 1995, 2000).
In addition, some clusters contain both activators and repressors
such as in the tylosin pathway that contains two activator and two
repressor-coding genes (Stratigopoulos et al., 2004). However, in
some pathways no regulatory genes have been identified, as is the
case of the erythromycin pathway (Rawlings, 2001). Moreover,
other regulatory genes generally located outside the biosynthetic
gene cluster may play a regulatory role in the cluster, in many cases
showing pleiotropic effects on the production of multiple second-
ary metabolites. The best-known example is S. coelicolor that
produces several antibiotics (actinorhodin, calcium-dependent
antibiotic, undecylprodigiosin and methylenomycin) and where
the onset of their biosynthesis is controlled by specific regulators
(actII-orf4, cdaR, redD and redZ), while there are several pleiotropic
genes (i.e. afs, abs and bld) affecting antibiotic production and, in
addition, the morphological development of the bacteria (Huang et
al., 2005). Taking into account all previous observations it seems
obvious that deregulation of the expression of secondary metabo-
lite pathways, by overexpression of pathway-specific positive
regulators or by inactivation of pathway repressors, is the most
intuitive approach for the improvement of their production (Fig. 1).
3.1. Up-regulation
The vast majority of the pathway-specific activators in
actinomycetes belong to the Streptomyces Antibiotic Regulatory
285
Protein family (SARP) characterized by the presence of a winged
helix-turn-helix (HTH) motif towards the N-termini (Wietzorrek
and Bibb, 1997). Overexpression of SARP positive regulators has
been reported to increase the production of different secondary
metabolites such as actinorhodin and undecylprodigiosin in S.
coelicolor by actII-orf4 and redD (Narva and Feitelson, 1990;
Fernández-Moreno et al., 1991), undecylprodigiosin in S. lividans
and S. parvulus by redD (Malpartida et al., 1990), nikkomycin in S.
ansochromogenes by sanG (Liu et al., 2005) and clavulanic acid in S.
clavuligerus by ccaR (Pérez-Llarena et al., 1997; Hung et al., 2007).
The production of other secondary metabolites such as tylosin,
daunorubicin and mithramycin was reported to be up-regulated
either using SARP activator coding genes or, in addition, other
pathway-specific positive regulators. The production of tylosin by
Streptomyces fradiae was reported to be increased by overexpres-
sion of tylS or tylR encoding a SARP regulator and a protein
containing a transposase domain, respectively. In both cases an
increase in tylosin production was obtained in the wild-type
strain and in a tylosin overproducing strain (Stratigopoulos et al.,
2004). Overexpression of locus dnrR1, encoding SARP regulator
DnrI, or dnrR2, encoding positive regulators DnrN and DnrO
containing LuxR and ArsR type HTH motifs, led to a 10–100-fold
increase in the production of e-rhodomycinone and 25-fold
increase in daunorubicin (Stutzman-Engwall et al., 1992; Otten
et al., 1995). Mithramycin production by Streptomyces argillaceus
has been improved by overexpression of two different regulatory
genes in multicopy plasmids: mtrY encoding a protein with a
PadR-like HTH motif (Garcı́a-Bernardo et al., 2000) and mtmR
encoding a SARP family regulatory protein (Lombó et al., 1999;
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Wohlert et al., 1999). In addition, mtmR was also able to activate
actinorhodin biosynthesis and complement actII-orf4 mutation in
S. coelicolor JF1 (Lombó et al., 1999; Blanco et al., 2000). Other
secondary metabolite biosynthetic pathways lacking SARP activa-
tors have been up-regulated using other kind of pathway-specific
regulators such as pimM encoding for a PAS/LuxR regulator that
enhance pimaricin production in Streptomyces nataliensis (Antón
et al., 2007) or rapH and rapG, encoding proteins with LuxR and
AraC-like HTH motifs respectively, that increase rapamycin
production in Streptomyces hygroscopicus (Kuscer et al., 2007).
