Introduction

Secondary metabolites (SM) are compounds with varied and sophisticated chemical
structures, produced by strains of certain microbial species, and by some plants. Although
antibiotics are the best known SM, there are other such metabolites with an enormous range
of biological activities, hence acquiring actual or potential industrial importance. These
compounds do not play a physiological role during exponential phase of growth. Moreover,
they have been described as SM in opposition to primary metabolites (like amino acids,
nucleotides, lipids and carbohydrates), that are essential for growth. A characteristic of
secondary metabolism is that the metabolites are usually not produced during the phase of
rapid growth (trophophase), but are synthesized during a subsequent production stage
(idiophase). Production of SM starts when growth is limited by the exhaustion of one key
nutrient source: carbon, nitrogen or phosphate. For example, penicillin biosynthesis by
Penicillium chrysogenum starts when glucose is exhausted from the culture medium and the
fungus starts consuming lactose, a less readily utilized sugar. Most SM of economic
importance are produced by actinomycetes, particularly of the genus Streptomyces, and by
fungi.

Biosynthetic Families

Microbial SM show an enormous diversity of chemical structures. However, their

biosynthetic pathways link them to the more uniform network of primary metabolism. It has
been shown that SM are formed by pathways which branch off from primary metabolism at a
relatively small number of points, which define broad biosynthetic categories or families:

(1) Metabolites derived from shikimic acid (aromatic amino acids). Examples are ergot
alkaloids and the antibiotics candicidin and chloramphenicol.
(2) Metabolites derived from amino acids. This family includes the ~-lactam antibiotics:
penicillin, cephalosporins and cephamycins, as well as cyclic peptide antibiotics such
as gramicidin or the immunosupressive agent cyclosporine.
(3) Metabolites derived from Acetyl-CoA (and related compounds, including Kreb's
cycle intermediates). This family can be subdivided into polyketides and terpenes.
Examples of the former group include the antibiotic erythromycin, the insecticidal-
antiparasitic compound avermectin and the anti tumour agent doxorubicin. An
example of the second group is the non citotoxic anti tumour agent taxol.
(4) Metabolites derived from sugars. Examples of SM in this group are streptomycin and
kanamycin
.
Since secondary biosynthetic routes are related to the primary metabolic pathways and use
the same intermediates, regulatory mechanisms i.e. induction, carbon catabolite regulation
and/or feedback regulation, apparently operate in conjunction with an overall control, which
is linked to growth.
Table 4.
Intermediate from primary metabolism and their secondary metabolite derivatives
S. No Intermediates from primary
Secondary metabolites derived
. metabolic pathway
1. Shikimic acid Ergot alkaloids, antibiotics: candicidin and chloramphenicol
Antibiotics: penicillin, cephalosporins and cephamycins,
2. Amino acids
and gramicidin, immunosuppressive cyclosporine
3. Acetyl-CoA and other Kreb’s Antibiotics: erythromycin, antiparasitic avermectin
S. No Intermediates from primary
Secondary metabolites derived
. metabolic pathway
cycle intermediates antitumor doxorubicin, taxol
4. Sugars Antibiotics: streptomycin and kanamycin.

Secondary metabolites of microorganisms

Microbial secondary metabolites are low molecular mass products with unusual structures.
The structurally diverse metabolites show a variety of biological activities like antimicrobial
agents, inhibitors of enzymes and antitumors, immune-suppressives and antiparasitic agents ,
plant growth stimulators, herbicides, insecticides, antihelmintics, etc. They are produced
during the late growth phase of the microorganisms. The secondary metabolite production is
controlled by special regulatory mechanisms in microorganisms, as their production is
generally repressed in logarithmic phase and depressed in stationary growth phases. The
microbial secondary metabolites have distinctive molecular skeleton which is not found in the
chemical libraries and about 40% of the microbial metabolites cannot be chemically
synthesized .

