Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407

https://doi.org/10.1007/s10295-018-02120-y

METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - ORIGINAL PAPER

Bioactive molecules from Nocardia: diversity, bioactivities

and biosynthesis
Dipesh Dhakal1 · Vijay Rayamajhi1 · Ravindra Mishra1 · Jae Kyung Sohng1,2 

Received: 22 August 2018 / Accepted: 19 December 2018 / Published online: 18 January 2019
© Society for Industrial Microbiology and Biotechnology 2019

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Abstract
Nocardia spp. are catalase positive, aerobic, and non-motile Gram-positive filamentous bacteria. Many Nocarida spp. have
been reported as unusual causes of diverse clinical diseases in both humans and animals. Therefore, they have been studied
for a long time, primarily focusing on strain characterization, taxonomic classification of new isolates, and host pathophysiol-
ogy. Currently, there are emerging interests in isolating bioactive molecules from diverse actinobacteria including Nocardia
spp. and studying their biosynthetic mechanisms. In addition, these species possess significant metabolic capacity, which has
been utilized for generating diverse functionalized bioactive molecules by whole cell biotransformation. This review sum-
marizes the structural diversity and biological activities of compounds biosynthesized or biotransformed by Nocardia spp.
Furthermore, the recent advances on biosynthetic mechanisms and genetic engineering approaches for enhanced production
or structural/functional modification are presented.

Keywords  Nocardia spp. · Bioactive compounds · Biosynthetic mechanism · Structural modifications · Biotransformation

Introduction pathogenic infections [29]. Natural products (NPs) obtained

Infectious diseases caused by drug-resistant bacteria, cardio-
vascular disease and cancer are the top cause of mortality
in the world. Infections due to pathogens, such as bacteria,
fungi, parasites, virus etc., are the most significant causes of
mortality in both developed and un-developed countries. In
case of low-income countries, there is also high prevalence
of HIV/AIDS, tuberculosis, pneumonia, malaria, and other
This article is part of the Special Issue “Natural Product Discovery
and Development in the Genomic Era 2019”.
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s1029​5-018-02120​-y) contains
supplementary material, which is available to authorized users.
* Jae Kyung Sohng
sohng@sunmoon.ac.kr
1 The genus Nocardia is one of the important members of
Department of Life Science and Biochemical
Engineering, SunMoon University, 70 Sunmoon‑ro
221, Tangjeong‑myeon, Asan‑si, Chungnam 31460,
Republic of Korea
2
Department of BT‑Convergent Pharmaceutical
Engineering, SunMoon University, 70 Sunmoon‑ro
221, Tangjeong‑myeon, Asan‑si, Chungnam 31460,
Republic of Korea
from plants or microorganism, native or structurally modi-
fied, and natural or synthetic are used for the treatment of
such infections and disease conditions [28, 59, 107]. It is
believed that these NPs are secondary metabolites (SMs)
that can support the survival of producer microorganism act-
ing as direct “defense weapon” against other microorganism.
These molecules either aid inter-species and intra-species
communication or provide an advantage in nutrient acquisi-
tion [106]. Actinobacteria are the important microrganisms
characterized as the most prominent sources of such SMs
with pharmaceutical values, such as antibacterial, anti-viral
agent, anticancer agent, anti-parasite, and immunosuppress-
ing activities [6, 21, 36]. The biosynthesis of these SMs is
governed by defined sets of genes called biosynthetic gene
clusters (BGCs), including polyketide synthase (PKS), non-
ribosomal peptide synthase (NRPS) gene, and hybrid of PKS
and NRPS.
the phylum actinobacteria. The most characterstic feature of
Nocardia spp. is that its members possess tuberculostearic
acids in their cell wall, like the members of Mycobacterium.
However, different from mycobacteria, Nocardia spp. con-
tain short chain (40–60 chain) mycolic acids and usually
exhibit branching on Gram staining [8]. They also possess

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numerous distinct morphological features, including the
presence of fragmenting hyphal forms, with characteris-
tic presence of short chain spores, cyclic menaquinones in
bacterial cell membranes, and distinct distribution of ara-
binose and galactose in their cell walls [37, 38, 84]. They
are catalase positive, aerobic, and non-motile [27]. They
produce primary mycelia and usually fragment into rod-
to coccoid-shaped hyphal forms. Nocardia spp. can show
diverse morphologies ranging from smooth to rough with
clear margined to irregular colonies. They also exhibit vari-
ous colors [27, 84]. These unique morphological features
make the members of genus Nocardia distinct from other
members in actinobacteria.
Nocardia species have been studied from a long time, but
most of the studies have focused on the characterization and
taxonomic classification of new isolates. They are frequently
characterized as causative agents for a wide spectrum of
infectious diseases in both humans and animals. They can
infect cattle, horses, dogs, and swine. Therefore, they are
mostly isolated from such hosts, and only few strains are iso-
lated from environmental samples as soil and water [8, 91].
However, recently Nocardia spp. has been more importantly
characterized as a source of diverse bioactive substances
with pharmaceutical value [27, 84]. In addition, they have
been utilized in whole-cell biotransformation of bioactive
compounds or industrial production of valuable chemicals
[15, 30]. Hence, there are burgeoning interests in studying
their biosynthetic mechanisms of bioactive compounds and
their discrete metabolic pathways for biotransformation. The
aim of this review is to summarize diversities and activi-
ties of bioactive compounds isolated from Nocardia spp.
This review also provides insight into their biosynthetic
mechanisms and their applicability in biotransformation
experiments for generating novel derivatives of bioactive
molecules.
Classification and taxonomy
Genus Nocardia belongs to family Nocardiaceae, suborder
Corynebacterineae, order Actinomycetales, class Actinobac-
teria, and phylum Actinobacteria. The family Nocardiaceae
includes other morphologically or biochemically related
genera, including Gordonia, Micropolyspora, Millisia, Rho-
dococcus, Skermania, Smaragdicoccus, and Williamsia in
addition to genus Nocardia. Actinoplanes, Actinomadura,
Micromonospora, Streptosporangium, Saccharopolyspora,
Streptoverticillium, and Nocardia are referred as “non-strep-
tomyces actinomycetes” or “rare actinomycetes” [25].
The first reported isolation of Nocardia strain was from
a case of bovine farcy in 1888 by Edmond Nocard. The
strain was later named as “Nocardia farcinica”. Later, many
species belonging to genus Nocardia were identified and
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Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
taxonomically classified by using biochemical tests such as
antimicriobial susceptibility tests, substrate decomposition
tets and drug resistance patterns. Hence the differentiation
and identification was dubious. Around the 1990s, molecular
identification methods started to be applied in taxonomy and
strain characterizations. Hereafter, taxonomic classifications
were based on new methods such as restriction endonuclease
analysis of the portion of heat shock protein gene or 16S
ribosomal RNA (rRNA) genes [18, 120]. However, it has
been observed that 16S rRNA gene sequencing is inefficient
in satisfactorily distinguishing species. Meantime, stochas-
tic genetic variation, horizontal gene transfer, and recom-
bination could significantly affect the identification based
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on DNA sequence of a single housekeeping gene. Hence,
multilocus sequence analysis (MLSA) has been used for the
identification of Nocardia species because it exhibits more
powerful differentiation between different Nocardia species
[92, 136]. Tamura et al. [136] have analyzed draft genome
sequences of 26 Nocardia spp. and constructed a phyloge-
netic tree based on MLSA of concatenated sequences of
12 genes (atpD-dnaJ-groL1-groL2-gyrB-recA-rpoA-secA-
secY-sodA-trpB-ychF). They found that the correlation
between whole genome-derived and multiple gene-derived
relationships was useful for the classification of Nocardia
spp [136]. Furthermore, by utilizing whole genome analysis,
MLSA, and DNA–DNA hybridization, phylogenetic analysis
of 72 validated Nocardia spp. has been performed along-
with reclassification of various Nocardia species [137]. To
date, more than 200 species of Nocardia have been reported
based on various isolation strategies, biochemical studies,
and taxonomical classification methods (National Center for
Biotechnology Information/Taxonomy).
Genomic diversity and metabolic features
of Nocardia spp
Various strains belonging to Nocardia spp. have been cat-
egorized as either pathogenic, or inhabiting harsh environ-
mental conditions. So, in particular, these types of strain
are considered biologically more active, and to possess
specialized metabolic pathways [96]. In addition, adapta-
tion to the host strain or hostile environmental conditions
renders them with diverse biochemical and metabolic fea-
tures [23]. Many Nocardia spp. possess unique capabilitities.
They can either produce bioactive molecules with unique
chemical structures or degrade toxic substances and bio-
transform to be useful substances for industrial uses [23,
83, 84]. The rapid development of genome sequencing has
unveiled genetic information of many Nocardia spp., provid-
ing information about their wide metabolic capabilities and
biosynthetic pathways. “Genomic mining” provides connec-
tion of genomic sequence (BGCs) with particular bioactive
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 387

