Metabolic Pathways: Production of Secondary Metabolites of Bacteria

Abstract

Bacterial secondary metabolites exhibit several biological properties and many among them are pharmacologically important. Secondary metabolites are assembled by a variety of enzymatic pathways using l-chiral, d-chiral, and nonproteinogenic amino acids, as well as by using acetate units derived from various acyl-CoA esters. The nascent secondary metabolites are further modified by tailoring enzymes for biotechnological applications to attain the intended function. The production of secondary metabolites via several biochemical pathways generally occurs during the late phase of bacterial growth. These compounds protect bacteria during harsh environmental conditions and also act as antibacterial agents against other environmental microorganisms during competition for nutrients. Indirectly, these metabolites have both deleterious and beneficial effects on human health, nutrition, and economy of our society. This review will emphasize the formation of secondary metabolites using nonribosomal peptide synthase, polyketide synthase, shikimate pathways, and β-lactam pathways for uses in the pharmaceutical, food, and agriculture industries.

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From Gokulan, K., Khare, S., Cerniglia, C., 2014. Metabolic Pathways: Production of
Secondary Metabolites of Bacteria. In: Batt, C.A., Tortorello, M.L. (Eds.), Encyclopedia
of Food Microbiology, vol 2. Elsevier Ltd, Academic Press, pp. 561–569.
ISBN: 9780123847300
Copyright © 2014 Elsevier, Ltd unless otherwise stated. All rights reserved.
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Production of Secondary Metabolites of Bacteria
K Gokulan, S Khare, and C Cerniglia, National Center for Toxicological Research, US Food and Drug Administration,
Jefferson, AR, USA
Ó2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by M.D. Alur, volume 2, pp 1328–1334, Ó1999, Elsevier Ltd.
Introduction
Metabolism is a constant and collective biochemical process
that occurs in every single or multicellular organism lifelong.
The biochemical process largely can be classified into catabo-
lism and anabolism. The end-products of these pathways are
used for the formation of intermediates and substrates for other
metabolic pathways and are known as ‘metabolites.’Metabo-
lites exhibit several biological properties, which are of phar-
maceutical, nutritional, and agricultural importance. On the
basis of functional properties and metabolic pathways, these
molecules are classified into primary and secondary metabo-
lites. The primary metabolites serve as a primary source of
energy to perform various biochemical and physiological
functions of live cells (e.g., amino acids, pyruvate, citric acid,
and lactic acid). In contrast, the secondary metabolites are not
essential for cell growth, but rather they serve as a survival
strategy for the organism during adverse conditions. The focus
of this chapter is on the production of secondary metabolites
by bacteria (Table 1).
The secondary metabolite-producing microorganisms
synthesize these bioactive and complex molecules at the late
phase and stationary phase of their growth (Figure 1). The
production of secondary metabolites is triggered during the
exhaustion of nutrients, environmental stress, and limited
growth conditions. The secondary metabolites frequently are
found in bacteria, fungi, plants, and marine organisms. These
organisms have the capability to produce several metabolites
with various biological functions, including antibacterial agents,
toxins, metal-transporting agents, sex hormones, pigments,
anticancer agents, pesticides, immunomodulating agents,
immunosuppressants, receptor agonists, and antagonists.
Secondary metabolic pathway reactions are conducted by
an individual enzyme or multienzyme complexes. Interme-
diate or end-products of primary metabolic pathways are
channeled from their systematic metabolic pathways that lead
to the synthesis of secondary metabolites (Figure 1(b)). The
genes encoding these synthetic pathway enzymes generally are
present in chromosomal DNA and often are arranged in clus-
ters. For example, Streptomyces griseus and Streptomyces glau-
cescens chromosomal DNA contains 30 or more str/sts and blu
genes that participate in streptomycin biosynthesis. Chapters
Release of Energy (Aerobic) to Production of Secondary
Metabolites –Fungi cover several aspects of metabolic path-
ways. This chapter discusses the production of bacterial
secondary metabolites, its application on food and pharma-
ceutical industries, and its harmful effects on humans and
animals after consumption of contaminated food products.