SARP family proteins generally appear to function as pathway-
specific regulators but, in some cases, they can work as pleiotropic
regulatory proteins that control the production of multiple
secondary metabolites and morphological differentiation. Such
is the case of the afsR gene from S. coelicolor able to increase
actinorhodin and undecylprodigiosin production through its
overexpression in S. lividans (Horinouchi et al., 1983). Others
SARP pleiotropic activators homologue to AfsR such as SsmA and
AfsR-p from Streptomyces noursei and S. peucetius have been
shown to enhance nystatin and doxorubicin biosynthesis, respec-
tively (Sekurova et al., 1999; Parajuli et al., 2005). In addition,
overexpression of afsR-p in S. lividans, S. clavuligerus, S. griseus and
S. venezuelae leads to overproduction of actinorhodin, clavulanic
acid, streptomycin and pikromycin, respectively (Parajuli et al.,
2005; Maharjan et al., 2008). Different pleiotropic activators have
been shown to increase production of actinorhodin such as afsS
(Vögtli et al., 1994), the two-component regulatory system afsQ1
and afsQ2 (Ishizuka et al., 1992), abaA (Fernández-Moreno et al.,
1992) and ptpA (Umeyama et al., 1996). The last gene, pptA,
encodes a phosphotyrosine protein phosphatase presumably a
member of the signal transduction network that controls the
production of actinorhodin and undecylprodigiosin (Umeyama
et al., 1996).
3.2. Down-regulation
The inactivation of pathway-specific or pleiotropic repressors
can also lead to overproduction of secondary metabolites. This is
the case of chromomycin that is overproduced when a pathway-
specific transcriptional repressor cmmRII is inactivated in S. griseus
subsp. griseus (Menéndez et al., 2007). A similar effect was
accomplished in tylosin biosynthesis by disruption of tylP, gene
that encode a g-butyrolactone receptor that negatively affects the
production of the antibiotic and, in addition, influences morpho-
logical differentiation in S. fradiae (Stratigopoulos et al., 2002). An
additional pathway-specific transcriptional repressor, tylQ, is
present in tylosin gene cluster but, in this case, its inactivation
does not produce an increase in antibiotic production but to an
early onset of tylosin production (Stratigopoulos and Cundliffe,
2002). In actinorhodin gene cluster there is also a gene, actVB-
orf10, encoding a LysR-type transcriptional regulator that has been
used for antibiotic overproduction through its inactivation in
S. lividans (Martı́nez-Costa et al., 1999).
A well-known pleiotropic repressor system related with
phosphate regulation of secondary metabolite production is the
two-component phoR–phoP system. Disruption of phoR or simul-
taneous deletion of both phoR and phoP has been recently shown
to increase pimaricin production in S. nataliensis (Mendes et al.,
2007). Deletion of the same system in S. lividans was reported to
boost actinorhodin and undecylprodigiosin production, 5- and
12-fold increase, respectively (Sola-Landa et al., 2003). In
S. coelicolor there is an additional pleiotropic gene, nsdA, that
negatively affects antibiotic production and whose disruption
leads to an increase in actinorhodin, calcium-dependent antibiotic
and methylenomycin biosynthesis. In the nsdA mutant the levels
of the pathway-specific regulator actII-orf4 mRNA increased
(Li et al., 2006). Other modifications, such as the inactivation of
polyphosphate kinase gene ppk, have been reported to induce the
expression of actII-orf4 activating the actinorhodin pathway
(Chouayekh and Virolle, 2002).
4. Engineering antibiotic resistance
Antibiotic biosynthetic gene clusters typically include one or
more genes encoding different resistance mechanisms for self-
protection to overcome the toxic effects of their products. These
systems include enzymes to modify the antibiotic target site,
antibiotic inactivating enzymes and transport systems (Cundliffe,
1989; Méndez and Salas, 2001). In some cases these resistance
systems are involved not only in self-protection but also in
antibiotic biosynthesis (Olano et al., 1995; Menéndez et al., 2007).
Consequently, it is not surprising that increased antibiotic
resistance has often been used to select for mutants with
increased levels of antibiotic production (Yanai et al., 2006).