compound [157]. For example, genome-based analysis has
been performed using in silico tools to predict PKS and
NRPS gene clusters in different strains of Nocardia spe-
cies [69, 152]. Such detailed analysis of genome can also
unveil information about the genomic architecture that is
responsible for their unique biochemical and metabolic prop-
erties, which in turn can be associated with their biodegrada-
tion/biotransformation capabilities. For example, pathways
and probable mechanism of rubber and gutta-percha (GP)
degradation by Nocardia nova SH22a have been proposed
based on analysis of its complete genome information [83].
Recently, approaches of introducing the environmental DNA
into suitable expression host to create metagenomic library
with diverse chemical structures such as beta-lactams, pol-
yketides, terpenoids, and siderophores. Their bioactivities
were evaluated and reported. Table 2 shows the major bio-
active chemical entities isolated from diverse Nocardia spp
during the past decades along with their most promising
biological importance.
Nocardicins
Nocardicins (Fig. 1) are antibacterial compounds isolated
from the fermentation broth of Nocardia uniformis subsp.
tsuyamanensis [2]. Structural elucidation studies confirmed

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have enabled exploration of biosynthetic capability for
non-cultivable or genetically intractable strains [25]. Such
approaches can be used to understand the biosynthesis of
bioactive molecules from these pathogenic strains that are
generally genetically intractable. Table 1 shows the general
information of the available complete genome of Nocardia.
Bioactive compounds from Nocardia spp.
Nocardia species are an important microbial reservoir for
the isolation of bioactive compounds with clinical impor-
tance [23, 27, 84]. Pathogenic species such as Nocardia have
attracted increasing interest for several reasons. The patho-
genic behavior is believed to be associated with competi-
tion mechanisms with their hosts, which in turn lead to the
discrete metabolic pathways that are divergent from those
of non-pathogenic producer strains. Thus they can generate
diverse molecules for their survival and maintenance [84,
96]. Hence, numerous antibacterial, antivirus, antifungal,
immunosuppressive, and cytotoxic agents have been isolated
from different species of Nocardia, including compounds
the presence of monocyclic beta-lactam ring, along with
the presence of p-hydroxyphenylglycine and oxime units.
Among all nocardicins, the major compounds are named
nocardicin A and B that share structural similarities [42].
Activities of nocardicin A are attributed to presence of the
syn-oxime and terminal D-configuration of the homoseryl
side chain. Similar to all beta-lactams, nocardicins works
by inhibiting the synthesis of peptidoglycan layer of bacte-
rial cell walls. However, unlike most of the bicyclic beta-
lactams, nocardicin A shows resistance to beta-lactamase
probably due to presence of more stable monocyclic beta-
lactam ring in its structure [67]. In addition, unlike other
beta-lactam antibiotics, the activity of nocardicin A is higher
in vivo than that in vitro experimental models [58].
Earlier studies on the biosynthesis of nocardicins were
based on feeding experiments with radiolabeled precursors
to N. uniformis tsuyamanensis [47]. Natural amino acid
l-serine and two units of non-proteinogenic amino acid,
p-hydroxyphenylglycine, have been established as direct pre-
cursors in biosynthesis of Nocardicin A by precursor incor-
poration studies [47, 146]. Its homoseryl side chains have
been reported to be derived from l-methionine [142, 144].

Table 1  Genomic features of Organism N. nova N. brasiliensis N. seriolae N. farcinica N. cyriacigeorgica

different Nocardia spp. SH22a HUJEG-1 UTF1 IFM 10152a GUH-2a

Chromosome size 8348,532 9,436,348 8,121,733 6,021,225 6,194,645

Plasmids No No No 4 1
Accession no CP006850 CP003876 AP017900 AP006618 FO082843
GC content 67.77 68.05 68.1 70.69 68.37
rRNA 9 9 12 9 9
tRNA 49 50 64 58 49
Other RNA 3 3 3 3 3
Gene 7565 8496 7751 6114 5605
Pseudogene 125 114 433 174 79
Protein 7379 8320 7239 5870 5465
BCGs# 28 44 26 16 19
References [83] [152] [156] [53] [161]
#
 The number of BGCs determined with antiSMASH with ClusterFinder OFF

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Table 2  Major bioactive molecules isolated from Nocardia spp

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389

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Table 2  (continued)
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Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407

Table 2  (continued)

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Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 391

Table 2  (continued)

H3C
OH H O
HO N S N O
NH CH3

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HO O N S O
O O N O H O
O HO O HN N N O N
OH N N
O H H O CH3
O O O H NH S NH3C OH
NH HO N OH
OH O S
H N O HO O HN O
O O NH NH H NH
O H O H3C N
N H O
NH O NH HN S
OH O N O N
H N CH3
OO HO H3C O
N NH2 O O H3C
S
HO O
a b c d

OH
OH H OH
NH2
N HN
N O S S N
O N
O O COOH N NH
OH O N
O O O H H OH N O
H2N O H O O HO HO
N H OH
HOOC O HO HO
O
e f g h i

Fig. 1  Representative bioactive compounds produced by different
Nocardia spp. a Nocobactin A, a siderophore produced by N. farci-
nica. b Brasilicardin A, an immunosupprasant produced by N. brasil-
iensis. c Nocavionin, an antimycobacterial roduced by N. terpenica. d
Nocardithiocin, an anti-mycobacterial produced by N. pseudobrasil-
iensis. e Nocardicin NA, an antibacterial produced by N. uniformis.
f Nargenicin A1, an antibacterial produced by Nocardia sp. CS682. g
Tubelactamicin A1, an antimycobacterial produced by N. vinacea. h
Intervenolin, an anti-Helicobacter produced by N. terpenica. i Formy-
cin A, an anti-retroviral produced by N. interforma 