The Effect of Secondary Metabolites on Food
Products and Foodborne Illness
The secondary metabolites exhibit several beneficial effects in
pharmaceutical, cosmetic, food agricultural, and animal food
industries, but certain secondary metabolites cause deleterious
effects in humans and animals and also destroy certain food
types. For example, several pathogenic bacteria have evolved
with synthesizing and secreting toxins (secondary metabolites)
in the immediate environments. The secreted toxin contaminate
foods, food products, and water that enter the food chain. In
addition, pathogenic bacteria also secrete toxins inside the host.
The other route of toxin contamination is poorly packed canned
foods, packed meats, and dairy products, in which certain
bacteria grow anaerobically and secrete toxins. The consum-
mation of toxin via contaminated food, food products, and
water causes severe illness. The secreted toxin either kills the
host or interferes with normal cellular functions. This toxic
substance can be classified into two types: (1) exotoxins that
usually are secreted by bacteria, and (2) endotoxins that are part
of the cellular component of the bacteria (cell wall component).
Some of the bacterial toxins are assembled by bacterial
secondary metabolic pathways. For example, in poorly packed
canned food products, Clostridium botulinum colonize anaerobi-
cally and secret exotoxins, which cause paralytic illness and
respiratory failure. The secreted toxin acts on the peripheral nerve
system. Vibrio cholerae is a Gram-negative and facultative bacteria
that colonizes in the small intestine. In the host, certain strains of
V. cholerae cause disease by secreting exotoxins and virulence
factors, which are toxic to intestinal mucosa and epithelial cells.
Most of the Escherichia coli strains are nonpathogenic bacteria,
but few serotypes secrete exotoxins and cause food poisoning by
ingesting the contaminated foods. Clostridium tetani is an
Table 1 Biochemical and physiological properties of primary and
secondary metabolites
Primary metabolites Secondary metabolites
Small molecules
Produces few intermediates or
end-products
End-products are building
blocks for macromolecules
Essential for growth and cell
viability
Known physiological function
Composed of simple chemical
structure
End-products are used for
Coenzyme synthesis
Production occurs at log phase
Primary metabolites are used in
food and feed industry
Provides the energy for cellular
activities
Small molecules
Produces array of molecules
Synthesize new compounds
Not vital for the cell growth
Analysis of physiological
function is difficult
Products of complex unusual
chemical structure
End-products are used an
antibacterial agent
Production occurs at late and
dormant phase
Secondary metabolites are used
in food, cosmetic, agricultural
and farming industry
Protects the organisms under
various harsh environment
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Figure 1 (a) Various phases of bacterial growth and production of metabolites. The primary metabolites production generally occurs at the late lag phase
and middle of exponential phase. The secondary metabolites production occurs at the end of the stationary phase and during the persistent phase. (b)
Various pathways responsible for the assembly of secondary metabolites. (c) Structural diversity of NRP molecules are generated by cyclization, het-
erocyclization, and macrocyclization mechanisms.
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obligatory anaerobic bacterium and is mostly found in deep
wounds or cuts. In infected individuals, these bacteria secrete
a powerful neurotoxin that causes uncontrolled contraction of
skeletal muscles, often leading to fatality.
Streptomyces species produce macrolide antibiotic bafilo-
mycin A and streptozotocin that cause glucose intolerance in
human, resulting in type I diabetes. These secondary metabo-
lites enter human through tuberous vegetables, in particular,
potatoes and beets. Bafilomycin and streptozotocin are toxic to
human pancreatic islet cells that lead to the secretion of low
levels of insulin, and the outcome is type I diabetics.
Microcytins, nodularin, cylindrospermopsin, anatoxin-a,
anatoxin-a(s), and saxitoxin are secondary metabolites
produced by several cyanobacterial species, which are highly
abundant in freshwater. These toxic metabolites have a great
impact on several living organisms, including humans. These
metabolites are toxic to human hepatocytes, liver, kidney,
lungs, spleen, intestine, and neuronal systems. In the natural
environment, the freshwater cyanobacterium enters humans
and animals through consumption of contaminated drinking
water. The ingested cyanobacteria may contain microcystins
and other secondary metabolites, which cause the fatality. The
freshwater cyanobacterial secondary metabolites, specifically,
microcystins, are still a threat to human health and life. Cya-
nobacterial toxins also enter the food chains through fish,
crustaceans, and crayfish and sometimes through plants that
grow in contaminated water (Figure 2). On the basis of target
cells, they have been classified into neurotoxin, hepatotoxins,
and dematotoxins.