4.1. Ribosome engineering
A dramatic activation of antibiotic production was observed in
S. lividans and S. coelicolor containing a mutation in rpsL gene,
encoding the ribosomal protein S12, which confers resistance to
streptomycin (Shima et al., 1996). This led to rationally improve
the production of actinorhodin and undecylprodigiosin by
introducing point mutations in chromosomal rpsL gene or by
using a single-copy-number plasmid to express rpsL mutant
versions (Shima et al., 1996; Okamoto-Hosoya et al., 2003). Since
then, ribosome engineering has been used as a rational approach
to enhance antibiotic production in different Streptomyces spp. by
conferring resistance to several antibiotics mediated by mutant
ribosomes (Fig. 1). The beneficial effect on antibiotic production of
mutations in rpsL gene conferring high-level resistance to
streptomycin have been confirmed by the isolation of mutants
in other Streptomyces spp. such as S. chattanoogensis, S. anti-
bioticus, S. lavendulae and S. albus producers of fredericamycin,
actinomycin, formycin and salinomycin, respectively (Hosoya
et al., 1998; Tamehiro et al., 2003). Overexpression in S. coelicolor
of genes such as frr encoding a ribosome-recycling factor whose
overproduction has been detected in streptomycin resistant rpsL
mutants, also leads to actinorhodin overproduction up to 10-fold
(Hosaka et al., 2006). On the other hand, low-level resistance to
streptomycin by mutation or deletion of rsmG gene, coding for a
SAM-dependent 16S rRNA methyltransferase, has been reported to
raise actinorhodin production in S. coelicolor (Nishimura et al.,
2007).
Apart from mutations conferring resistance to streptomycin,
enhancement of antibiotic production can be also achieved by
mutations conferring resistance to other antibiotics such as
gentamicin, rifampin, paromomycin, geneticin, fusidic acid,
thiostrepton and lincomycin. The simultaneous introduction of
several resistant mutations to streptomycin, gentamicin and
rifampin has a cumulative effect on antibiotic production leading
to improvements in actinorhodin, salinomycin and thiazolylpep-
tide GE2270 production in S. coelicolor, S. albus and Planobispora
rosea strains, respectively (Hu and Ochi, 2001; Tamehiro et al.,
2003; Beltrametti et al., 2006). In addition, an increase in
actinorhodin production up to 180-fold has been recently
described in a S. coelicolor strain resistant to seven of the eight
antibiotics mentioned above, depending on the culture medium
used (Wang et al., 2008). These mutations produce ribosomes
with aberrant protein and hyperphosphorylated guanosine
nucleotide (ppGpp) synthesis activities (Okamoto-Hosoya et al.,
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C. Olano et al. / Metabolic Engineering 10 (2008) 281–292
2003; Wang et al., 2008). There is a positive correlation between
ppGpp and antibiotic biosynthesis since ppGpp triggers bacterial
secondary metabolism when cells enter into stationary phase
(Chakraburtty and Bibb, 1997). On the other hand, in all these
mutant strains overproduction of actinorhodin correlates to
higher expression of the actII-orf4 gene probably due to the
enhanced protein synthesis (Hu and Ochi, 2001; Okamoto-Hosoya
et al., 2003; Wang et al., 2008).
4.2. Self-resistance improvement
Increasing self-resistance levels in producing organisms has
been also used as an approach for increasing production yields.
This strategy was used in the producers of two aminoglycosides,
kanamycin and neomycin, and consisted in the introduction
in the producer strains of the gene encoding an aminoglycoside
60 -N-acetyltransferase derived from Streptomyces kanamyceticus
that confers resistance to aminoglycoside antibiotics. This re-
sulted in a substantial increase in production of these antibiotics,
especially neomycin (Crameri and Davies, 1986). Similar results
were described in several antibiotic overproducing organisms
such as S. aureofaciens, 6-demethylchlortetracycline producer,
through the overexpression of a self-defense gene involved in
drug efflux (Dairi et al., 1995), or the S. kanamyceticus industrial
kanamycin producer strain where duplication of the entire
kanamycin gene cluster, including its three self-resistance genes,
leads to enhanced kanamycin resistance in addition to antibiotic
overproduction (Yanai et al., 2006).