Subsequently, it has been observed that amine oxidation can
lead to the formation of oxime while its monocyclic beta-
lactams is formed simply and directly through nucleophilic
displacement of activated seryl hydroxyl by amide nitrogen
without requiring a change of oxidation state [143, 145].
Gunsior et al. [39] have reported the BGC for nocardicin
A, which includes fourteen open reading frames as shown
in Table S1. NocF, NocG and NocN have been predicted to
be involved in biosynthesis of the nonproteogenic l-p-hy-
droxyphenylglycine (pHPG) precursor. NocA and NocB
belong to a nonribosomal peptide synthetase that is respon-
sible for assembly of two units of l-pHPG, and one unit of
serine to yield a d, l, d-tripeptide, or possibly nocardicin
G by concerted action of five domains of NocA and NocB
[20]. In vitro reaction studies showed that Nat catalyzes the
addition of the homoserine sidechain which is crucial to the
transfer of 3-amino-3-carboxypropyl group from S-adeno-
syl-l-methionine to the diverse substrates such as nocardicin
E, F, and G, although nococardicin G is the most preferred
substrate [119]. Nocardicin G is the first beta-lactam anti-
biotics containing intermediate in the biosynthetic path-
way. Further, amine oxidation to generate C-2′ oxime, the
attachment of homoserine residue from methionine, and an
epimerization event in which C-9′ undergoes L–D configu-
ration leads to the formation of nocardicin A. In vivo gene
inactivation and in vitro enzymatic studies have revealed
that C-9′ epimerization is catalyzed by NocJ [65], whereas
N oxygenation is catalyzed by NocL, leading to the forma-
tion of oxime in nocardicin A [63]. It was observed that
nocardicin A cluster did not possess an NAD(P)H or fer-
rodoxin. It was assumend that the electron transport protein
was recruited elsewhere from cellular mechanism. Hence,
an in vitro enzymatic assay using spinach ferredoxin and
spinach ferredoxin-reductase was performed. It established
that Nocardin C was the most suitable substrate for NocL.
Meanwhile, the conversion of nocardicin G to E or F was not
significantly observed. Hence, it was established that NocL
could act on 2′ amine of nocardicin C to produce oxime in