Bacillus cereus is a Gram-positive bacterium, which cau-
ses foodborne illness after consumption of spores or
vegetative cells. Bacillus cereus secretes toxins (secondary
metabolites) that cause diarrhea, nausea, and vomiting.
These toxins cause illness by two ways: one way is through
the secretion of heat-labile peptide by multiplying bacteria
in the small intestine, and another way is by ingestion of
heat-labile peptide cereulide. This secondary metabolite is
synthesized by nonribosomal peptide synthase (NRPS). The
cereulide toxins have been detected in several rice dishes,
sweetened dairy-based desserts, and Camembert cheese. In
addition, B. cereus also secretes food spoilage and virulence
factors, phospholipase, protease, hemolysins, and
enterotoxins.
Beneficial Effects of Bacterial Secondary Metabolites
The shelf life of foods depends upon several factors,
including microbial growth. Microbial proliferation
contributes to modification of food products, which is
unacceptable for consumption. The microbial reductions are
achieved by using bacteriocins and other antibacterial agents
in food products. Bacteriocins are assembled by NRPS
(Lactobacillus sake and Carnobacterium piscicola), and they
have been used as a food additive agents to reduce the
pathogenic bacterial load (Listeria monocytogenes,B. cereus,C.
botulinum,orStaphylococcus aureus) and to improve the
quality and safety of foods. Bacteriocins act as antibacterial
agents for Gram-positive as well as Gram-negative bacteria.
Lactococcus lactis is a Gram-positive bacterium used exten-
sively in the dairy industry. This bacterium assembles nisin
and subtilin antibiotics, which belong to the nonribosomal
peptide (NRP) family. Nisin is used as a food preservative,
because it has bactericidal properties and prevents the pore
formation in C. botulinum and B. cereus. Recently, nisin-
producing bacteria have been isolated from human milk.
Subtilin shares structurally and functionally with nisin
molecule. Streptomycin and oxytetracycline are used to treat
bacterial infections in fruits and vegetables. Cylin-
drospermopsis raciborskii belongs to cyanobacteria family, and
it naturally produces butylated hydroxtoluene. This metab-
olite has been used as an antioxidant, food additive,
cosmetic, pharmaceuticals, rubber and electrical transformer
oil, and industrial chemical. In agricultural setup, in partic-
ular, in animal farming and aquaculture, antibiotics are
largely used in feed to prevent the infection as well as to
promote growth. The discoveries of bacterial secondary
metabolites have revolutionized human and animal health
Humans, consumption by
drinking water and food
products from freshwater
Consumption of
contaminated water by
wild and domestic animals
Cyanobacteria/secondary
metabolites in freshwater
Irrigation of
plants
Accumulation of cyanobacteria
or secondary metabolites in fish,
clams, crabs, mussels, and
crayfish
Figure 2 Cyanobacterial secondary metabolites enter humans by various routes of the food chain.
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by advancing our knowledge for treatment and prevention of
several infectious diseases.
Nonribosomal Peptide Synthesis Pathways
NRPs are natural products assembled by NRPS enzymes. The
antimicrobial peptides –bacitracin, ramicidin, polymyxin B,
and vancomycin –are products of nonribosomal peptide
synthesis pathways (NRPSP). The NRPS enzymes generate NRP
molecules containing unique structures by using building
blocks of L-chiral and D-chiral centers and nonproteinogenic
and modified amino acids as substrates. NRPS enzymes
generate structural diversity by modifying NRP molecules by
linking fatty acids, methyl groups, phosphate groups, and
oligosaccharides at the N-terminal end. Furthermore, to
generate sophisticated structural diversity and rigidity, NRPS
employs three basic mechanisms: (1) cross-linking, (2) heter-
ocyclization, and (3) macrocyclization (Figure 1(c)).
Assembly of NRP Molecules
A majority of the lipopeptide containing NRP molecules are
produced by Streptomyces spp. The largest genes to produce
antibiotics against bacteria, fungi, and parasitic infections.
