Microbial products often are classified as primary and secondary metabolites.
primary metabolites consist of compounds related to the synthesis of microbial cells in the growth phase.
Secondary metabolites usually accumulate during the period of nutrient limitation or waste product accumulation that follows the active growth phase.
Bacterial growth is graphed over time as cell number versus time (this called growth curve)
Bacteria are becoming "acclimated" to the new environmental conditions to which they have been introduced (pH, temperature, nutrients, etc.). There is no significant increase in numbers with time.
Exponential Growth Phase:
The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth.
With the exhaustion of nutrients and build-up of waste and secondary metabolic products, the growth rate has slowed to the point where the growth rate equals the death rate. Effectively, there is no net growth in the bacteria population.
The living bacteria population decreases with time, due to a lack of nutrients and toxic metabolic by-products.
Naturally occurring compounds may be divided into three broad categories. Firstly. there are those compounds which occur in all cells and play a central role in the metabolism and reproduction of those cells. These compounds include the nucleic acids and the common amino acids and sugars. They are known as primary metabolites. Secondly, there are the high molecular-weight polymeric materials such as cellulose. the lignins and the proteins which form the cellular structures. Finally, there are those compounds that are characteristic of a limited range of species.
These are the secondary metabolites. Most primary metabolites exert their biological effect within the cell or organism that is responsible for their production Secondary metabolites. on the other hand. Have often attracted interest because of their biological effect on other organisms. The biologically active constituents of medicinal, commercial and poisonous plants have been studied throughout the development of organlc chemistry. Many of these compounds are secondary metabolites It has been estimated that over 40% of medicines have their origins in thes natural products. A number of screening programmes for bioactive compounds exist and have lead to new drugs, for example taxol. which is used for the treatment of various cancers. Natural products often have an ecological role in regulating the interactions between plants, microorganisms, insects and animals. They can be defensive substances. Antifeedants, attractants and pheromones. Chemotaxonomy provides another reason for examining the constituents of plants.
Phytochemical surveys can reveal natural products that are "markers" for botanical and evolutionary relationships
Microbial products often are classified as primary and secondary metabolites. As shown in figure primary metabolites consist of compounds related to the synthesis of microbial cells in the growth phase. They include amino acids, nucleotides, and fermentation end products such as ethanol and organic acids. In addition, industrially useful enzymes, either associated with the microbial cells or exoenzymes, often are synthesized by microorganisms during growth. These enzymes find many uses in food production and textile finishing.
Secondary metabolites usually accumulate during the period of nutrient limitation or waste product accumulation that follows the active growth phase. These compounds have no direct relationship to the synthesis of cell materials and normal growth.
Most antibiotics and the mycotoxins fall into this category. (Prescott−Harley−Klein:Microbiology, Fifth Edition 2002)
Microbial secondary metabolites include antibiotics, pigments, toxins, effectors of ecological competition and symbiosis, pheromones, enzyme inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, antitumor agents and growth promoters of animals and plants. They have a major effect on the health, nutrition and economics of our society. They often have unusual structures and their formation is regulated by nutrients, growth rate, feedback control, enzyme inactivation, and enzyme induction. Regulation is influenced by unique low molecular mass compounds, transfer RNA, sigma factors and gene products formed during post-exponential development. The synthases of secondary metabolism are often coded by clustered genes on chromosomal DNA and infrequently on plasmid DNA. Unlike primary metabolism, the pathways of secondary metabolism are still not understood to a great degree and thus provide opportunities for basic investigations of enzymology, control and differentiation.
Secondary metabolism is brought on by exhausion of a nutrient, biosynthesis or addition of an inducer, and/or by a growth rate decrease. These events generate signals which effect a cascade of regulatory events resulting in chemical differentiation (secondary metabolism) and morphological differentiation (morphogenesis). The signal is often a low molecular weight inducer which acts by negative control, i.e. by binding to and inactivating a regulatory protein (repressor protein/receptor protein) which normally prevents secondary metabolism and morphogenesis during rapid growth and
nutrient sufficiency. Nutrient/growth rate/inducer signals presumably activate a “master gene” which either acts at the level of translation by encoding a rare tRNA, or by encoding a positive transcription factor. Such master genes control both secondary metabolism and morphogenesis. At a second level of regulatory hierarchy genes could exist which control one
branch of the cascade, i.e. either secondary metabolism or morphogenesis but not both. In the secondary metabolism branch, genes at a third level could control formation of particular groups of secondary metabolites. At a fourth level there may be genes which control smaller groups, and finally,
fifth level genes could control individual biosynthetic pathways; these are usually positively acting but some act negatively.
There are also several levels of hierarchy on the morphogenesis branch. The second level could include genes which control aerial mycelium formation in filamentous organisms plus all the sporulation genes lower in the cascade. Each third level locus could control a particular stage of sporulation. Some of these loci code for sigma factors. Feedback regulation also is involved in secondary metabolite control. This contribution focuses on the role of low molecular weight compounds which act to induce secondary metabolism.