5. Engineering biosynthetic structural genes
Usually structural genes involved in the biosynthesis of
secondary metabolites have been targeted for the generation of
new compounds through their inactivation or deletion. In
addition, these mutants can be used as hosts for the expression
of heterologous genes that can lead to the generation of new
compounds. There are a number of examples in the literature
where gene dose alteration, modification and heterologous
expression of biosynthetic structural genes have been used to
boost production of secondary metabolites (Fig. 1).
5.1. Gene dose increase
S. pristinaespiralis is the producer of pristinamycin II, strepto-
gramin antibiotic produced as a mixture of two compounds PIIA
and PIIB in a ratio 80:20. The complete conversion of compound
PIIB into PIIA was achieved by integration into the chromosome of
an additional copy of snaA and snaB genes coding for a
heterodimeric monooxygenase catalyzing this conversion. The
duplication of snaA/snaB gene dosage did not increase the total
amount of pristinamycins but enhanced the ratio of pristinamycin
PIIA leading to a better production of this compound (Sezonov
et al., 1997). A similar approach has been used to boost the
production of tetracenomycin D3, intermediate in the biosynth-
esis of tetracenomycin C by Streptomyces glaucescens. In this case
overexpression of tcmM, coding for an acyl carrier protein (ACP) of
a type II polyketide synthase (PKS), cloned into a high copy vector
leads to increase up to 30-fold the production of this biosynthesis
intermediate (Decker et al., 1994).
Carbon flux from glucose can be channeled to enhance the
production of secondary metabolites by manipulating glucose
catabolism as showed in Section 2.1 and, in addition, by altering
the expression of structural genes specifically involved in the
biosynthesis of a particular metabolite. Among these, genes
287
belonging to 6DOH pathways are good candidates for this kind
of manipulation. This was the case of gene sgcA1 encoding a NDP-
glucose synthase, involved in the biosynthesis of 6DOH 4-deoxy-
4-(dimethylamino)-5,5-dimethyl-D-ribopyranose, that has been
used to engineer the production of the enediyne antitumor
antibiotic C-1027 in S. globisporus. The overexpression of sgcA1
alone resulted in a two-fold improvement for C-1027 production,
and up to four-fold if cagA gene coding for C-1027 apoprotein was
coexpressed (Murrell et al., 2004). It is noteworthy that genes
coding for enzymes involved in the biosynthesis of 6DOH, such as
sgcA1, are usually clustered together with the rest of secondary
metabolite biosynthetic genes involved in the biosynthesis of the
aglycone (Salas and Méndez, 2005). However, there are a few
examples where genes involved in the biosynthesis of 6DOH are
found outside of the biosynthetic gene cluster and shared
between primary and secondary metabolism. This is the case for
genes involved in the biosynthesis of L-rhamnose usually found
outside of the cluster in all cases reported (Madduri et al., 2001;
Gullón et al., 2006; Luzhetskyy et al., 2007; Ramos et al., 2008).
The duplication of L-rhamnose biosynthetic genes gtt and gdh
encoding NDP-glucose synthase and NDP-glucose dehydratase
respectively, have been used for the improvement of spinosyn
production in Sac. spinosa. These enzymes are the two first
activities responsible for channeling G1P to the biosynthesis of L-
rhamnose and L-dimethyl-forosamine, 6DOHs present in spinosyn
(Madduri et al., 2001).
Other primary metabolism precursors such as G3P, lysine or
2-oxoglutarate can be channeled to enhance the production of
several secondary metabolites by using specific biosynthetic
structural genes (Fig. 2). Overexpression or integration into the
chromosome of clavaminate synthase gene cas2, resulted in up to
five-fold increase in clavulanic acid production in S. clavuligerus.
An additional improvement, up to 23-fold, was achieved by the
simultaneous integration into the chromosome of pathway-
specific activator ccaR and the clavaminate synthase gene cas2
(Hung et al., 2007). In addition, the duplication of pah2 gene,
encoding a proclavaminate amidino hydrolase, has been recently
reported to improve clavulanic acid production (Song et al., 2008).