4.3.1. Features of microbial secondary metabolites

 The principle and process of natural fermentation product synthesis can be

successfully scaled up and employed to maximize its application in the field of
medicine, agriculture, food, and environment.
 The metabolite can serve as a starting material for deriving a product of interest,
extended further through chemical or biological transformation.
 New analog or templates in which secondary metabolite serve as lead compounds will
lead discovery and design of new drugs.

4.4. Applications of microbial secondary metabolites

4.4.1. Antibiotics

The discovery of penicillin initiated the researchers for the exploitation of microorganisms
for secondary metabolite production, which revolutionized the field of microbiology . With
the advent of new screening and isolation techniques, a variety of β-lactam-containing
molecules and other types of antibiotics have been identified. About 6000 antibiotics have
been described, 4000 from actinobacteria. In the prokaryotic group, unicellular
bacteria Bacillusand Pseudomonas species are the most recurrent antibiotic producers.
Likewise in eukaryotes, fungi are dominant antibiotic producers next to plants . In the recent
years, myxobacteria and cyanobacteria species have joined these distinguished organisms as
productive species.

Name of secondary metabolites Source of secondary metabolites Biological activities

Secondary metabolites of Actinobacteria
Resistomycin S. corchorusii HIV-1 protease inhibitor
Himalomycins A and B Streptomyces sp. B6921 Antimicrobial
Bonactin Streptomyces sp. BD21–2 Antibacterial
Name of secondary metabolites Source of secondary metabolites Biological activities
Trioxacarcins S. ochraceus and S. bottropensis Antitumor and antimalarial
Chinikomycins A and B Streptomyces sp. Antitumor and antiviral
Daryamides Streptomyces sp. CNQ-085 Cytotoxic polyketides
Resistoflavine S. chibaensis Antibacterial
Chalcomycin A and terpenes Streptomyces sp. M491 Antibacterial
Napyradiomycin (C-16
S. antimycoticus Antibacterial
stereoisomers)
Streptomyces sp. RM17;
Oxohexaene and Cephalaxine Antibacterial
Streptomyces sp. RM42
Citreamicin θ A, Citreamicin θ B,
S. caelestis Antibacterial
and Citreaglycon A
Spiramycin Streptomyces sp. RMS6 Antibacterial
N-isopentyltridecanamide Streptomyces labedae ECR 77 Antibacterial
Staurosporine Streptomyces champavatii KV2 Antimicrobial
Secondary metabolites of Bacillus spp.
Coagulin B. coagulans Bactericidal, Bacteriolytic
Fungicidal sub
Bacthurucin f4 B. thuringenesis sp.
sp., kurstaki BUPM4
Cerein B. cereus Bactericidal, bacteriolytic
Megacin B. megaterium ,
Thuricin S B. thuringenesis ,
B. thuringenesis DPC6431
Thuricin CD 19 ,
B. anthracis
Halobacillin 5b B. licheniformis Hemolytic, cytotoxic
B. amyloliquefacins FZB42,
Bacillomycin Antifungal hemolytic
B. subtilis
Bacilysocin B. subtilis Fungicidal, antibacterial
B. subtilis 168, B. pumilus
Bacilysin 1 Antifungal, antibacterial
B. amyloliquefaciens GSB272
Secondary metabolites of Pseudomonas spp.
Competitive inhibition of
Pseudomonine P. stutzeri KC ]
phytopathogens
Hydrogen cyanide P. pseudoalcaligenes P4 Antifungal
Secondary metabolites of Fungi
Monascus ruber;
Lovastatin Enzyme inhibitor
Aspergillus terreus
Limonene and guaiol Trichoderma viride Antimicrobial
Tuberculariols Tubercularia sp. TF5 Anticancer
Oxaline Penicillium raistricki Anti-cell proliferation
Penicillium
Benzomalvin C Antimalarial
raistrickii, Penicillium sp. SC67
Roquefortine C P. roqueforti; P. crustosum Neurotoxin
Pravastatin Penicillium citrinum Anticholesterolemics
Table 3.
Secondary metabolites produced by microorganisms.
The pharmaceutical product, especially anti-infective derivatives comprise 62%
antibacterials, 13% sera, immunoglobulins, and vaccines, 12% anti-HIV antivirals, 7%
antifungals, and 6% nonHIV antivirals. There are over 160 antibiotics. Streptomyces
hygroscopicus with over 200 antibiotics, Streptomyces griseus with 40 antibiotics,
and Bacillus subtilis with over 60 compounds are the major contributors to the antibiotic
market .