( Arnold L. Demain, 1998)
secondary metabolites are produced only by special groups of organisms through specialized pathways. Their chemical structure tends to be complex, and they are often produced only during the special growth phase, most often during the stationary phase. The most important of these secondary metabolites are the antibiotics.
Many different classes of antibiotics were discovered among secondary metabolites since then, as will be discussed below. However, it is not clear whether all of these natural antibiotics are synthesized by the producing organisms for the purpose of killing off their neighbors and competitors. Many of these compounds show only marginal antimicrobial activity and have tobe altered by chemical modification to produce semi synthetic agents that can be used in therapy. Furthermore, some of them are produced only at very low levels. It is thus likely that many of the “natural antibiotics”
are produced for other purposes, for example, as signaling molecules. This hypothesis fits with the observation that there are a great many secondary metabolites that do not show antimicrobial activity even with the very sensitive assays available. On the other hand, compounds like penicillin probably
have served as an antibiotic even in natural microbial communities, because genes coding for resistance against β-lactams (including penicillins) are found on the chromosomes of many bacterial species( Alexander N.Glazer et al Hiroshi Nikaido, September 1981.)
In order to grow, any living organism needs a supply of nutrients that will feature as, or go on to form, the building blocks from which that organism is made. These nutrients must also provide the energy that will be needed by the organism to perform the functions of accumulating and assimilating those nutrients, to facilitate moving around, etc. The four elements required by organisms in the largest quantity (gram amounts) are carbon, hydrogen, oxygen and nitrogen. This is because these are
the elemental constituents of the key cellular components of carbohydrates , lipids , protein, and nucleic acids
food is broken down so as to extract energy in the form of adenosine triphosphate (ATP), as well as reducing power in the form of nicotinamide adenine dinucleotide (NADH, reduced form) but utilised as nicotinamide adenine dinucleotide phosphate (NADPH, reduced form) to fuel the reactions (anabolism) on those that use sugars as the main source of carbon and energy, we must first consider the Embden–Meyerhof–Parnas (EMP) This is the most common route by which sugars are converted into a key component of cellular metabolism, pyruvic acid. In this pathway, the sugar is ‘activated’ to a more reactive phosphorylated state by the addition of two phosphates from ATP. There follows a splitting of the diphosphate to two three-carbon units that are in equilibrium. It is the glyceraldehyde 3-phosphate that is metabolized further, but as it is used up, the equilibrium is strained and dihydroxyacetone phosphate is converted to it. we are in dealing with two identical units proceeding from the fructose diphosphate. The first step is oxidation, the reducing equivalents (electrons, hydrogen) being captured by NAD. En route to pyruvate are two stages at which ATP is produced by the splitting off of phosphate – this is called substrate-level phosphorylation. As there are two three-carbon (C3) fragments moving down the pathway,
this therefore means that four ATPs are being produced per sugar molecule. As two ATPs were consumed in activating the sugar, there is a net ATP gain of two.
In certain fermentations, the Entner–Doudoroff pathway is employed by the organism, a pathway differing in the earliest part as only one ATP is used. in certain lactic acid bacteria, there is the quite different phosphoketolase pathway
Amajor outlet for pyruvate is into the Krebs cycle (tricarboxylic acid cycle) this cycle is important in aerobically growing cells.
There are four oxidative stages with hydrogen collected either by NAD or flavin adenine dinucleotide (FAD). When growing aerobically, this reducing power can be recovered by successively passing the electrons across a sequence of cytochromes located in the mitochondrial membranes of eukaryotes or the plasma membrane of prokaryotes , with the resultant flux of protons being converted into energy collection as ATP through the process of oxidative phosphorylation . In aerobic systems, the terminal electron acceptor is oxygen, but other agents such as sulphate or nitrate can serve the function in certain types of organism.
The above-named pathways are examples of how cells deal with sugars, thereby obtaining carbon, hydrogen and oxygen. As observed earlier, cells must also secure a supply of other elements from the medium. Nitrogen may be provided as amino acids (e.g. in the case of brewing yeast), urea or inorganic nitrogen forms, primarily as ammonium salts (often used in wine fermentations).
Sulphur can variously be supplied in organic or inorganic forms. Brewing yeast, for example, can assimilate sulphate, but will also take up sulphurcontaining amino acids .
The major structural and functional molecules in cells are polymeric. These Include:
(1) Polysaccharides – notably the storage molecules such as glycogen in yeast, which has a structure closely similar to the amylopectin fraction of starch and the structural components of cell walls, for example, the mannans and glucans in yeast and the complex polysaccharides in bacterial cell walls.
(2) Proteins – notably the enzymes and the permeases.
(3) Lipids – notably the components at the heart of membrane structure.