By expressing lat gene, encoding a lysine e-aminotransferase, in a
high-copy-number plasmid in S. clavuligerus, cephamycin C
production was greatly enhanced (Malmberg et al., 1993, 1995).
A similar approach has been used in order to increase nikkomycin
X production in S. ansochromogenes by the overexpression of sanU
and sanV genes coding for the two subunits of a glutamate mutase
involved in the conversion of glutamate into nikkomycin pre-
cursor 3-methylaspartate (Li et al., 2005). Duplication of sanO
gene coding for a non-ribosomal peptide synthetase (NRPS)
responsible for the formation of the 4-formyl-4-imidazolin-2-
one moiety present in nikkomycin X leads to an equivalent
increment in antibiotic production (Wang and Tan, 2004).
5.2. Gene inactivation or deletion
A different approach for improving metabolite production is to
remove genes coding for activities that transform the metabolite
into a different one. Such is the case of dnrX and dnrH genes
identified in S. peucetius and involved in the transformation of
daunorubicin and doxorubicin into their polyglycosylated forms
known as baumycins. Inactivation of dnrX and dnrH represent
improvements of 3- and 8.5-fold in the biosynthesis of doxor-
ubicin and daunorubicin, respectively (Lomovskaya et al., 1998;
Scotti and Hutchinson, 1996). In addition, disruption of dnrU,
involved in the transformation of daunorubicin into 1,3-dihydro-
daunorubicin, leads to a similar improvement of doxorubicin
production (Lomovskaya et al., 1999). Production of doxorubicin
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288 C. Olano et al. / Metabolic Engineering 10 (2008) 281–292
was further improved by disruption of several genes in the same
strain, which lead to increases up to seven-fold in the dnrU/dnrX
double mutant and to 26-fold in the dnrU/dnrX/dnrH triple
mutant. An additional 1.3–2.8-fold raising was obtained by
overexpressing dnrV and doxA genes, involved in late steps during
doxorubicin biosynthesis, in the double or triple mutants
(Lomovskaya et al., 1999). Following a similar approach, over-
expressing dnmT, involved in the biosynthesis of daunorubicin
deoxysugar L-daunosamine, in the dnrH mutant led to an
improvement in daunorubicin production (Scotti and Hutchinson,
1996).
Channeling of carbon flux by modification of amino acid-
related precursor supplies has been applied to increase clavulanic
acid production in S. clavuligerus, strain that also produces
cephamycin C and other clavams. Production of cephamycin C
can be abolished by inactivation of lat gene coding for a lysine e-
aminotransferase. This inactivation leads in turn to an increase in
clavulanic acid production (Paradkar et al., 2001). A similar effect
can be achieved if production of non-clavulanic acid clavams is
depleted by inactivation of cvm1 (Mosher et al., 1999). Both
approaches, inactivation of lat and cvm1 applied at the same
organism (Fig. 2), in this case an industrial strain of S. clavuligerus,
resulted in a significant improvement of clavulanic acid produc-
tion, several orders of magnitude higher than in the wild-type
strain (Paradkar et al., 2001).
Deletion of biosynthetic genes can lead occasionally to raise
secondary metabolite production. Such is the case of nysF
inactivation that increases nystatin production in S. noursei. nysF
encodes a putative 40 -phosphopantetheinyl transferase that is
supposed to be involved in post-translational modification of the
ACP domains in nystatin type I PKS, but it was shown to negatively
regulate nystatin production (Volokhan et al., 2005). In the case of
nanchangmycin production by Streptomyces nanchangensis, this
can be improved by selective deletion of other PKS-containing
clusters found in the same organism. Eight of these clusters were
found in S. nanchangensis and the inactivation of only one of them
led to a three-fold increase in nanchangmycin yield. This deletion
probably affects precursor supply for the rest of PKSs (Sun et al.,
2002).