For example, Streptomyces coelicolor,Streptomyces roseoporus,
Streptomyces fradiae, and Actinoplanes friuliensis produce
calcium-dependent antibiotic, daptomycin, A54145, and
amphomycins, respectively. Daptomycin has been approved by
the Food and Drug Administration (FDA) to treat skin-related
infections caused by Gram-negative bacteria and endocarditis
caused by S. aureus. The presence of daptomycin genes also is
observed in other bacterial species, including S. coelicolor and
Streptomyces ambofaciens. The bioinformatics and molecular
biology approaches reveal that 12 genes are responsible for the
assembly of functional daptomycin. Among them, three genes
encode proteins for peptide backbone assembly, and the
remaining gene products take part in structural modification,
generating constraints, and cyclization.
NRPS is a multimodular enzyme; each module requires at
least three catalytic domains to initiate or to add one building
block in the growing intermediate chain. The catalytic domains
are the adenylation domain (A), peptide carrier protein (PCP)
domain, and condensation (C) domain (Figure 3). Each
domain catalyzes a specific function on the assembly line of the
growing NRP molecule. The adenylate domain is responsible
for the recognition of related building blocks for the
Figure 3 Assembly of secondary metabolites by NRPS, type I PKS, type II PKS, and type PKS enzymes. (a) The NRPS multimodular enzyme, which
synthesizes the peptide molecules by generating peptide bonds between newly recruited amino acids with the growing intermediate. (b) Type I PKS enzyme
is similar to NRPS, but it employs C–C condensation to assemble the secondary metabolite. (c) Type II enzymes are discrete enzymes and synthesize the
aromatic molecules by the Clasien condensation method. (d) Type III enzymes are ketosynthase units and perform various functions at a single active site.
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adenylation process, and it also is responsible for transferring
the building blocks into a PCP domain of the same module.
This process leads to the formation of a thioester (TE) bond
between the substrate and phosphopantethiene group present
in the PCP domain. The peptide bond formation takes place in
the C-domain (Figure 3(a)). It catalyzes the formation of
a peptide (C–N) bond between newly recruited amino acid
present upstream of the PCP domain with the growing peptide
intermediate holding the downstream of the PCP domain.
Apart from these three catalytic domains, the NRPS multi-
enzyme complex also possesses additional domains in few of
the modules. For example, in daptomycin synthesis, the first
module of the dptA enzyme has a C
III
domain that catalyzes the
addition of a long fatty acid chain to the N-terminal amino
acid. Attached fatty acid facilitates the interaction between NRP
molecules and hydrophobic cell walls of target microbes.
Similarly, few modules have an epimerase domain, which
accounts for and reevaluates the D-chirality of amino acids
during extension. The last module contains an additional
domain known as the TE domain (Figure 3(a)), where the
nascent peptide undergoes cyclization, ring formation, and
reduction, and releases the NRP from the enzyme complex. The
sequential structural organization of NRP modules reflects the
order of amino acid incorporation in the end-product.
Nascent NRP molecules are further modified by chemical
cross-linkage, heterocyclization, and macrocyclization to
generate structurally complex molecules. In addition, NRP
molecules also undergo oxidation and reduction reactions to
attain the intended biological function. For example, the
vibriobactin molecule (a siderophore from V. cholerae)is
generated by a heterocyclization mechanism, which contains
two oxazoline rings, both of which are synthesized by utilizing
threonine. The oxazoline ring is further oxidized to form oxa-
zole, which is a component of the potent telomerase inhibitor,
telomestatin. Telomestatin also harbors a thiazole ring, which
is generated by cyclization process using cysteine. The macro-
cyclization reaction generates covalent linkages between linear
nascent NRP molecules that lead to cyclization. This cyclization
process alters the part of the nascent linear peptide and also
generates constraints on the NRP molecule. The direction of
nonribosomal peptide synthesis always starts from the
N-terminal to the C-terminal, like ribosomal peptide synthesis.
Polyketide Synthase Pathways
Polyketides (PK) are natural products that display diverse
functions with clinical applications. Polyketides are assembled
by polyketide synthase (PKS) enzymes. PKS enzymes operate
similarly to fatty acid synthase to generate a diverse range of
PKs. PKS enzymes begin the PK assembly by priming the starter
molecule to the catalytic residue, and then it employs an
extender unit for the chain elongation. On the basis of struc-
tural architecture and variation in enzymatic mechanism, PKS
enzymes have been classified into three types: (1) type I PKS,
(2) type II PKS, and (3) type III PKS. This section describes all
three types of PKS enzymes (Table 2).