(4) Nucleic acids – DNA and RNA. (Bull, A.T., Ward, A.C., Goodfellow, M. 2000) 
Products of Primary Metabolism
Primary metabolism is the inter-related group of reactions within a microorganism which are associated with growth and the maintenance of life. Primary metabolism isessentially the same in all living things and is concerned with the release of energy, andthe synthesis of important macromolecules such as proteins, nucleic acids and other cell constituents. When primary metabolism is stopped the organism dies.
Products of primary metabolism are associated with growth and their maximum production occurs in the logarithmic phase of growth in a batch culture. Primary catabolic products include ethanol, lactic acid, and butanol while anabolic products include amino-acids, enzymes and nucleic acids. Single-cell proteins and yeasts would also be regarded as primary products (Table 1)
2. Amino acids
5. Yeast cells
6. Single cell protein
7. Nucleic acids
8. Citric acid
1. Ethanol and ethanol-containing products, e.g. wines
4. Lactic acid
5. Acetic acid (vinegar)
Table (1) Some industrial products resulting from primary metabolism
Products of Secondary Metabolism
In contrast to primary metabolism which is associated with the growth of the cell and the continued existence of the organism, secondary metabolism, which was first observed in higher plants, has the following characteristics:
(i) Secondary metabolism has no apparent function in the organism. The organism continues to exist if secondary metabolism is blocked by a suitable biochemical means. On the other hand it would die if primary metabolism were stopped.
(ii) Secondary metabolites are produced in response to a restriction in nutrients. They are therefore produced after the growth phase, at the end of the logarithmic phase of growth and in the stationary phase (in a batch culture). They can be more precisely controlled in a continuous culture.
(iii) Secondary metabolism appears to be restricted to some species of plants and microorganisms (and in a few cases to animals). The products of secondary metabolism also appear to be characteristic of the species. Both of these observations could, however, be due to the inadequacy of current methods of recognizing secondary metabolites.
(iv) Secondary metabolites usually have ‘bizarre’ and unusual chemical structures and several closely related metabolites may be produced by the same organism in wild-type strains. This latter observation indicates the
existence of a variety of alternate and closely-related pathways.
(v) The ability to produce a particular secondary metabolite, especially in industrially important strains is easily lost. This phenomenon is known as strain degeneration.
(vi) Owing to the ease of the loss of the ability to synthesize secondary metabolites, particularly when treated with acridine dyes, exposure to high temperature or other treatments known to induce plasmid loss secondary metabolite production is believed to be controlled by plasmids (at least in some cases) rather than by the organism’s chromosomes.
TROPHOPHASE-IDIOPHASE RELATIONSHIPS IN THE PRODUCTION OF SECONDARY PRODUCTS:
From studies on Penicillium urticae the terms trophophase and idiophase were introduced to distinguish the two phases in the growth of organisms producing secondary metabolites. The trophophase (Greek, tropho = nutrient) is the feeding phase during which primary metabolism occurs. In a batch culture this would be in the logarithmic phase of the growth curve. Following the trophophase is the idio-phase (Greek, idio = peculiar) during which secondary metabolites peculiar to, or characteristic of, a given organism are synthesized. Secondary synthesis occurs in the late logarithmic, and in the stationary, phase. It has been suggested that secondary metabolites be described as ‘idiolites’ to distinguish them from primary metabolites. .( Nduka Okafor,2007)
ACTIVITIES OF SECONDARY METABOLITES
SECONDARY METABOLITES WITH USEFUL NONANTIBIOTIC ACTIVITIE
culture filtrates of microorganisms are screened not only for antimicrobial activity, but also for the presence of other activities, such as inhibition of particular animal enzymes or binding to specific receptors on animal cell surfaces .By this means , anumber of compounds with interesting activity were discovered . These compounds do not fit the traditional definition of antibiotics.
Perhaps the most important agents in cancer chemotherapy are secondary metabolites of microorganisms belonging to the anthracycline family. These compounds, including doxorubicin and daunorubicin, contain a reduced naphthacene ring in which four benzene rings are fused together
These compounds intercalate between the bases in double-helical DNA and are thought to act by inhibiting the topoisomerase reaction. Another family, including dactinomycin, contains three fused rings also intercalates into double-stranded DNA, and inhibits both the transcription and synthesis of DNA.
Bleomycin, which also has a high affinity for DNA, has a totally different structure and destroys DNA by generating oxygen free radicals (catalyzed by a ferrous ion that is coordinated by this antibiotic)
Finally, zinostatin (neocarzinostatin), is composed of a chromophore that is protected by its binding to an apoprotein. When the chromophore leaves the protective protein and is reduced by sulfhydryl agents such as glutathione, it becomes converted to a free radical, and because its naphthalene ring system intercalates into double-stranded DNA, the free radical produces oxidation of the deoxyribose in DNA, followed by the cleavage of DNA strand(s). All of these compounds are products of Streptomyces species, the most important source of traditional antibiotics.