5.3. Gene modification
Doramectin (also named CHC-B1) is an antihelmintic polyke-
tide compound produced by a S. avermitilis mutant strain, which
lacks a branched-chain a-keto acid dehydrogenase activity coded
by bkdF. This strain produces two related compounds, doramectin
and CHC-B2. The conversion of CHC-B2 into doramectin is a
process that remains to be clarified but it involves in some way
aveC, a gene with unknown function. Improvement of production
ratios for doramectin was achieved by generating several modified
versions of aveC by site-directed mutagenesis and error-prone
PCR, screening for the best variants and chromosomal insertion of
the refined gene in the producer strain where the wild-type aveC
gene was previously inactivated (Stutzman-Engwall et al., 2003).
Further enhancements of doramectin production were accom-
plished by aveC semi-synthetic DNA shuffling and chromosomal
insertion of the best versions (Stutzman-Engwall et al., 2005).
5.4. Expression of heterologous genes
In S. spiramyceticus F21 the production of shengjimycin (also
named 400 -isovalerylspiramycin) was improved by inserting in its
chromosome a copy of the 400 -O-acyltransferase gene, ist, from S.
mycarofaciens 1748 (Guangdong et al., 2001) (Fig. 3). On the other
hand, productivity of erythromycin has been improved in a Sac.
erythraea industrial strain by a chromosomally integrated copy of
a bacterial hemoglobin gene vhb, originally isolated from
Vitreoscilla spp. (Brünker et al., 1998; Minas et al., 1998). This
system was previously used for the enhancement of cephalospor-
in C in the fungus Acremonium chrysogenum (DeModena et al.,
1993). Improvement of erythromycin production in that strain
may be the consequence of an increase in erythromycin
biosynthetic flux as a result of the increased activity of an
oxygen-dependent step in erythromycin synthesis, most likely
hydroxylation steps (Brünker et al., 1998). However, it is uncertain
how the presence of the oxygen-binding heme protein affects
erythromycin production (Minas et al., 1998).
6. Genome shuffling
Genome shuffling has been described and demonstrated
as a new method for rapid enhancement of secondary metabolite
production. Using this approach six- to eight-fold increase in
tylosin production in S. fradiae were obtained by two rounds of
genome shuffling over a population of classically improved
strains. Similar titers were obtained by classical improvement
methods but along 20 rounds of mutagenesis and screening
(Zhang et al., 2002). Following the same method Streptomyces spp.
U121 has been engineered for the overproduction of (2S,3R)-
hydroxycitric acid by three rounds of genome shuffling after
one round of mutagenesis using nitrosoguanidine (Hida et al.,
2007).
7. Heterologous expression of entire gene clusters
Advances in developing DNA manipulation tools and the
improvement in genome sequencing technologies have proved
fruitful for the isolation of many gene clusters involved in natural
products biosynthesis. However, in some cases production titers
of the encoded compound are low, and there are no genetic tools
optimized for metabolic engineering in the particular producer
strain. The consequence of these problems is an increased interest
for transferring secondary metabolite pathways to new hosts
where genetic tools are available or they have been previously
engineered for the heterologous production of bioactive com-
pounds.
7.1. Plasmids for heterologous expression
Heterologous production of secondary metabolites in S.
coelicolor can be significantly enhanced by using specific plasmids
designed for that purpose. This is the case of plasmid pSMALL that
was obtained by co-integration of plasmids pJRJ2 and SCP2@ both
derived from plasmid SCP2*. The utilization of pSMALL for the
production of 15-methyl-6-deoxyerythronolide B (15-methyl-6-
dEB) resulted in four-fold increase in both S. coelicolor and S.
lividans, probably due to increased gene dosage and higher
plasmid stability. This production can be further enhanced until
25-fold if plasmid pBOOST is used for co-integration instead of
SCP2@ (Hu et al., 2003). Other plasmids have been developed for
its use in the erythromycin producer Sac. erythraea and other
actinomycetes. Such is the case of pCJR24, integrative vector
containing the pathway-specific activator actII-orf4 and promoters
from actI and actIII, that lead to increments in erythromycin
production up to five-fold compared to wild-type strain after
placing the entire PKS under the control of the actI promoter
(Rowe et al., 1998).