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nocardicin A whereas oxidation of nocardicin G was infea-
sible. Therefore, it was established that the biosynthesis
of nocardicin A from nocardicin G first required attache-
ment of homoseryl side chain, followed by inversion at the
C9′ stereocenter and oxime formation [64]. Based on these
information, biosynthetic steps for biological production of
nocardicin A1 are assigned in order as shown in Fig. 2.
NocD with similarity to the superfamily of acetyltrans-
ferase and NocH showing similarity to membrane transport
protein have been suggested to be involved in resitance
mechanism against nocardicins [107]. Acetyltransferase
is the predominant mechanism of antibiotic resistance by
pathogenic bacteria against aminoglycosides. Membrane
transport proteins are involved in exporting the antibiotics
outside the cell and generally associated with self-resistance
mechansim [39]. NocR is related to the family of pathway-
specific activators as SARPs (Streptomyces antibiotic regu-
latory proteins). Gene inactivation, transcriptional analysis,
and DNA binding assays have established that NocR is a
positive transcriptional regulator [19].
Nocobactin
Nocardia spp. produces structurally diverse siderophores
(Table 1) to facilitate the uptake of metal ions such as iron.
Nocardia spp. are highly related to mycobacteria, so they
produce numerous mycobactin-like siderophores [113],
called nocobactin. The basic difference between mycobac-
tin and nocobactin is that nocobactin has an oxazole ring
whereas the mycobactin has an oxazoline ring [118]. Noco-
bactin A (Fig. 1) is mycobactin-like siderophores produced
by Nocardia farcinica ATCC 3318, with cytotoxic activities
[121].
Fig. 2  Biosynthetic mechanisms of nocardicin A. l-tyrosine is first
converted to l-pHPG, which is condensed with l-Ser and l-Arg to
form nonribosomal peptide synthase, peptide product. It eventually
forms Nocardicin G and the addition of the homoseryl sidechain is
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Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
Analysis of the complete genome sequence of Nocar-
dia farcinica IFM 10152 [53] has revealed the presence
of 11 NRPS genes and 7 PKS genes. Among them, gene
cluster I has been predicted to be involved in biosynthe-
sis of nocobactin A [45]. The gene cluster I includes eight
genes with significant homologies with biosynthetic genes
for mycobactin, as shown in Table S2. NbtA encodes thi-
oesterases, with significant homology with MbtB from M.
tuberculosis. NbtB and NbtC are involved in the formation
of core polyketide backbone. Both NbtB and NbtC contain
required domains for polyketide biosynthesis viz keto acyl
synthase (KS), ketoreductase (KR), acyltransferase (AT),
and acyl carrier protein(ACP). NbtD, NbtE and NbtF are
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NRPS modules. Each of them possesses peptidyl carrier
protein (PCP), and domains as adenylation (A) and conden-
sation (C). NbtG catalyzes the N6-hydroxylation of lysine
and NbtH transfers an acyl chain to the ε-amino group of
lysine. Structurally, nocobactin NA contains hydroxyben-
zoate, methyloxazoline, N-hydroxy-N-acetyl-lysine, a fatty
acyl chain, and N-hydroxy-ɛ-caprolactam moieties. Genes
in cluster I are coherent with the structure of nocobactin I
except for the hydroxybenzoate moiety that can be derived
from salicylate. Another cluster (cluster II) has been found
180 kb apart from cluster I which includes two genes des-
ignated nbtS and nbtT. NbtS has high homologies to the
bifunctional salicylate synthetases. NbtT is presumed to be
a discrete NRPS. The heterologous expression of nbtS into S.
avermitilis, lacking a salicylate synthase gene in its genome
has been reported to produce salicylate. This confirms role
of nbtS as salicylate synthase. Similarly, unmarked in-frame
deletion of nbtA and nbtS can lead to reduction of noco-
bactin production to level of 1% or less than the wild type.
However, in case of deletion mutant of nbtE, the production
of nocobactin has been complete abolished, whereas new
compounds equivalent to nocobactin NA derivatives missing
catalyzed by Nat. The subsequent N-oxygenation by NocL leads to
formation of nocardicin A. l-pHPG: l-p-hydroxyphenylglycine; PLP:
pyridoxal 5′-phosphate
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 393
the N-hydroxy-ɛ-caprolactam moiety has been detected [45].
Based on these biochemical and genetic studies, the biosyn-
thesis mechanism for nocobactin A is proposed as shown
in Fig. 3.
Nocardithiocin
Thiopeptides (thiazolyl peptides) are sulfur rich peptides
synthesized ribosomally.They usually contain a six-mem-
bered ring with a central nitrogen that can serve as scaf-
fold for at least one macrocycle and a tail [57]. Thiopeptide
compounds have a broad spectrum of biological activities,
including antimicrobial, antiviral, anticancer, and antiplas-
modial activities. Ribosome pathway or NRPS pathways can
synthesize these peptide-based natural products. In case of
thiopeptide compounds, the biosynthesis begins with ribo-
somally-synthesized precursor peptide followed by several
modifications such as cyclilization and dehydration to form
the macrocycle structure. In addition, several side chain
Fig. 3  Biosynthetic mechanism of nocobactin A. NbtS converts the
chorismate to salicylate. Subsequently, salicylate is adenylated and
cyclilized by NbtT and NbtF respectively. NbtB and NbtC results in
formation of core polyketide backbone. NbtA is thioesterases and
modifications can lead to final products. Of all of the thio-
peptide gene clusters reported, core sets of genes involved in
heterocyclilization and dehydration of core peptides along-
with sets of genes encoding precursor peptide have been
observed [80].
Nocardithiocin (Fig.  2) is a thiopeptide produced by
Nocardia pseudobrasiliensis that exhibits prominent activ-
ity against acid-fast bacteria, including clinical pathogens,
such as rifampicin-resistant M. tuberculosis [100]. Before
identifying the nocardithiocin BCG, the production of nocar-
dithiocin has been confirmed by metabolite profiling of
fourteen different N. pseudobrasiliensis strains. The highest
production has been observed in N. pseudobrasiliensis IFM
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0761. Sequencing of whole genome N. pseudobrasilensis
and comparative analysis of the BGC of thiopeptide com-
pounds in diverse strains have led to the identification of the
BGC of nocardithiocin [122]. Gene inactivation and com-
plementation studies of notL (the putative cyclodehydratase)
have been performed to confirm the genomic loci of BGC.
Comparative transcriptional analysis and gene expression
NbtD catalyzes the condensation. NbtG catalyzes the N6-hydroxyla-
tion of lysine whereas; NbtH transfers an acyl chain to the ε-amino
group of lysine leading to formation of nocobactin as final product
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394
profiling have been performed for N. pseudobrasiliensis IFM
0761 under nocardithiocin-producing and non-producing
conditions. Results showed that the addition of fetal bovine
serum (FBS) played a significant role in elevating the pro-
duction titer of nocardithiocin. So, RPMI 1640 medium sup-
plemented with 10% FBS has been used as nocardithiocin
producing condition, whereas RPMI 1640 medium without
FBS has been used for non-producing conditions. Expres-
sion levels have been compared and quantified by reads per
kilobase of exon per million-mapped sequence read (RPKM)
values by RNA-seq analysis. Fold changes in RPKM val-
ues have been compared between nocardithiocin-producing
and non-producing conditions (production/no-production).
It has been observed that RPKM fold changes of predicted
genes in the biosynthetic gene cluster (notA-notL) were
higher whereas fold changes for flanking regions were very
small [122]. Hence, the BGC has been predicted based on
the catalogue of genes only over-expressed under the nocar-
dithiocin-production condition (Table S3). The biosynthetic
Fig. 4  Biosynthetic mechanism of nocardithiocin. Cascades of
enzymes resulting in stepwise dehydration and dehydrogenation cata-
lyzes the modification of the precursor peptide. Finally, the notE and
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Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
mechanism has been proposed based on the biosynthetic
genes. Figure  4 shows their putative functions of genes
encoded in BGC of nocardithiocin in N. pseudobrasiliensis
IFM 0761.
Nargenicin A1
Nargenicin A1 (Fig. 1) belongs to group of naturally occur-
ring reduced alicyclic polyketides with characteristic pres-
ence of a cis-fused octalin ring system. It has been first
reported from the cultures of N. argentinensis in 1977 and
named as CP-47444 [13]. It also also isolated and charac-
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terized from Nocardia sp. CS682 (KCTC 11297BP), a soil
isolate from Jeonnam, South Korea [131]. Nargenicin A1
is significantly effective against Gram-positive bacteria,
including methicillin-resistant Staphylococcus aureus. It
possesses stronger activity and lower cytotoxicity than com-
monly used drugs such as erythromycin, spiramycin, and
notH are responsible for characteristic methylation and hydroxylation
reaction, to generate nocardithiocin as the final product
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 395
vancomycin [68, 131]. Recently, it has been characterized as
the first identified natural product inhibiting DnaE, a crucial
enzyme involved in DNA replication [112].
The biosynthetic mechanism for nargenicin has been
studied and confirmed through different feeding experi-
ments with different radiolabeled precursors. The polyke-
tide backbone of nargenicin is derived from five acetate and
four propionate building blocks. Radiolabeled precurosrs
such as [1-l3C]-, [2-I3C]-, and [1,2-13C2]acetate and [1-13C]-
and [2-13C] propionate have been directly administered to
cultures of Nocardia argentinensis Huang, ATCC 31306.
Experimental results indicated that the carbon pairs, such
as ­C1–C2, ­C3–C4, ­C5–C6, ­C7–C8, and ­C11–C12 of nargenicin
A1, are derived from acetate. In addition, the ­C17–C18–C19,
­C15–C16–C20, ­C13–C14–C21, and ­C9–C10–C22 triads of nar-
genicin A1 are derived from propionate. Thus, advanced
di-, tri-, and tetraketide fatty acid precursors are incorpo-
rated directly into the nargenicin A1. In addition, incorpora-
tions of [1-18O2,1-13C]acetate and [l-18O2,1-13C]-propion-
ate have been studied. Labeling studies with 18O-labelled
propionate-acetate precursors have indicated that oxygen
atoms at ­C1, ­C9, ­C11, and ­C17 are derived from propion-
ate and acetate. Fermentation studies conducted in an 18O2
atmosphere has indicated that the ­C2 and ­C18 oxygen sub-
stituents as well as the C ­ 8–C13 ether bridge of nargenicin
A1 has originated from molecular oxygen [10–12]. Thus,
the formation of nargenicin A1 has been hypothesized to
take place with three distinct phases: (1) biosynthesis of
a branched chain, partially saturated polyketide chain, (2)
cyclilization and generation of macrolide and ring system,
and (3) post modifications such as oxidations, metylations
and addition of pyrrole moiety. Moreover, the demonstration
Fig. 5  Proposed biosynthetic mechanism of nargenicin A1.The pro-
posed pathway includes there major biosynthetic stages: (1) conden-
sation of short chain fatty acid precursors to form long chain pol-
of origins of the various oxygen atoms rules out plausible
epoxy-olefin cyclization mechanisms, suggesting that the
characteristic octalin ring system is generated by an intramo-
lecular Diels–Alder reaction [11]. The presence of pyrrole as
characterstics ester of polyketide backbone is an interesting
feature of nargenicin A1. The formation of pyrrole moiety
has been confirmed to be derived from l-proline based on
in vitro enzymatic assay using three proteins, NgnN4 (pro-
line adenyltransferase), NgnN5 (proline carrier protein),
and NgnN3 (flavine-dependent acyl-coenzyme A dehydro-
genases) from Nocardia sp. CS682 [89]. Similarly, a path-
way engineering approach has been employed to enhance
the biogenesis of pyrrole moiety by overexpression of these
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gene sets, subsequently leading to enhanced production of
nargenicin A1 [22]. Based on these inferences from different
experimental feeding studies and in vitro enzymatic studies,
the proposed biosynthetic pathway is shown in Fig. 5.
Brasilicardin A
Brasilicardin A has been reported to be produced by Nocar-
dia brasiliensis IFM 0406 (currently referred as N. terpen-
ica). It has a unique structure, including a diterpene skeleton
attached with N-acetylglucosamine, amino acid, l-rhamnose,
and 3-hydroxybenzoate moieties [129]. Brasilicardins
B–D has been later isolated from Nocardia brasiliensis
IFM0406, and their structures have been determined [70].
Brasilicardin A (Fig. 1) exhibits potent immunosuppressive
activities. Hence, there is high interest in biosynthesis and
production of this molecule. Due to its high potency as an
effective immunosuppressant, different chemical synthesis
yketides, (2) cyclization to generate macrolide systems, and (3) final
post-modifications by oxidations and introduction of the ­C23 methyl
and ­C9-pyrrole-groups of nargenicin A1
13