Some chemotherapeutic agents used for the treatment of tumors are totally synthetic. These include antimetabolites (e.g., methotrexate and fluorouracil), alkylating agents (e.g., cyclophosphamide), and DNA-crosslinking agents (e.g., cisplatin). However, even these classes include secondary metabolites of microorganisms – for example, pentostatin (a purine Nucleoside analog produced by Streptomyces spp. Streptozocin (an alkylating agent produced by Streptomyces spp and mitomycin C (another Streptomyces product that becomes an alkylating agent after reduction in cells Some others are either plant products or synthetic compounds whose structures are based on these natural products for example, the antitubulin drugs vinblastine, vincristine, and paclitaxel and the DNA topoisomerase inhibitor etoposide.
Proteases are essential for many normal physiological functions (e.g., the blood-clotting cascade) but also are implicated in many pathological states (e.g., elastase in emphysema). Furthermore, proliferation of many animal viruses requires proteolytic processing of viral polyprotein precursors by virally encoded proteases (e.g., the aspartyl protease of the AIDS virus). protease inhibitors is isolated from culture filtrates of Streptomyces
Inhibitor of Cholesterol Biosynthesis
Limiting dietary intake of foods rich in cholesterol lowers blood cholesterol levels significantly in some individuals, but in others high cholesterol levels result from elevated endogenous synthesis of cholesterol rather than from dietary intake. There are products from cultures of Penicillium species that inhibited cholesterol biosynthesis by rat liver extract. thesecompounds, including compactin competitively inhibit the enzyme that catalyzes the first unique step of cholesterol synthesis, hydroxymethylglutaryl-CoA (HMG-CoA) reductase. Compactin was also effective in lowering the serum cholesterol levels in experimental animals and in humans. isolated a closely related compound, mevinolin, from culture filtrate of another fungus, Aspergillus. This compound is now successfully marketed
as lovastatin .Several other semi synthetic or synthetic “statins” are now available, and these belong to the drugs with the highest market values; for example, atorvastatin (Lipitor)( In 1980, scientists atMerck, Sharpe & Dohme)
Some Other Inhibitors
Some microbial products act as powerful immunosuppressants. Treatment of patients with these compounds is therefore useful in decreasing the incidence of allograft rejection in organ transplantation operations. The best known among these compounds is cyclosporin A . a cyclic peptide produced by a fungus. this compound was caught in the screening net originally because of its antifungal activity. Cyclosporin forms a complex with a peptidyl prolyl cis–trans isomerase, cyclophilin, in eukaryotic cytoplasm. The cyclosporin–cyclophilin complex binds to and inhibits the action of a phosphoprotein phosphatase known as calcineurin. Inhibition of this phosphatase prevents a dephosphorylation event that is required for the nuclear entry of a transcription factor necessary for the expression of the autocrine lymphokine interleukin 2 in T cells, and in this manner prevents T cell activation. More recently, rapamycin (now called sirolimus) was identified through its antifungal activity, and FK-506 (now called tacrolimus) was isolated by screening microbial products for the activity to suppress interleukin 2 production. Both of these compounds are important agents in the clinic.
Amino acids such as lysine and glutamic acid are used in the food industry as nutritional supplements in bread products and as flavor enhancing compounds such as monosodium glutamate (MSG).
Amino acid production is typically carried out by means of regulatory mutants, which have a reduced ability to limit synthesis of an end product. The normal microorganism avoids overproduction of biochemical intermediates by the careful regulation of cellular metabolism. Production of glutamic acid and several other amino acids in large quantities is now carried out using mutants of Corynebacterium glutamicum that lack, or have only a limited ability to process, the TCA cycle intermediate ketoglutarate to succinyl-CoA as shown in figure . A controlled low biotin level and the addition of fatty acid derivatives results in increased membrane permeability and excretion of high concentrations of glutamic acid. The impaired bacteria use the glyoxylate pathway to meet their needs for essential biochemical intermediates, especially during the growth phase. After growth becomes limited because of changed nutrient availability,
an almost complete conversion of isocitrate to glutamate occurs.
Lysine, an essential amino acid used to supplement cereals and breads, was originally produced in a two-step microbial process. This has been replaced by a single-step fermentation in which the bacterium Corynebacterium glutamicum, blocked in the synthesis of homoserine, accumulates lysine.
In organic acid synthesis and excretion. Citric, acetic, lactic,
fumaric, and gluconic acids are major products .
Until microbial processes were developed, the major source of citric acid was citrus fruit. Today most citric acid is produced by
microorganisms; 70% is used in the food and beverage industry, 20% in pharmaceuticals, and the balance in other industrial applications.
The essence of citric acid fermentation involves limiting the
amounts of trace metals such as manganese and iron to stop Aspergillus niger growth at a specific point in the fermentation. The medium often is treated with ion exchange resins to ensure low and controlled concentrations of available metals. Citric acid fermentation, The success of this fermentation depends on the regulation and functioning of the glycolytic pathway and the tricarboxylic acid cycle . After the active growth phase, when the substrate level is high, citrate synthase activity increases and the activities of aconitase and isocitrate dehydrogenase decrease. This results in citric acid accumulation and excretion by the stressed microorganism.