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C. Olano et al. / Metabolic Engineering 10 (2008) 281–292
7.2. Using Streptomyces hosts
In some cases heterologous expression has being used as a
genetic tool for the identification of a particular gene cluster that
resulted in the improvement of the production yields. This was
the case of the anthracycline tetracenomycin C produced by S.
glaucescens and the indolocarbazole rebeccamycin produced by
Lechevaleria (formerly Saccharothrix) aerocolonigenes. Tetraceno-
mycin C cluster was identified by its expression in S. lividans,
which resulted in the overproduction of pigmented intermediates
of the biosynthetic pathway (Motamedi and Hutchinson, 1987),
while rebeccamycin gene cluster was identified through the
expression of L. aerocolonigenes DNA containing cosmids into S.
albus, which led to production levels several folds higher that
those of the original strain (Sánchez et al., 2002).
In other cases, a set of genes was heterologously expressed. An
example is the production of megalomycin in Sac. erythraea.
Megalomycin was originally produced by Micromonospora mega-
lomicea and it is structurally very similar to erythromycin but
differs in the presence of an additional deoxysugar, megosamine,
attached at the C-6 hydroxyl group of the aglycone. Since the
production of megalomycin in M. megalomicea is quite poor, the
expression of the megosamine pathway in Sac. erythraea was
attempted. This led to a significant increment in megalomycin
production and opened the possibility for further improvement
both in production and in new compound development (Volche-
gursky et al., 2000). In a similar way, production of the tylosin
aglycone tylactone and its glycosylated derivative desosaminyl
tylactone was achieved by expressing the tylosin PKS genes into a
S. venezuelae strain where the pikromycin PKS genes were
previously deleted to avoid competition for the acyl-CoA pre-
cursors (Jung et al., 2006). Furthermore, 2.7- and 17.1-fold
increments in production of these compounds were accomplished
by introducing an additional copy of the pikromycin pathway-
specific positive regulator gene pikD (Jung et al., 2008).
Expression of different secondary metabolite gene clusters in
heterologous hosts usually is accompanied by approaches pre-
viously described in this review such as overexpression of genes
involved in 6DOH metabolism, fatty acid precursors supply or
pathway-specific regulatory genes. In the case of the aminocou-
marins novobiocin and clorobiocin produced by S. spheroides and
S. roseochromogenes, respectively, the entire gene clusters were
expressed independently in S. coelicolor M512 leading to equiva-
lent production titers of the respective compounds as in the
original strains (Eustáquio et al., 2005a). However, in the
heterologous host these pathways can be more easily engineered
than in their wild-type strains. Metabolic engineering of these
pathways in S. coelicolor has led to enhance novobiocin production
by introducing the regulatory gene novG in a multicopy plasmid
leading to a three-fold overproduction (Eustáquio et al., 2005b)
and to increase 8–26-fold the production of novclobiocin 122 by
the introduction of genes involved in the biosynthesis of
L-rhamnose from the elloramycin pathway (Freitag et al., 2006a).
Production of novclobiocin 122 was attempted before in
S. coelicolor expressing the novobiocin gene cluster, by inactivation
of methyltransferase gene cloU, but only small amounts of the
desired L-rhamnoside derivative were obtained (Freitag et al.,
2006b).
A good example on how to engineer fatty acid precursor supply
in order to enhance the production of a secondary metabolite in a
heterologous host is the production of 6-deoxy-erythronolide B
(6-dEB) in S. coelicolor. This compound is produced from
propionyl-CoA and methylmalonyl-CoA. Production titers of this
compound were increased four-fold by the heterologous expres-
sion of genes matB and matC genes from Rhizobium trifolii,
involved in the generation of malonyl-CoA and methylmalonyl-
289
CoA from exogenous malonate and methylmalonate, in a S.
coelicolor strain expressing erythromycin PKS from Sac. erythraea
(Lombó et al., 2001).
7.3. Using industrially optimized strains
A further increase in production can be achieved by expression
of the newly engineered pathways into industrially optimized
strains. Using this approach, high erythromycin titers were
obtained in the industrial Sac. erythraea K41-135 strain after
expressing a genetically modified PKS. This is possible because it
has been demonstrated that the overproduction phenotype is due
to mutations in non-PKS genes (Rodrı́guez et al., 2003).