Type I Polyketide Synthases
Type I PKS is a multienzyme complex with several modules,
which is similar to NRPS. The type I PKS, however, assembles
PKs by catalyzing C–C condensation (Figure 3(b)). These are
multienzyme complexes and are known as modular enzymes.
The mega enzyme contains several modules and each module is
folded into domains and subdomains. The individual domain
achieves a specific function in the line of product formation. For
example, Saccharopolyspora erythraea produces erythromycin that
functions as an antibacterial agent. Its base structure is synthe-
sized by 6-deoxyerythronolide B synthase (DEBS), which is
encoded by three genes: eryAI,eryAII, and eryAIII. This enzyme
first uses propionyl as a starter unit and then it uses six mole-
cules of methyl-MCoA as an extender unit for the condensation
reaction, which results in the formation of 6-deoxyery-
thronolide B (6dEB). Each enzyme harbors two modules, and
each module completes one condensation reaction.
Type I enzymes are multimodules, and each module is
organized into several domains and subdomains that include
ketosynthase (KS), acyltransferase (AT), ketoreductase, and acyl
carrier protein (ACP) domain. The modules are numbered
according to the order of reaction. The KS domain binds with
an extender unit and performs a decarboxylation reaction, and
then catalysis occurs by a Claisenlike condensation between
growing intermediates that are attached to a Cys thiol group
with two carbon units of an extender unit. The AT domain
selects an appropriate extender unit and chiral center for chain
elongation except the first AT domain. Generally, the first AT
domain has the capacity to recognize the range of acyl-CoA as
a starter molecule. The ACP domain contains a phospho-
pantethiene (40-ppt) arm that forms a TE linkage with the
growing chain. The ACP domain presents an elongated chain to
a reductase or dehydration domain through the 40-ppt arm.
A TE domain occupies the end of the last module and releases
the nascent PK by a hydrolysis mechanism. The nascent
product is further modified by the tailoring enzyme to achieve
bioactive molecules.
Table 2 Classification of polyketide synthase enzymes and the functional and the mechanistic differences between them
Type I or modular PKS Type II or discrete PKS Type III or Ketosynthase PKS
Multifunctional enzymes that are organized
into modules
Each modules bears a specific function
Uses acyl carrier protein (ACP) domain to
activate acyl-CoA substrates
Malonyl-CoA or methylmalonyl-CoA or
ethylmalonyl-CoA an extender unit
Consists of a series of singular modular
heterodimeric enzymes
Each enzyme has a specific function
Uses ACP domain to transfer activate
acyl-CoA substrate
Malonyl-CoA, an extender unit
Homodimeric ketosynthase enzyme
Performs various biochemical reactions
at single active site
Acts without ACP or directly recognizes
the acyl-CoA molecules
Malonyl-CoA or methylmalonyl-CoA, an
extender unit
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A type I PKS assembles a wide array of PKs, but the struc-
tural diversities are introduced at different phases in the line of
synthesis. The variations are introduced by priming of various
starter molecules and number of extension by selecting
different extender units, chirality, and number of modules. The
final variations are introduced by tailoring enzymes, which
include cyclase, dehydratase, and aromatase; they are respon-
sible for the final modification of bioactive molecules. In type I
PKS, variation is also introduced by gene duplication, losses of
modules and domain, recombination, and horizontal gene
transfer among different bacterial species.
Type II Polyketide Synthase
Type II PKS mostly contains discrete enzymes, and each
enzyme executes a specific function in the assembly line of the
PK synthesis. Type II PKS enzymes produce several aromatic
compounds that include anthracyclines, aureolic acids, tetra-
cyclines, tetracenomycins, pradimicin-type polyphenols, and
benzoisochromanquinones. Anthracycline’s basic structure
contains four rings with additional methoxyl and carbonyl
groups at various positions. The modified rings have an impact
on the biological function. Some PKs are capable of targeting
one particular cancer cell, some have antibacterial activity, and
some specifically act on a particular cell type.