In comparison, the production of gluconic acid involves a
single microbial enzyme, glucose oxidase, found in Aspergillus
niger. A. niger is grown under optimum conditions in a corn-steep liquor medium. Growth becomes limited by nitrogen, and the resting cells transform the remaining glucose to gluconic acid in a single-step reaction. Gluconic acid is used as a carrier for calcium and iron and as a component of detergents
Specialty Compounds for Use in Medicine and Health
In addition to antibiotics, amino acids, and organic acids, microorganisms are used for the production of nonantibiotic specialty compounds. These include sex hormones, antitumor agents, ionophores, and special compounds that influence bacteria, fungi, amoebae, insects, and plants . In all cases, it is necessary to produce and recover the products under carefully controlled conditions to assure that these medically important compounds reach the consumer in a stable, effective condition.
Inhibitor of Cholesterol Biosynthesis
Limiting dietary intake of foods rich in cholesterol lowers blood cholesterol levels significantly in some individuals, but in others high cholesterol levels result from elevated endogenous synthesis of cholesterol rather than from dietary intake. In the 1970s, scientists at Sankyo Co. in Tokyo isolated
several novel products from cultures of Penicillium species that inhibited cholesterol biosynthesis by rat liver extract. Further study established that these compounds, including compactin ,competitively inhibit the enzyme that catalyzes the first unique step of cholesterol synthesis,
hydroxyl methyl glutaryl-CoA (HMG-CoA) reductase.
Compactin was also effective in lowering the serum cholesterol levels in experimental animals and in humans. a closely related compound, mevinolin, from culture filtrate of another fungus, Aspergillus. This compound is now successfully marketed as lovastatin .Several other semi synthetic or synthetic “statins” are now available, and these belong to the drugs with the highest market values; for example, atorvastatin (Lipitor) (1980, scientists atMerck, Sharpe & Dohme)
antibiotics are chemicals produced by microorganisms and which in low concentrations are capable of inhibiting the growth of, or killing, other microorganisms. Antibiotics may be wholly produced by fermentation they are increasingly produced by semi-synthetic processes, in which a product obtained by fermentation is modified by the chemical introduction of side chains. Some wholly chemically synthesized compounds are also used for the chemotherapy of infectious diseases e.g. sulfonamides and quinolones. But these will not be considered since they are not produced wholly or partially by fermentation. Some antibiotics e.g. chloramphenicol were originally produced by fermentation, but are now more cheaply
produced by chemical means.
Most of the antibiotics that are now in commercial production are compounds active against bacteria. It is important to distinguish between the agents that act against Gram-positive bacteria only and those that are also active against Gram-negative bacteria. The cells of Gram-negative organisms have the added protection of an outer membrane
the diffusion of water-soluble antibacterial agents larger than 1000 daltons through the outer membrane is severely limited. Not surprisingly, the permeability of these water-filled channels to lipophilic solutes is also poor. Furthermore, the lipid bilayer region of the outer membrane has an unusually low permeability toward lipophilic molecules apparently because its outer leaflet is composed exclusively of an unusual lipid molecule, the lipopolysaccharide (LPS). Consequently, the larger and more lipophilic antibiotics tend to have significant activity against Gram positive bacteria only.
Classification and Nomenclature of Antibiotics
they have been classified on the basis of the producing organisms, but the same organism may produce several antibiotics, e.g. the production of penicillin N and cephalosporin by a Streptomyces sp. The same antibiotics may also be produced by different organisms. Antibiotics have been classified by routes of biosynthesis; however, several different biosynthetic routes often have large areas of similarity. The spectra of organisms attacked have also been used, e.g. those affecting bacteria, fungi, protozoa, etc. Some antibiotics belonging to a well known group e.g. aminoglycosides may have a different spectrum from the others. The classification to be adopted here therefore is based on the chemical structure of the antibiotics and classifies antibiotics into 13 groups. This enables the accommodation of new groups as they are discovered (Table 2)
The nomenclature of antibiotics is also highly confusing as the same antibiotic may have as many as 13 different trade names depending on the manufacturers. Antibiotics are therefore identified by at least three names: the chemical name, which prove long and is rarely used except in scientific or medical literature; the second is the group, generic, or common name, usually a shorter from of the chemical name or the one given by the discoverer; the third is the trade or brand name given by the manufacturer to distinguish it from the product of other companies.
The Beta-lactam antibiotics are so-called because they have in their structure the fourm embered lactam ring.
The Beta-lactam antibiotics include the well-established and clinically important penicillins and cephalosphorins as well as some relatively newer members: cephamycins, nocardicins, thienamycins, and clavulanic acid. Except in the case of nocardicins these antibiotics are derivatives of bicyclic ring systems in which the lactam ring is fused through a nitrogen atom and a carbon atom to ring compound. This ring compound is five-membered in penicillins (thiazolidine), thienamycins (pyrroline) and clavulanic acid (oxazolidine); it is six-membered (dihydrothiazolidine) in cephalosporins and cephamycins (Fig. 24.1).