396
approaches have been attempted. Anada et al. [1] have suc-
cessfully achieved the total synthesis of brasilicardins A and
C for the first time. Similarly, Jung et al. [56] have synthe-
sized brasilicardin A analogue, in which the natural tricyclic
skeleton has been replaced with a synthetically more acces-
sible substituted tetrahydronaphthalene core.
To understand the biosynthesis of brasilicardin, a feeding
experiment with radiorabelled glucose has been performed.
D-[1-13C] glucose was added to growing culture of N. bra-
siliensis IFM 0406. Clear increment of the signals of ­C2,
­C6, ­C11, ­C15, ­C19, ­C21, ­C22, and ­C23 in the perhydrophen-
anthrene skeleton was observed. Thus it was hypothesized
that the perhydrophenanthrene skeleton in the compound
was derived from geranylgeranyl phosphate. In addition, the
increment of a signal of C ­ 16 suggested that the amino acid
moiety ­(C16-C18) might be derived from [3-13C] pyruvate.
The feasibility of incorporating the label into these posi-
tions might be due to glycolysis of the labeled glucose to
[3-13C] pyruvate and [3-13C] glyceraldehyde 3-phosphate
to form [l,5-13C2] isopentenyl diphosphate (IPP) through the
non-mevalonate pathway [128]. Geranylgeranyl diphosphate
synthase (GGPPS) has been characterized as a direct pre-
cursor to diterpene compounds. Hence, genes responsible
for GGPPS have been cloned, heterologously expressed in
E. coli, and functionally characterized. In addition, bra4
encoding a cyclase gene has been disrupted in the producer
strain. Based on these observation and analysis of sequence
alignment, the BGC has been predicted (Table S4) and bio-
synthetic pathway proposed [43].
Fermentation-based bio-production of natural products
is often considered as being more cost effective and less
hazardous than the chemical synthesis [55]. Heterologous
expression is one of the effective bioprocesses, where a sin-
gle gene, a cassette of genes, or an entire BGC is introduced
into a genetically tractable or metabolically efficient host to
identify and engineer the corresponding natural products [5,
28, 85]. Schwarz et al. [125]. have attempted the expression
of gene cluster of brassilicardin, and performed the second-
ary metabolite profiling using Amycolatopsis japonicum as
a heterologous host. They confirmed the production of bra-
silicardin congeners (BraC and BraD) and determined the
borders of the brasilicardin gene cluster. Meanwhile, Bra12
(a positive transcriptional regulator) and Bra0 (a putative
dioxygenase involved in methoxylation of brasilicardin) have
been identified in their study. Hayashi et al. [43] have sug-
gested that Bra6 or Bra8 is responsible for the hydroxyla-
tion reactions at ­C2 and at C ­ 16 of the brasilicardin diterpene
backbone, whereas Bra11 attaches methyl group at hydroxyl
group at ­C16. The proposed biosynthetic pathway was in the
order as shown in Fig. 6. However, Schwarz et al. [125] have
reported that levels of unmethoxylated BraD are increased
when bra0 is deleted while levels of methoxylated BraC
are increased when bra0 is overexpressed. Based on these
13
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
observations, they suggested that methoxylation at C ­ 16 of the
brasilicardin backbone starts with an oxidation performed
by Bra0, catalyzed by an incorporation of dioxygen. Subse-
quently, this intermediate is methylated by Bra11 to gener-
ate methoxy group. Thus, the extension of the brasilicardin
BGC by bra0 envisioned a revised biosynthetic pathway for
brasilicardin. In addition, different recombinant strains of A.
japonicum have been generated by deletion or overexpres-
sion of bra12. It has been observed that the production was
enhanced in case of overexpression, whereas the deletion
strain could not produce any brasilicardin congeners. Fur-
ther analysis of transcription level for all bra genes (includ-
ing bra0 to bra12) was performed in different recombinant
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strains by RT-PCR. Results of analysis confirmed that bra12
is a positive regulator crucial for the production of brasilicar-
din [125]. Based on the secondary metabolite profiling, the
revised biosynthetic pathway has been proposed as shown
in Fig. 6.
Other metabolites from Nocardia spp.
Most of the bioactive compounds from Nocardia spp. have
not been characterized and studied properly. Biosynthetic
mechanism is known for even fewer of them. For a few bio-
active molecules derived from Nocardia spp., their biosyn-
thetic steps have been studied in detail, which has been dis-
cussed already. However, for a few other compounds, their
biosynthetic steps have been partially studied.
Intervenolin (Fig. 1) is an interesting compound with
multiple biological activities. It was isolated from Nocardia
sp. ML96-86F2. It has unique structure possessing peculiar
side chain containing (–C–N=C–(SCH3)2). It can inhibit the
growth of human gastric and colorectal cancer cell lines. It
also shows antitumor activity against a xenograft model of
human colorectal cancer cells in vivo. Most interestingly, it
possess selective anti-Helicobacter pylori activity [62]. Its
unique structural features along with its bioactivities make
this molecule interesting to both biologists and chemists.
Nocavionin (Fig. 1) is a lanthipeptide compound pro-
duced by Nocardia terpenica [153]. Lanthipeptides are
major classes of ribosomally synthesized and post-trans-
lationally modified peptides (RiPPs) [3]. Their common
structural features include noncanonical thioether amino
acids lanthionine (Lan) or labionin (Lab), that can be enzy-
matically installed as post-translational modifications. The
structural features confer proteolytic stability that play cru-
cial roles in a variety of its functions, includimg antimi-
crobial and antiretroviral activities. [153]. Microvionin is
a lanthipeptide isolated from a culture of Microbacterium
arborescens. It has unique chemical structure including
decarboxylated labionin. Therefore, it is the first N-termi-
nally lapidated RiPP. In addition, it possesses promising
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 397
H
Bra2 Bra5
IPP and
DMAPP
OPP OPP
O H
OCH3
H O
HO O H
O Bra0
O O OH
Bra9 Bra8 Bra7 Bra10 HO
OH NH2
O OH
O HO H L-Rhamnose,
HO 3-hydrobenzoate
HO
NH N-acetylglucosamine
O
Fig. 6  Proposed biosynthetic mechanism of Brasilicardin A. Bra2
starts the synthesis by catalyzing the condensation of geranylge-
ranyl diphosphate (GGPP). In the later steps, for formation of char-
acteristic diterpene backbone of brasilicardin continue with Bra5
(epoxidation), Bra4 (diterpene cyclisation), and Bra3 (prenylation of
the amino acid moeity). Further tailoring reactions are catalyzed by
activity against different Gram-positive pathogenic bacte-
ria, including methicillin-resistant S. aureus (MRSA) [153].
Nocavionin is also structurally similar to microvionin due
to presence of characterstic avionin motif. However it pos-
seses a shortened unsaturated bismethylated guanidine fatty
acid (MGFA) moiety. Such disparity in nocavionin fatty acid
moiety compared to microvionin might be due to shorter
elongation cycle and incomplete enoyl reduction during
MGFA biosynthesis [153].
Tubelactomicin A (Fig. 1) is a macrolide antibiotic iso-
lated from Nocardia sp. MK703-102F1. It shows strong and
specific activity against various Mycobacterium spp, includ-
ing drug-resistant strains [49]. Due to its potency as an anti-
tuberculosis drug, different chemical synthesis approaches
have been utilized for the total synthesis of this compound
[48, 98, 99].
Formycin (Fig. 1), an effective antiretroviral compound
consists of the C-ribosides of pyrazolopyrimidine deriva-
tives [73]. By feeding diverse precursors, the key steps in
the biosynthesis mechanism of formycin A produced from
Nocardia interforma have been determined [109, 123]. Che-
momicin is an angucyclinone antibiotic with potent immu-
nosupprasant activities and anticancer properties. It was
extracted from the fermentation broth of Nocardia mediter-
ranei subsp. kanglensis 1747-64 [79, 134]. Diverse antibac-
terial lipopeptide, peptidolipins B-F have been isolated from
marine Nocardia sp. (strain WMMB215), cultivated from
the ascidian Trididemnum orbiculatum. It has been reported
that peptidolipins have significant antibacterial effect against
both methicillin-sensitive S. aureus (MSSA) and methicil-
lin-resistant S. aureus (MRSA) [155]. A novel anticancer
and antifungal phenazine derivative N-(2-hydroxyphenyl)-
2-phenazinamine (NHP) and six known antibiotics have
H H O
Bra4 OH Bra3 OH
OPP OP O
HO HO
H
Bra6 Hydroxylation
Bra1 Amination
OCH3
O H O
HO
OH Bra11 OH
NH2 Dioxygenation NH2
HO HO
H Methoxylation H
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Bra1 (amination), Bra6 (hydroxylation) and Bra11 (methoxylation).
Bra0 is a dioxygenase, which catalyzes the incorporation of dioxygen
at ­C16 which is directly methylated by Bra11. Bra7, Bra8 and Bra9
(3-hydroxybenzoate activation, transfer) and Bra10 (l-rhamnose
transfer) are the final tailoring steps
been isolated from Nocardia dassonvillei [34]. A novel
siderophore compound named JBIR-16 has been isolated
from Nocardia tenerifensis NBRC 101015 [102]. These
results strongly support that strains belonging to Nocardia
spp. have huge potential to produce compounds with diverse
chemical structures and biological properties. Hence, it will
be very interesting to unravel the biosynthetic mechanism
of such compounds and develop more effective derivatives
using different chemical and biological approaches [25, 28].
Metabolic engineering for enhancing
the production of bioactive compounds
from Nocardia spp.
Natural products derived from plant or microbial sources
are appropriate starting point in drug discovery and devel-
opment. These molecules of natural origin are produced
by primary or secondary metabolic pathways of producer
hosts [6, 14, 21, 26]. For commercial use, large-scale pro-
duction by microbial fermentation, chemical synthesis, or
semisynthetic processes is required. The production through
biological processes can be superior in terms of cost effec-
tiveness, environmental safety, and scale-up possibilities.
Major advantages of biosynthesis over chemical synthesis
include its chemical selectivity, environmental friendliness,
molecular diversity, and less costly process of scale-ups [14,
28, 154].
However, most of Nocardia spp. isolated from nature
are usually fastidious. They produce only trace amounts
of a particular secondary metabolite, making it difficult to
isolate and produce such compounds for widespread clini-
cal use. In addition, the Nocardia spp. are biosafety level 2
13