Tetracycline is another important antibiotic molecule that is
commonly used for the treatment of food poisoning, which is
assembled by type II PKS. This antibiotic interferes with tRNA-
binding site at the 30S ribosomal unit, which results in protein
synthesis blocking. Streptomyces resistomycificus produces resis-
tomycin that has the ability to inhibit HIV protease and
bacterial RNA and DNA polymerase activity. Aromatic poly-
ketides are assembled by several type II PKS enzymes in
a stepwise manner. This family of enzymes performs chemical
reactions similar to type I PKS enzymes. For example, type I and
type II enzymes use ACP domains for the transfer of active-
acetyl-CoA for the condensation reaction. In type II enzymes,
however, each reaction is catalyzed by a modular enzyme rather
than a multienzyme protein complex.
The basic architecture of type II PKS enzymes is the
composition of two domains. All aromatic PK synthetic path-
ways require two ketosynthase units (KSaand KSb) and one
ACP domain (Figure 3(c)). KS is a heterodimeric enzyme; both
KSaand KSbhave sequence similarity, with the exception that
the KSbdomain lacks active cysteine. The KSadomain is
responsible for the formation of Claisen-type C–C bonds
between activated acyl-intermediates (starter-acyl molecule)
with decarboxylated MCoA. Mutational studies have shown
that the KSbdomain facilitates MCoA binding with the ACP
domain. In addition, it plays a part in the formation of acetyl-
KS intermediates from the decarboxylated MCoA of the ACP
domain. KSbdomain also is known as chain-length factor
(CLF), because it determines the length of carbon chain elon-
gation during the iterative cycle. Type II PKS enzymes employ
MCoA only as an extender unit for chain elongation. In the type
II synthetic pathway, there is an involvement of MCoA ACP
transferase enzyme for the M-CoA source.
The aromatic PK synthesis begins with acetate. Few other
type II enzymes also use propionyl, butyrate, malonate, and
benzoate acyl esters as starter molecules. Furthermore, few type
II PKS have two additional domains to select a starter molecule
and initiate a chemical reaction, which include a FabH-like KS
domain and an AT domain. In the fatty acid biosynthetic
pathway, DpsC operates as a KSIII domain and makes the
condensation reaction between propionyl-CoA with MCoA.
The presence of KSIII genes has been identified in frenolicin,
hedamycin, and R1128 biosynthetic enzyme-encoding gene
clusters.
In type II polyketide biosynthesis, the number of iterative
cycles is determined by the KSbdomain. To assemble actino-
rhodin type II enzymes, use 16 molecules of malonyl-CoA,
20 cycles for tetracenomycin, and 24 cycles for pradimicin. The
KSbdomain operates as a gatekeeper during polyketide
assembly, and it occupies the tunnel entrance. In addition,
other enzymes in this pathway, including cyclases, also
contribute to determine the chain length.
Ketoreductase enzymes modify the keto group of metabo-
lites into secondary alcohol through nicotinamide adenine
dinucleotide phosphate (NAD(P)H). The ketoreductase reac-
tion is essential for the ring-closure reaction. This enzyme is the
first one to react with nascent metabolites before the cyclization
reaction. Ketoreduction is essential to orient the poly-b-keto
chain for favored alcohol condensation, rearranging the elec-
tron orbitals from Sp
2
to Sp
3
hybridization to make well-
defined structural intermediates.
Type III Polyketide Synthase
Flavonoids and phenylpropanoids are common plant natural
products that exhibit numerous pharmacological properties.
Chalcone synthase enzyme (CHS) initially was identified in
plants and involved in biosynthesis of flavonoids. The struc-
tural and functional characterization of CHS revealed that it
belongs to the type III PKS family. Total genome sequencing of
bacteria reveals the presence of several plantlike PKS
genes, which encode CHS-like enzymes. Streptomyces coelicolor
contains three copies of genes for type III PKSs, which assemble
1,3,6,8-tetrahydroxynaphthalene. The mycobacterium genome
sequence reveals more than 18 PK enzymes and three of them
(PKS10, PKS11, and PKS18) belong to the type III PKS.