The Beta-lactam antibiotics inhibit the formation of the structure-conferring petidoglycan of the bacterial cell wall. As this component is absent in mammalian cells, Beta-lactam antibiotics have very low toxicity towards mammals.
) Nduka Okafor,modern industrial microbiology and biotechnology, 2007) 
Theβ-lactams inhibit the synthesis of the eubacterial cell wall, which is composed of a polymer unique to the bacterial world, called peptidoglycan. Peptidoglycan is a network of polysaccharide (or glycan) chains composed of alternating N-acetylglucosamine and Nacetylmuramicacid Residues Thepolysaccharide chains are cross-linked to each other via short peptide chains, which include some d-amino acids and are attached to the N-acetylmuramic acid residues This structure gives peptidoglycan exceptional chemical stability, mechanical strength, and rigidity. The addition of new glycan chains to the cell wall in growing cells takes place by the cross-linking of the peptide side chain of a new unit to the preexisting peptidoglycan structure The cross-linking reaction is catalyzed by a dd-transpeptidase. This enzyme cleaves the peptide bond between the two d-alanine residues in the side chain of a newly made glycan chain and transfers the glycan–peptidyl complex to the free amino group of the diamino acid residue in the side chain of the preexisting peptidoglycan, thereby cross-linking different chains. a β-lactam is an example of a “suicide inhibitor” because it interacts with its target enzyme like a substrate and then undergoes chemical reaction with the enzyme, thereby causing its permanent inactivation. Suicide inhibitors are more desirable antimicrobial agents than are competitive inhibitors (such as sulfonamides) because complete inhibition of the target is achieved.
Penicillin G is very effective in killing most Gram-positive bacteria but is ineffective against most Gram-negative bacteria because of its lipophilicity.
However, the benzyl side chain at the 6-position can be
chemically replaced with other substituents. Some of the resulting compounds show significant activity against Gram-negative bacteria and thus are “broad-spectrum” antibiotics
The basic macrolide structure – seen, for example, in erythromycin is synthesized by bacterial species related to Streptomyces
it characterized by presence of alarge lactone ring These macrocyclic compounds are sufficiently large and hydrophobic that their inhibitory action is largely limited to Gram-positive bacteria. However, more recently, semisynthetic macrolides (such as azithromycin) have been introduced that show significant anti–Gram-negative activity.
Ansamycins also have a macrocyclic structure, but the ring differs from that of the macrolides because it contains an aromatic chromophore as well as an amide (lactam) linkage
These compounds are isolated from Amycolatopsis species, which (like Streptomyces), are members of the actinomycete branch of eubacteria Rifamycins inhibit prokaryotic RNA polymerase. Natural rifamycins, like rifamycin SV have significant activity only against Gram-positive bacteria Thesecompoundsareimportant incombating
a special class of Gram-positive pathogens including Mycobacterium tuberculosis (cause of tuberculosis) and Mycobacterium leprae (cause of leprosy).
Tetracyclines, produced by several species of Streptomyces, contain a fused four-ring system
These compounds are inhibitors of prokaryotic protein synthesis. Because these agents are relatively hydrophilic due to the presence of several hydroxyl groups, an amide moiety, and a tertiary amine substituent, they can cross the outer membrane of Gram-negative
bacteria efficiently through porin channels. Tetracyclines are thus broadspectrum antibiotics active against both Gram-positive and Gram-negative bacteria.
Chloramphenicol was originally isolated from the filtrate of Streptomyces venezuelae cultures. because it is a small molecule withasimplestructure, it is more economic also produce by chemical synthesis than by fermentation. Chloramphenicol inhibits prokaryotic protein synthesis. It penetrates through the outer membrane porin channels of Gramnegative bacteria easily because of its small size and is therefore another broad-spectrum antibiotic. However, chloramphenicol can enter eukaryotic cells and inhibit mitochondrial protein synthesis. Thus, use of chloramphenicol may prevent growth of rapidly proliferating cells and this may be related to at least one of its side effects, the suppression of bone marrowcells.
Because of this and other side effects, chloramphenicol is not widely used today, except in the treatment of diseases in which the pathogen survives within human cells, such as infection by Salmonella typhi (typhoid fever). ( Alexander N.Glazer et al Hiroshi Nikaido, September 1981.)
MICROBIAL TRANSFORMATION OF STEROIDS AND STEROLS
A retrospective look at the contribution of enzymes to the production of therapeutically valuable steroids, such as cortisone, demonstrates that the power of biocatalysis was already fully appreciated more than 50 years ago.