398
organisms. Hence, it can be labor intensive and time-con-
suming to optimize the genetic manipulation in the native
producer to understand their biosynthetic mechanism and
industrial level production [23, 84, 85, 126]. Heterologous
expression is a key strategy to gain access to the promising
secondary metabolite gene cluster of actinomycetes. Heter-
ologous expression of the biosynthetic pathway of brasili-
cardins has been employed in Amycolatopsis to gain insight
on biosynthetic mechanism [125]. Similarly, Bra12 has been
identified as a positive regulator. The production level of
brasilicardin congeners from A. japonicum::bcaAB01_bra12
(over-expression strain) were significantly enhanced (+ 63%)
compared to A. japonicum::bcaAB01 (strain containing
empty vector). Furthermore, optimization of the expression
of its precursor pathway was regulated in heterologous host
bacterium A. japonicum. First, heterologous genes encod-
ing the MVA pathway were optimized to elevate the iso-
prenoid precursor IPP in A. japonicum. Overexpression of
mva genes increased production of BraC and BraC-agl by
27%. This clearly indicates that MVA pathway can provide
alternative route for producing isoprenoid precursor iso-pen-
tenyl diphosphate (IPP). In the next step, precursors were
channeled into the direction of diterpenoid synthesis by
expressing diterpenoid specific prenyltransferases. Finally,
the production of BraC and BraC-agl by the combination
of overexpression of the respective genes of the MVA path-
way (idi) and the positive regulator gene (bra12), along with
geranylgeranyl diphosphate synthase (ggpps) and farnesyl
diphosphate synthase (fpps) amounted to 794 mg/l. This
titer is comparable to production from the native producer
N. terpenica IFM0406 [126]. Figure S1A depicts different
strategies employed to enhance the production of brasilicar-
din congomers by tuning regulation and precursor pathway.
Recently, the application of synthetic biology and meta-
bolic engineering has shown potential to revolutionize the
biological production of natural products in desired indus-
trial titer [60]. Nevertheless, very few studies have used
metabolic engineering approaches and synthetic biological
tools to rationally engineer Nocardia spp. However, there
have been successful reports of using these approaches to
enhance the production of nargenicin A1 (Fig. S1B). Pol-
yketides such as Nargencin A1 are assembled by a series
of decarboxylative condensations of malonyl-CoA and
methylmalonyl-CoA. These short chain fatty acid esters
can be directly incorporated into polyketide backbone.
Thus, nargenicin A1 has been successfully enhanced 3.8-
fold by overexpression of acetyl-CoA carboxylase, which
is involved in the biosynthesis of malonyl-CoA [90]. Pri-
mary metabolite precursors, such as acetate, are directed to
the pool of malonyl-CoA, while propionate contributes to
13
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
methylmalonyl-CoA [132]. Hence, the production titer of
nargenicin A1 was further enhanced to sevenfold by using
methyl oleate, sodium propionate, and sodium acetate as car-
bon sources to enhance the pool of short fatty acids [68], as
shown in Fig. S1B. Different cheap materials such as etha-
nol, glucose, glycerol, and propanol as source of fatty acid
precursors, and l-proline forming pyrrole moiety have been
used in feeding experiment to recombinant Nocardia sp.
CS682 (Fig. S1B). A combination of glucose and glycerol
exhibited the best yield of nargenicin A1. Feeding of such
combination to recombinant strains, such as Nocardia sp.
metK1 and Nocardia sp. ACC18 enhanced the production
titer of nargenicin A1 by (6.3 and 7.1)-fold, respectively, in
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comparison to the wild type Nocardia sp. CS682 without
feeding [24].
Similarly, a synthetic biology approach was used to
develop a multi-monocistronic expression vector, pNV18L2.
Vector pNV18L2 contained a hybrid promoter (derived from
ermE* and promoter region of neor), ribosome binding sites
(RBS), and restriction sites for cloning. In this vector each
gene could be cloned in such way that each cloned gene
could express under its own promoter and RBS. Multiple
genes involved in the biogenesis of pyrrole moiety (ngnN2,
ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glu-
cose utilization (glf and glk from Zymomonas mobilis), and
malonyl-CoA synthesis (accA2 and accBE from Strepto-
myces coelicolor A3 (2)), were cloned in pNV18L2. This
construct using combinatorial assembly of genes involved in
the biosynthesis of specific precursors as pyrrole moiety and
acyl-CoA esters has been used to enhance the production of
nargenicin A1 (Fig. S1B). Further statistical optimization of
precursor feeding conditions led to ~ 24-fold higher produc-
tion than the control strain [22].
Generating structurally modified molecules
from Nocardia spp
Most NPs exhibit a broad range of pharmacophores with
a high degree of stereochemistry. Still there is a need for
new NPs with better properties. Thus, NPs have been used
as starting materials for structural modification to amelio-
rate their biological or chemical properties for therapeutic
uses [6, 25, 26, 28]. Different production host-based bio-
logical engineering and chemical synthesis platforms have
been used to facilitate such structural modification. Particu-
larly, the biosynthesis of a few secondary metabolies from
Nocardia spp. has been characterized, including nocardicin,
nocobactin, nocardithiocin, brasilicardin and nargenicin as
described earlier. Novel derivatives can be obtained from
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407 399
the deletion mutants or overexpression mutants. This has
already been discussed in earlier part of biosynthesis. Hence,
in the section, approaches particularly used for targeted
diversification of the secondary metabolites from Nocardia
spp. are discussed.
Heterologous expression of brasilicardin gene cluster has
been successfully accomplished in A. japonicum, resulting
in production of two brasilicardin congeners: BraC and
BraD. In addition, two new non-glycosylated brasilicardin
congeners, BraC-agl and BraD-agl have also been charac-
terized from same heterologous expression system [125]
as shown in Fig. S2A. A one-pot in vitro system combin-
ing the biosynthesis of NDP-sugar with a substrate-flexible
Bacillus glycosyltransferase named YjiC has been used
to generate novel glucosides of nargenicin A1. Structural
elucidation studies have confirmed that the new glyco-
sides are nargenicin A ­ 1 11-O-β-d-glucopyranoside, nar-
genicin ­A1 18-O-β-D-glucopyranoside, nargenicin ­A1 11,
18-O-β-d-diglucopyranoside, and nargenicin 11-O-β-d-2-
deoxyglucopyranoside (Fig. S2B). Assessment of their phys-
iochemical activities showed that these glycosylated prod-
ucts exhibited higher solubility than the parental compound
[24]. The chance of interaction between water molecule
and multiple -OH present in glucose leads to an increase in
solubility for glucosides [7]. Hence, the attachment of glu-
cose provides a benefit of higher solubility. In another study,
the construct containing combinatorial assembly of genes
involved in the biosynthesis of pyrrole moiety and acyl-CoA
esters has been supplemented with substrate flexible enzyme
PikC (a cytochrome P450 from Streptomyces venenzuelae).
The construct has been heterologously expressed in Nocar-
dia sp. CS682 to generate Nocardia sp. PikC. A novel mol-
ecule named as nargenicin A1 acid has been produced from
Nocardia sp. PikC. Its chemical structure has been depicted
and biological properties has been assessed (Fig. S2C). The
compound exhibits lower antibacterial activity against S.
aureus than its parental compound [22]. The possible reason
for this loss of activity may be the loss of lactone ring struc-
ture which might play a vital role in antibacterial activities.
Thus the aforementioned examples illustrates the feasibil-
ity of successful modification strategies by in vitro reaction,
heterologous gene expression in native host, and expression
of complete biosynthetic pathway in heterologous host. Such
rational strategies have potential to develop novel derivatives
of bioactive molecules from other Nocardia spp.
Biotransformation using Nocardia spp.
Microbial biotransformation catalyzed by microbial cells
(growing or resting) is an efficient technique for facilitating
chemical reactions. These techniques can frequently lead to
the formation of metabolites with improved pharmacological
activities or chemical properties. These kinds of reactions
are highly sensitive and specific, resulting in higher produc-
tion yields but lower byproducts than the chemical synthesis
[115, 151]. Such biocatalysts utilizing microbial biotransfor-
mation can be better than traditional chemical synthesis, as
they can be operated at non-extreme reaction conditions such
as near neutral pH, ambient temperature, and atmospheric
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pressure. Moreover, in most cases, predicted reaction prod-
ucts can be generated due to regio-selective and enantio-
specific conversions [33, 140]. Hence, microbial biotrans-
formation has been an active area of research [15, 30, 44].
Due to their adaptation to pathogenic life cycle or sustained
growth features under harsh conditions, Nocardia species
are associated with different biochemical features and meta-
bolic pathways, making them well equipped for performing
diverse chemical transformations, including those that are
infeasible by chemical synthesis or biotransformations from
other microbial taxa.
Nocardia spp. have been successfully employed for
biotransformation of diverse compound classes, including
saponins, terpenes, polyketides, flavonoids, and steroids as
shown in Fig. S3A-I. In most cases, bioactive molecules are
transformed to their new derivatives with interesting chemi-
cal structures or novel activities. For example, quinovic
acid glycosides (saponins with antiviral activities) has been
converted to their aglycon quinovic acid and its biogenetic
counterpart, cincholic acid via an unprecedented carbon
skeleton rearrangement involving a methyl group migration
by Nocardia sp. NRRL 5646 [15].
Vetramine, an alkaloid with antihypertensive and sero-
tonin agonist activity, has been used for biotransformation
with Nocardia species ATCC 21145. There was formation of
oxidation product as (23R)-12,13,14,15,16,17-hexadehydro-
23-hydroxy-5R-veratranin-3-one. Reduction products such
as 4, 5-didehydro and 1, 2-didehydro derivative were also
obtained. Assessement of biological activities showed that
all of these biotransformed products retained antimalarial
activities [30].
Echinocystic acid, a pentacyclic triterpene with diverse
bioactivities, has been oxidated and/or glycosylated by
Nocardia coralline CGMCC4.1037 [32]. Diverse pen-
tacyclic triterpenoids with anti cancer activities such as
ursolic acid, oleanic acid, betulinic acid, and 23-hydroxy-
betulinic acid have been methylated in biotransformation
13