7.4. Using other microorganisms as hosts
In the production of secondary metabolites not only important
is the final yield but also the fermentation process. Heterologous
expression of gene clusters in Escherichia coli to overproduce
desired compounds would first reduce fermentation times and
would allow industrial scale fermentations since there are well-
established scalable protocols. In addition, the genetic tools for
engineering E. coli strains are highly developed. Following these
criteria during the last years important efforts have been made for
the production of erythromycin in E. coli. These efforts started by
expressing erythromycin PKS in an E. coli strain genetically
modified to facilitate (i) post-translational PKS modifications by
expressing sfp phosphopantetheinyl transferase gene from Bacillus
subtilis and (ii) methylmalonyl-CoA production by expressing PCC
genes (pccA and pccB) from S. coelicolor. The activity of the
biotinylated subunit PccA was enhanced by overexpression of E.
coli birA biotin ligase gene. The resultant strain was able to
produce 6-dEB at levels equivalent to a Sac. erythaea industrial
strain (Pfeifer et al., 2001). Further metabolic engineering of that
strain such as the expression of metK (Fig. 2) from S. spectabilis
encoding a SAM synthetase, led to a two-fold improvement in
6-dEB production (Wang et al., 2007). All accumulated knowledge
has led to the final production of erythromycin C and D in E. coli by
expression of erythromycin PKS and 17 additional genes from
megosamine pathway coding for enzymes involved in 6DOH
biosynthesis and other tailoring modifications (Peirú et al., 2005).
Similar approaches are underway for the production of polyke-
tides in other heterologous hosts such as Saccharomyces cerevisiae,
where different pathways, propionyl-CoA ‘‘dependent and inde-
pendent’’ for the production of methylmalonyl-CoA have been
introduced (Mutka et al., 2006).
In addition to polyketides, other secondary metabolites such as
non-ribosomal peptides are produced in E. coli. In these cases, the
strain also has to be engineered by introducing sfp gene for post-
translational modification of the NRPS. In that way antitumor
echinomycin and its intermediate triostin A were produced in
E. coli by expressing the entire gene cluster isolated from
Streptomyces lasaliensis (Watanabe et al., 2006).
8. Conclusions
The future success of the pharmaceutical industry depends on
the identification or development of new compounds with novel
activities or directed to more specific targets. The rapidly growing
amount of secondary metabolite gene clusters identified and
characterized provides new genetic tools for the generation of
novel compounds by combinatorial biosynthesis. In addition,
these clusters contain elements that can be used to increase
the production yields. As shown in this review, significant
 ARTICLE IN PRESS
290 C. Olano et al. / Metabolic Engineering 10 (2008) 281–292
improvement of secondary metabolite production has been
obtained during recent years applying different methods of
genetic and metabolic engineering both of wild-type producer
organisms and heterologous host after expression of biosynthetic
gene clusters. These genetic methods include the deletion and
overexpression of genes directed to up- or down-regulate the
expression of secondary metabolite gene clusters, or to override
bottlenecks in the biosynthesis of these compounds. Particular
attention has been drawn to alter carbon flux by redirecting the
central primary metabolic networks toward specific parts of the
metabolism. Following these approaches, further improvements
in the production of the compounds reviewed here and new ones
should be expected in the future, with special attention to the
efforts in the development of heterologous hosts for the expres-
sion of secondary metabolites.
Acknowledgments
Research in the authors’ laboratory has been supported by
grants from the Spanish Ministry of Education and Science
(BFU2006-00404 to J.A.S. and BIO2005-04115 to C.M.), the Red
Temática de Investigación Cooperativa de Centros de Cáncer to
J.A.S. (Ministry of Health, Spain; ISCIII-RETIC RD06/0020/0026)
and from the UE FP6 (Integrated Project no. 005224). We thank
Obra Social Cajastur for financial support to Carlos Olano and
Felipe Lombó.
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