The oxidation and reduction reactions that microorganisms perform on steroid and sterol substrates provide particularly impressive examples of regioselective and stereospecific biotransformations and also showcase the ability of enzymes to promote reactions at unactivated centers in hydrocarbons.
Virtually any position in the carbon skeleton of a steroid nucleus can be hydroxylated stereospecifically by enzymes present in some microorganism. Steroid hydroxylases are named according to the
position they attack on the rings or the side chain of the steroid nucleus.
There are three primary carbon atoms (C18, C19, and C21). An enzyme that catalyzes hydroxylation at C21, for example, is designated as 21-hydroxylase. There are 18 secondary carbon atoms. At the secondary carbon atoms within the ring system, there are two alternative ways, designated α and β, to attach the −OH group. The α (equatorial) position lies below the plane of the
steroid ring, and the β (axial) position lies above the plane Every one of the 18 secondary carbon atoms can be hydroxylated, in either the α or β configuration, each by a different known microbial hydroxylase In addition to hydroxylations, certain microbial enzymes can aromatize ring A, reduce double bonds in the rings, and reduce specific ketone substituents. The microbial transformations of steroids and sterols A major complication in the synthetic route fromdeoxycholic acid to cortisone is the need to shift the C12β hydroxyl in deoxycholic acid to C11. In the chemical synthesis, this required nine steps. In 1952, however, researchers at Upjohn Company discovered that an aerobically grown bread mold, Rhizopus
arrhizus, could hydroxylate progesterone (another steroid and an early intermediate in cortisone synthesis) at C11α, and workers at the Squibb Institute found that another common mold, Aspergillus niger, carried out the same reaction. By exploiting microbial hydroxylation at C11, industrial cortisone synthesis was shortened from 31 to 11 steps.Moreover, the microbial hydroxylation of progesterone had economic benefits beyond those resulting from the abbreviation of the chemical synthesis. This biotransformation takes place at 37◦C in aqueous solution at atmospheric pressure, conditions that aremuchless expensive than the high temperature and pressure
and nonaqueous solvents required for the equivalent steps in chemical synthesis. Further reductions in the cost of cortisone came from introducing inexpensive sterols, instead of deoxycholate, as the starting material. Two such sterols, stigmasterol and sitosterol, are generated in large amounts as byproducts in the production of soybean oil; a third, diosgenin, comes from the roots of theMexican barbasco plant. To make steroids from these plant sterols, the side chain beyond C21 must be removed. Although chemical
degradation can accomplish this step, it is achieved much more economically by mycobacteria, aerobic Gram-positive eubacteria that can utilize sterols as a carbon and energy source. To prevent mycobacteria from breaking the sterols down totally, mutant strains have been developed that are unable to degrade the sterols beyond the desired stage. In addition to being used to treat rheumatoid arthritis, steroids are prescribed for allergies and other inflammatory diseases (especially of the skin), contraception, and hormonal insufficiencies. Various steroids useful for these purposes are produced with the aid of microbes capable of modifying the steroid nucleus in specific ways.Worldwide bulk sales of the four major
steroids – cortisone, aldosterone, prednisone, and prednisolone amount to more than 700,000 kg/year. pharmaceuticals, herbicides, and pesticides. Faber, K. (2004). Biotransformation in Organic Chemistry, Berlin: Springer-Verlag. 
Applications of secondary metabolites on industrial procceses
the microbes in industrial fermentation produce either primary metabolites, such as ethanol, or secondary metabolites, such as penicillin. A primary metabolite is formed essentially at the same time as the new cells, and the production curve follows the cell population curve almost in
parallel, with only minimal lag Secondary metabolites are not produced until the microbe has largely completed its logarithmic growth phase, known as the trophophase, and has entered the stationary phase of the growth cycle. The following period, during which most of the secondary metabolite is produced, is known as the idiophase. The secondary metabolite may be a microbial conversion of a primary metabolite. Alternatively, it may be a metabolic product of the original growth medium that the microbe makes only after considerable numbers of cells and a primary metabolite have accumulated.
Amino acids have become a major industrial product from microorgan isms. For example, over 600,000 tons of glutamic acid (L-glutamate), used to make the flavor enhancer monosodium glutamate, arc produced every year. Certain amino acids, such as lysine and methionine, cannot be synthesized by animals and are present only at low levels in the normal diet. Therefore, the commercial synthesis of lysine and some of the other essential amino acids as cereal food supplements is an important industry. More than 70,000 tons each of lysine and methionine arc produced every year.
Two microbially synthesized amino acids, phellylalanil1e and aspartic acid (L-aspartate), have become important as ingredients in the sugar-free sweetener aspartame (NutraSweet). Some 3000 to 4000 tons of each of these amino acids are produced annually in the United States. In nature, microbes rarely produce ami no acids in excess of their own needs because feedback inhibition prevents wasteful production of primary metabolites .Commercial microbial production of amino acids depends on specially selected mutants For example, in applications in which only the L-isomer of an amino acid is desired, microbial production, which forms only the L-isomer, has an advantage over chemical production, which forms both the D-isomer and the L-isomer
Citric acid is a constituent of citrus fruits, such as oranges and lemons, and at one time these were its only industrial source.