Table 3  Different bioactive compounds used for biotransformation by Nocardia spp

400

Substrate Compound class Substrate bioactivities Strain Biotransformations Product bioac- References
tivities

13
Quinovic acid glyco- Saponins Antiviral Nocardia sp. NRRL 5646 Quinovic Acid Glycosides converted to their aglycon – [15]
sides quinovic acid and its biogenetic counterpart, cincholic
acid via an unprecedented carbon skeleton rearrange-
ment involving a methyl group migration
Echinocystic acid Pentacyclic Anticancer, anti-HIV, anti- Nocardia corallina Oxidation to generate 3-oxo-16-hydroxy-olean-12-en-28- – [32]
triterpene fungal, immunostimula- CGMCC4.1037 oic acid
tory etc. Glycosylation to generate 3β,16α-dihydroxy-olean-12-en-
28-oic acid 28-O-β-D-glucopyranoside
Oxidation and glycosylation to generate 3-oxo-
16α-hydroxy-olean-12-en-28-oic acid 28-O-β-D-
glucopyranoside
Veratramine Alkaloid Antihypertensive and sero- Nocardia species ATCC Generation of oxidation products (23R)- Antimalarial [30]
tonin agonist activity. 21145 12,13,14,15,16,17-hexadehydro-23-hydroxy-5R-ver-
atranin-3-one, generation of reduction products as
4,5-didehydro and 1,2-didehydro derivative
Artemisinin Sesquiterpene Antimalarial Nocardia corallina ATCC Reduction reaction to generate deoxyartemisinin – [74]
lactone 19070
Ursolic acid Pentacyclic Anti-cancer and anti-HIV Nocardia sp. 5646 Methylation at C-28 to form ursolic acid methyl ester – [158]
triterpenoids Generation of skeleton rearranged product oleanolic acid
and its metyl ester
Oleanic acid Pentacyclic Anti-cancer and anti-HIV Methylation to form olenolic acid metyl ester –
triterpenoids
Betulinic acid Pentacyclic Anti-cancer and anti-HIV Metylated to form betulinic methyl ester –
triterpenoids
23-hydroxybetulinic Pentacyclic Anti-cancer and anti-HIV Methylated to form 23-hydroxybetaulinic acid methyl –
acid triterpenoids ester
Glycyrrhetinic acid Pentacyclic Selective inhibitor of Methylated to form glycyrrhetinic acid methyl ester –
triterpenoids 11-β-hydroxysteroid
dehydrogenase
Senegenin Pentacyclic Neuroprotective Dechlorination and metylation to generate senegenic acid –
triterpenoids 28-methyl ester
Ursolic acid Pentacyclic Anti-cancer and anti-HIV Nocardia sp. 44000 Methylation to ursolic acid methyl ester – [76]
triterpenoids Generation of 3-oxoursa-1,12-dien-28-oic acid and its
methyl ester
Nocardia sp. 45077 Generation of ursonic acid and 3-oxoursa-1,12-dien-28- –
oic acid
Nocardia sp. 46002 Generation of ursonic acid –
Betulonic acid pentacyclic Anticancer, anti-HIV and Nocardia sp. NRRL 5646 Metylation to generate betulonic acid methyl ester – [116]
triterpenoids anti-inflammatory α-hydroxylation and acetylation to generate methyl
2a-acetoxy-3-oxo-lup-20(29)-en-28-oate
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 Table 3  (continued)
Substrate Compound class Substrate bioactivities Strain Biotransformations Product bioac- References
tivities