Type III PKS varies mechanistically from the other two types
in the following manner: (a) type III enzymes function inde-
pendently of the ACP domain; and (b) type III enzymes have
acquired substrate priming, iterative decarboxylation, exten-
sion, intramolecular, and cyclization reactions at a single active
site. Type III PKS enzymes differ in their choice for selecting
starter molecule to initiate PK synthesis. For example, RppA
and SrsA of S. griseus utilize C
2
to C
24
acyl-CoA as starter
molecule, Mtb PKS18 primes with C
6
to C
20
acyl-CoA, and
Azetobacter vinelandii accepts C
22
to C
26
acyl-CoA as a starter
molecule. These enzymes differ from each other, however, by
the number of iterative reactions and ring formations (Claisen
or aldol). The type III PK enzyme superfamily utilizes MCoA as
a universal extender unit (Figure 3(d)). A few bacterial type III
enzymes use both MCoA and methyl-MCoA as extender units
for the synthesis of alkylresorcinols. The order of selection of
the extender unit or condensation is important to synthesize
the final products.
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Type III PKS enzymes harbor two cavities: one cavity for the
starter molecule and the other cavity for the extender unit. Type
III PKS enzymes first select a starter molecule (acyl-CoA), which
binds the substrate-binding pocket. The nucleophilic Cys
attacks the starter acyl-CoA and forms a covalent TE linkage
with the substrate. The extender unit binds in the CoA-binding
tunnels, and then the active site residues decarboxylate and
transfer two carbon units to the growing intermediates. The
interactive condensation reaction varies for each type III PKS.
These enzymes employ more than one type of acyl-CoA
(MCoA or methyl-MCoA) for the condensation reaction. After
the completion of condensation reaction, the end-products
undergo cyclization and aromatization before being released
from the catalytic triad. Some type III enzymes employ aldol
cyclization and some modify the products by Claisen-
cyclization.
Shikimate Pathway
The shikimate pathway contributes to assemble the basic
building blocks for the range of aromatic metabolites and
aromatic amino acids. Metabolites that are derived from
aromatic compounds provide ultraviolet protection, electron
transport, and signaling molecules, and they serve as antibacte-
rial agents. The shikimate pathway enzymes employ erythrose-
4-phosphate and phosphoenol pyruvate (primary metabolites)
as substrates to initiate the aromatic building block synthesis. In
this pathway, the first seven enzymes catalyze the chemical
reactions in a sequential manner to generate chorismate. In the
bacterial system, two enzymes have the capacity to transfer
a complete enolpyruvoyl moiety to a metabolic pathway. In the
shikimate pathway, 5-enolpyruvoyl shikimate 3-phosphate
synthase is one of them. The next enzyme in this pathway is
chorismate synthase, which requires a reduced cofactor, flavin
mononucleotide, for its activation.
Certain microorganisms have evolved to assemble various
secondary metabolites by employing aromatic building
blocks. Pseudomonas,Burkholderia,Brevibacterium,andStrepto-
myces have the capacity to synthesize phenazine compounds.
Phenazine serves as a virulence factor and undergoes
oxidation–reduction reactions, which result in the accumula-
tion of toxic-free radicals in the target cells. This compound
also is used as an antifungal agent. Earlier biochemical studies
demonstrated the relationship between phenazine synthesis
and the shikimate metabolic pathway. A gene cluster
(phzABCDEFG) is responsible for the assembly of phenazine-
1-carboxylic acid (PCA) in Pseudomonas fluorescens strain 2-79.
Phzc, PhzD, and PhzE have significant sequence homology
with known and functionally well-characterized shikimate
pathway enzymes. On the basis of the protein sequence, PhzD
and PhzE gene products possibly can modify the chorismate
before entering into the formation of PCA. The C-terminal
region of PhzE has similarity with anthranilate synthase. PhzD
has high homology to bacterial 2,3-dihydro-2,3-dihy-
drobenzoate synthase.
The Gram-positive, filamentous Streptomyces venezuelae
(soil bacterium) and other actinomycetes assemble chloram-
phenicol using aromatic precursors. Aromatic building blocks
that are derived from the shikimate pathway serve as
precursors for the phenylpropanoid unit of chloramphenicol.