4.4.2. Antitumor agents

Natural product and its derivatives account for more than 60% of anticancer formulations.
Actinobacteria derived antineoplastic molecules currently in use are actinomycin D,
anthracyclines (daunorubicin, doxorubicin, epirubicin, pirarubicin, and valrubicin),
bleomycin, mitosanes (mitomycin C), anthracenones (mithramycin, streptozotocin, and
pentostatin), enediynes (calicheamicin), taxol, and epothilones .

Taxol is the nonactinobacterial molecule derived from plant Taxus brevifolia and endophytic

fungi Taxomyces andreanae and Nodulisporium sylviforme. It interferes with microtubule
breakdown, an essential event leading to cell division, thereby inhibiting rapidly dividing
cancer cells. It is effective against breast and advanced form Kaposi’s sarcoma. It is also
found to exhibit antifungal activity against Pythium, Phytophthora, and Aphanomyces.

4.4.3. Pharmacological and nutraceutical agents

One huge success was the discovery of the fungal statins, including compactin, lovastatin,
pravastatin, and others which act as cholesterol-lowering agents. Lovastatin is produced
by A. terreus. Of great importance in human medicine are the immunosuppressants such as
cyclosporin A, sirolimus (rapamycin), tacrolimus, and mycophenolate mofetil. They are used
for heart, liver, and kidney transplants and were responsible for the establishment of the
organ transplant field. Cyclosporin A is made by the fungus Tolypocladium niveum.
Mycophenolate mofetil is a semisynthetic product of the oldest known antibiotic,
mycophenolic acid, and is also made by a fungus. Sirolimus and tacrolimus are products of
streptomycetes . Metabolites of probiotic bacteria are considered as a remedy for controlling
weight gain, preventing obesity, increasing satiety, prolonging satiation, reducing food
intake, reducing fat deposition, improving energy metabolism, treating and enhancing insulin
sensitivity, and treating obesity. Firmicutes and Bacteroidetes are the dominant beneficial
bacteria present in the normal human gastrointestinal tract, and the latter was reported in
lower numbers in constipation-predominant irritable bowel syndrome patients . Carotenoids
of microbial origin are used as food colorant, fish feeds, nutraceuticals, cosmetics, and
antioxidants. Food colorant widely used is carotene derived from Blakeslea trispora,
Dunaliella salina and lycopene from B. trispora and Streptomyces chrestomyceticus,
subsp. rubescens. Astaxanthin produced from Xanthophyllomyces dendrorhous is an
approved fish feed. Astaxanthin, lutein, β-carotene, zeaxanthin, and canthaxanthin are used as
nutraceuticals due to their excellent antioxidant property. Docosahexaenoic acid (DHA) used
in infant formula as nutritional supplements is derived from microalgae Schizochytrium spp. .

4.4.4. Enzymes and enzyme inhibitors

Enzymes produced from microorganism have annual sales of US $ 2.3 billion enzymes that
find application in detergents (34%), foods (27%), agriculture and feeds (16%), textiles
(10%), and leather, chemicals, and pulp and paper (10%). The protease subtilisin used in
detergents has an annual sale of $ 200 million. The other major enzymes include glucose
isomerase (100,000 tons) and penicillin amidase (60,000 tons). Nitrilase (30,000 tons) and
phytase are amounting for $135 million worth of production. Streptomyces glucose isomerase
is used to isomerize D-glucose to D-fructose, to make 15 million tons per year of high
fructose corn syrup, valued at $1 billion .