Sinenxan A Diterpenes Anticancer Nocardia purpurea Generation of demethylated and acetylated product as: Reversal activi- [81]
CGMCC 4.1182 10β-hydroxy2α,5α,14β-triacetoxytaxa-4(20),11(12)- ties against
diene, A549/taxol,
10β-methoxy-2α,5α,14β-triacetoxytaxa-4(20), 11(12)- upon on co-
diene, 10β-methoxy-2α,5α,14β-trihydroxytaxa-4(20), adminstration
11(12)-diene and 5α,10β,14β-trihydroxy-2α- with paclitaxel
acetoxytaxa-4(20),11(12)- diene
Oleanolic acid Triterpenes Anticancer, antibacterial, Nocardia iowensis Generation of oleanolic metyl ester and oleanonic acid Anti-cancer, [4, 82]
antiviral etc. (DSM 45197, NRRL 5646) methyl ester by whole cell biotransformation by sus- antidiabetic,
pended and immobilized cells of Nocardia antimicrobial
etc.
Cholesterol Sterol Human body metabolite Nocardia sp. cholesterol to 1,4-androstadiene-3,17-dione – [127, 135]
Nocardia sp. NCIB 10554 cholesterol to cholest-4-ene-3-one Anti-obesity [9]
agent
Diadzein Flavonoid Anticancer, antioxidant, N. farcinica IFMA10152 Generated mono hydroxylated derivatives as : 3′,4′,7-tri- Anticancer and [17]
antithrombotic, etc. hydroxyisoflavone (3′-ODI), 4′,6,7-trihydroxyisoflavone anti-melano-
(6-ODI) and 4′,7,8-trihydroxy-isoflavone (8-ODI) genic
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407

Generated subsequent O-methylated products of hydroxy-

lated products: 4′,7-dihydroxy-3′-methoxyisoflavone,
4′,7-dihydroxy-6-methoxy isoflavone(glycitein) and
4′,7-dihydroxy-8-methoxyisoflavone
Nocardia sp NRRL 5646 Generated hydroxylated and methylated derivatives as Anticancer and [86]
7-methoxy-4′-hydroxyisoflavone and 7, 8-dimethoxy-4′- anti-melano-
hydroxyisoflavone. genic
Linaloyl acetate Acetate of Antiinflammatory Nocardia sp. TB 1 Nocar- Enantiometric deacetylation to generate linalool Antinflammatory [110]
allylic tertiary dia sp.H8
alcohol
Thymol Monoterpenoid antibacterial, antifungal, N. cyriacigeorgica Generation of 1,4 hydroxyquinone, 5-Hydroxymethyl- – [40]
phenol insecticidal, antioxidant, 2-isopropylphenol, 3-Hydroxy-4-isopropylbenzoic acid
etc. and thymobenzoquinone
Simvastatin Polyketide Anticholesterol compound Nocardia autotropica Hydroxylated to generate 6-hydroxy-simvastatin [35]
Hydroxylation and methylation to generate 6-hydroxyme-
thyl-simvastatin
Resibufogenin, Cinob- Steroids Cytotoxic Nocardia sp. NRRL 5646 Resibufogenin was converted to 3-acetyl 15β-hydroxyl Cytotoxic [159]
ufagin, Bufalin bufotalin, via an unprecedented 14β,15β-epoxy ring
cleavage and a regio-selective acetoxylation.
Cinobufagin was acetylated to form 3-acetyl cinobufagin.
Bufalin was acetylated to form 3-acetyl bufalin.

13
401

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402
with Nocardia sp. 5646. Interestingly, skeleton rearranged
product oleanolic acid and its metyl ester have been gen-
erated during biotransformation of ursolic acid. In another
biotransformation of ursolic acid by Nocardia sp. 44000,
ursolic acid methyl ester, 3-oxoursa-1,12-dien-28-oic acid
and its methyl ester have been generated. However, ursonic
acid and 3-oxoursa-1,12-dien-28-oic acid have been gen-
erated by Nocardia sp. 45077, whereas ursonic acid has
been generated in case of Nocardia sp. 46002 from bio-
transformation of ursolic acid [76]. Betulonic acid, a pen-
tacyclic triterpenoids substrate with anticancer properties,
has been biotransformed by Nocardia sp. NRRL 5646 to
generate betulonic acid methyl ester. In addition, there was
unique α-hydroxylation and acetylation to generate methyl
2α-acetoxy-3-oxo-lup-20(29)-en-28-oate [116].
Sinenxan A, an anticancer diterpene compound has been
biotransfomed to demethylated and acetylated products
by Nocardia purpurea CGMCC 4.1182. All the generated
novel products exhibited better anticancer reversal activities
against A549/taxol upon co- adminstration with paclitaxel
[81]. Similarly, diadzein, a flavonoid has been biotrans-
formed to its hydroxylated and/or methylated derivatives by
N. farcinica IFMA10152 [17].
Biotransformation with Nocardia species can generate
novel derivatives of bioactive substrates such as alkaloids,
flavonoids, and terpene compounds. Generally, modifica-
tions of different chemical structures by oxidations, meth-
ylations, and acetylations have been successful. In most of
the cases, generated products can either retain the biologi-
cal potency or attain new activities. Thus, by appropriate
selection of Nocardia strain, it is feasibile to obtain diversi-
fied modification for compounds sharing identitical/similar
chemical structures. Tuning by strain selection provides
possibility for combinatorial biotransformation approach,
whereas the product from one biotransformation experi-
ment can be used as substrate for next biotransformation
experiment. Structural diversification of already bioactive
molecule has potential to develop new molecules with bet-
ter activities or novel activities. Table 3 depicts different
examples of biotransformation using different bioactive
compounds (e.g., steroids, flavonoids, polyketides, etc.) and
various Nocardia strains to diversify chemical structure or
functional properties.
Conclusion and future perspectives
Recently, Nocardia species have been characterized as an
important source of effective bioactive molecules, or bio-
catalytically efficient strain with potential industrial applica-
tion. The whole cell biotranformation has been effectively
utilized to obtain structural/functional diversities to different
13
Journal of Industrial Microbiology & Biotechnology (2019) 46:385–407
compound classes. In most of these reports, prior genomic
information is not mandatory. Moreover, the availability of
detailed genomic information by whole genome sequenc-
ing and application of advanced genetic engineering tools
has advanced our understanding about their metabolism and
biosynthesis mechanism [16, 22, 23, 83, 111–113]. Precise
pathway engineering using multiplexed genome editing
techniques such as the CRISPER-Cas9 technique can be
utilized to activate silent BGCs or study the biosynthetic
mechanisms [141, 160]. By utilizing these information,
techniques and tools, it is feasible to reprogram the genetic
architecture and discrete metabolism, or biosynthetic ability
of Nocardia spp. This knowledge can be utilized for tuning
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the biochemistry, metabolism, and physiology of Nocardia
spp to attain higer biocatalysis or generate molecules with
enhanced phramceutical values.
Acknowledgements  This work was supported by the National
Research Foundation of Korea (NRF) grant funded by the Korea gov-
ernment (MEST) (NRF-2014R1A2A2A01002875, JKS) and (NRF-
2017R1D1A1B03036273, DD). The authors want to dedicate this
manuscript in honor of Professors Heinz Floss and Chris Walsh for
inspiring research in the field of natural products.
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