First, chorismic acid branches out from the shikimate pathway
to generate p-aminophenylalanine, which is further converted
into a p-nitrophenylserinol component by an enzymatic
reaction. The molecular genetics and mutational studies have
demonstrated that 4-amino-4-deoxychorismic acid (ADC) is
a common precursor for both PABA and PAPA pathways. The
genetic map reveals that pabAB genes encode enzymes for ADC
biosynthesis that are clustered in a discrete region of the
S. venezuelae chromosome. Echinosporin functions as an
antibacterial and anticancer agent that was isolated from
Saccharopolyspora erythraea. This molecule has a unique tricy-
clic acetal-lactone structure, and the basic structure does not
reveal its biosynthetic pathway. The shikimate pathway
intermediate is channeled to assemble echinosporin by
enzymatic reactions.
b-Lactam Ring Synthetic Pathways
Cephalosporins belong to the b-lactam family of antibiotics.
These antibiotics have been used to treat bacterial infections for
more than 40 years. Gram-positive bacteria, Gram-negative
bacteria, and fungi are the major sources of b-lactam antibi-
otics. The Gram-positive Streptomyces clavuligerus is capable of
producing both clavulanic acid and cephamycin. The Gram-
negative bacterium Lysobacter lactamgenus produces cepha-
bacins. Two hypotheses have been put forward for b-lactam
biosynthesis: (1) horizontal gene transfer (HGT) from bacteria
to fungi and (2) vertical descent (originated from a common
ancestor). Bioinformatics, genetic approaches, and sequence
identity are more in favor of HGT.
The b-lactam antibiotic formation takes place in three
different steps; early biosynthetic steps, intermediate
formation steps, and late steps (also known as decorating
steps). Building blocks for b-lactam biosynthesis include
L-a-aminoadipic acid, L-cysteine, and L-valine. L-a-amino-
adipic acid is a nonproteinogenic amino acid that is formed
from L-lysine. In actinomycetes, lysine 6-aminotransferase
converts L-lysine into L-a-aminoadipic acid (Figure 4). The
initial two enzyme reactions are common in fungi and
cephalosporin biosynthesis. d-(L-aminoadipyl)-L-cysteinyl-D-
valine synthase is the first enzyme, which uses all three
amino acids and assembles them into a tripeptide by
condensation reaction. This enzyme is encoded by the acvA
(pcbAB) gene and it is NRPS. The next step is the formation
of a bicyclic ring (a four-member b-ring is fused with a five-
member thiazolidine ring) by an oxidative reaction, which
is catalyzed by isopencillin N-synthase and results in the
formation of isopenicillin N. Cephalosporin–cephamycin
biosynthesis is the expansion of the five-member thiazoli-
dine ring into a six-member dihydrothiazine ring. Several
enzymes sequentially participate in this ring conversion.
Genes that are responsible for b-lactam biosynthesis always
are clustered in the DNA of all reproducing bacteria.
Bacterial species capable of producing b-lactam antibiotics
have an ecological advantage. In contrast, b-lactam–
producing bacteria show low sensitivity to b-lactams on
their own, or they have evolved to inactivate b-lactam
antibiotics by b-lactamase enzymes.
METABOLIC PATHWAYS jProduction of Secondary Metabolites of Bacteria 567
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Figure 4 Classification of b-lactam antibiotics and synthesis of penicillin and Cephalosporin biosynthesis.
568 METABOLIC PATHWAYS jProduction of Secondary Metabolites of Bacteria
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Conclusion
Microorganisms have the capability to produce a number of
antibiotics and other pharmaceutically important drugs to treat
bacterial and fungal infections, cancer, and heart-related
diseases. Bacterial species exhibit a complex life cycle with
a physiological and biochemical adaptability, with the capa-
bility to synthesize a great variety of secondary metabolites
with complex structures using different metabolic pathways.
Understanding the secondary metabolite biosynthesis and
pathways will lead to progress in combinatorial biosynthesis in
the pharmaceutical and biotechnology industries.
Disclaimer
The views expressed in this review do not necessarily reflect
those of the U.S. Food and Drug Administration.
Acknowledgments
We thank Dr. J. B. Sutherland and Dr. J. Kanungo for the
review of this document.
See also: Metabolic Pathways: Release of Energy (Aerobic);
Metabolic Pathways: Release of Energy (Anaerobic); Metabolic
Pathways: Nitrogen Metabolism; Lipid Metabolism; Metabolic
Pathways: Metabolism of Minerals and Vitamins.
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Encyclopedia of Food Microbiology, Second Edition, 2014, 561–569
Author's personal copy