The most important enzyme inhibitors are clavulanic acid, synthesized by Streptomyces
clavuligerus, the inhibitor of β-lactamases. Some of the common targets for other inhibitors
are glucosidases, amylases, lipases, proteases, and xanthine oxidase. Amylase inhibitors
prevent absorption of dietary starches into the body, and hence can be used for weight loss .

4.4.5. Agricultural and animal health products

Secondary metabolites find wide applications in the field of agriculture and animal health:
kasugamycin and polyoxins are used as biopesticides; Bacillus thuringiensis crystals,
nikkomycin, and spinosyns are used as bioinsecticides; bioherbicides (bialaphos) find
application as bioherbicides; ivermectin and doramectin as antihelmintics and endectocides
against worms, lice, ticks, and mites; ruminant growth promoters in the form of coccidiostats;
plant hormones like gibberellins as growth regulators are the most common application [7].

Production

Liquid Fermentation

Secondary metabolites are generally produced in industry by submerged fermentation (SmF)

by batch or fed-batch culture. An improved strain of the producing microorganism is
inoculated into a growth medium in flasks and then transferred to a relatively 323 small
fermenter or "seed culture". This culture, when in rapid growth phase, is used to inoculate a
fermenter tank, in the range of 30,000 to 200,000 litres, with production medium. Several
parameters, like medium composition, pH, temperature, agitation and aeration rate, are
controlled. The different regulatory mechanisms mentioned previously are bypassed by
environmental manipulations. Hence, an inducer such as methionine is added to
cephalosporine fermentations, phosphate is restricted in chlortetracycline fermentation, and
glucose is avoided in penicillin or erythromycin fermentation. The fermentation processes of
antibiotics regulated by carbon are now conduced with slowly utilized sources of carbon,
generally lactose. When glucose is used, it is usually fed at a slow, continuous rate to avoid
catabolite regulation. Also nitrogen sources like soybean meal are used to avoid nitrogen
(ammonium) regulation. In some cases, a precursor is used to increase one specific desirable
metabolite, for example lysine is added as precursor and cofactor to stimulate cephamycin
production by Streptomyces clavuligerus. Agitation is provided by turbine impellers at a
power input of 1-4 W/litre and air has to be supplied at flow rates of 0.5-1.0 v/v per min. Exit
gas is generally analyzed to monitor O2 and CO2 concentrations. This can provide metabolic
information to regulate the feeding rates of precursors and nutrients. Some natural antibiotics
and other SM are chemically modified, in a subsequent stage to produce semisynthetic
derivatives.

Solid-state Fermentation
Solid-state fermentation (SSF) holds an important potential for the production of secondary
metabolites. This fermentation system has been used in several oriental countries since
antiquity, to prepare diverse fermented foods from grains like soybeans or rice. However,
different SSF systems, that could be called nontraditional have been developed in the last 15
years. A modem SSF definition is the one proposed by Lonsane et al (1985)-a microbial
culture that develops on the surface and at the interior of a solid matrix and in absence of free
water. Today, two types of SSF can be distinguished, depending on the nature of solid phase
used.

(a) Solid culture of one support-substrate phase solid phase is constituted by a material that
support and of nutrients. source. Agricultural or even animal goods or wastes are used as
support substrate.

(b) Solid culture of two substrate-support phase solid phase is constituted by an inert support
impregnated with a liquid medium. Inert support serves as a reservoir for the nutrients and
water. Materials as sugarcane bagasse pith or polyurethane can be used as inert support.
Fungi and actinomycetes, the main microorganisms producer of SM grow well is SSF,
because the conditions are similar to their natural habitats, such as soil, and organic waste
materials. The advantages of SSF in relation with SmF include: energy requirements of the
process are relatively low, since oxygen is transferred directly to the microorganism. SM are
often produced in much higher yields, often in shorter times and often sterile conditions are
not required