However, over 100 years ago, citric acid was identified as a product of mold metabolism .
Enzymes arc widely used in different industries. For example, amylases are used in the production of syrups from corn starch, in the production of paper sizing (a coating for smoothness, as on this page), and in the production of glucose from sta rch. The microbiological production of amylase is considered to be The basic process by which molds were used to make an enzyme preparation used for make fermented soy products., either rice or a wheat-soybean mixture, with a filamentous fungus (Aspergillus). Primarily, the amylases change starch into sugars, but preparations also contain proteolytic enzymes that convert the protein in soybeans into a more digestible and flavorful form. It is the basis of soybean fermentations
that are staples of the Japanese diet, such as soy sauce and miso (a fermented paste of soybeans with a meaty flavo r). Sake, the well-known Japanese rice wine, makes use of amylases to change the carbohydrates of rice into a form that yeasts can use to produce alcohol. This is roughly the equivalent of the
barley malt used in beer brewing. Gillcose isomerase is an important enzyme; it converts the glucose that amylases form from starches into fructose, which is used in place of sucrose as a sweetener in many foods. Probably
half of the bread baked in this country is made with the aid of proteases, which adjust the amount of glutens (protein) in wheat so
that baked goods are improved or made uniform. Other proteolytic enzymes are used as meat tenderizers or in detergents
as an additive to remove proteinaceous stains. About a third of all industrial enzyme production is for this purpose. Rennin, an enzyme used to form curds in milk, is usually produced commercially by fungi but more recently by genetically modified bacteria.
Vitamins arc sold in large quantities combined in tablet form and arc used as individual food supplements. Microbes can provide an inexpensive source of some vitamins. Vitamin BI ] is produced by Pseudomonas and Propionibacterium species. Riboflavin (B2) is another vitamin produced by fermentation, mostly by fungi such as Ashbya gossypii . Vitamin C (ascorbic acid) is produced at the rate of 20,000 tons per year by a complicated
modification of glucose by Acetobacter species.
Modern pha rmaceutical microbiology developed after World War II, when production of antibiotics was introduced. All antibiotics were originally the products of microbial metabolism. Many are still produced by microbial fermentations, and work continues on the selection of more productive mutants by nutritional and genetic manipulations. At least 6000 antibiotics have been described. One organism, Streptomyces hygroscopius,
has different strains that make almost 200 different antibiotics. Antibiotics are typically made industrially by inoculating a solution of growth medium with spores of the appropriate mold or streptomycete and vigorously aerating it. Vaccines are a product of industrial microbiology. Many antiviral vaccines arc mass-produced in chicken eggs or cell cultures.(microbiology an introduction 10th edition , Gerard J. Tortora, Berdell R. Funke et al Christine L. Case, © 2010)
 (Prescott−Harley−Klein:Microbiology, Fifth Edition 2002)
 INTERNATL MICROBIOL (1998) Arnold L. Demain Department of Biology, Massachusetts Institute
of Technology, Cambridge, Massachusetts, USA
 Alexander N.Glazer et al Hiroshi Nikaido, September 1981.
 Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies for Biotechnology:
The Paradigm Shift Microbiology and Molecular Biology Reviews 64, 573 – 606.
Demain, A.L. 1998. Induction of microbial secondary Metabolism International Microbiology 1, 259–264.
Herrmann, K.H., Weaver, L.M. 1999. The Shikimate Pathway. Annual Review of Plant
Physiology and Plant Molecular Biology. 50, 473–503.
Madigan, M.T., Martinko, J.M. 2006. Brock Biology of Micro-organisms. Pearson Prentice Hall
Upper Saddle River, USA.
Meurer, G., Hutchinson, C.R. 1999. Genes for the Synthesis of Microbial Secondary M etabolites.
In: Manual of Industrial Microbiology and Biotechnology. A.L. Demain and J.E. Davies, (eds).
ASM Press. 2nd Ed. Washington, DC, USA pp. 740-758.
Zahner, H. 1978. In: Antibiotics and other Secondary Metabolites. R. Hutter, T. Leisenger, J.
Nuesch, W. Wehrli (eds). Academic Press, New York, USA, pp. 1-17.
 Nduka Okafor,2007
 In 1980, scientists atMerck, Sharpe & Dohme
 Nduka Okafor,modern industrial microbiology and biotechnology, 2007
Page, M. I. (ed.) (1992). The Chemistry of β-Lactams, Glasgow, UK: Blackie Academic and Professional. Matagne, A., Lamotte-Brasseur, J.,Dive, G.,Knox, J. R., and Fr`ere, J.-M. (1993). Interactions between active-site-serine β-lactamases and compounds bearing a methoxy side chain on the α-face of the β-lactam: kinetic and molecular modelling studies. Biochemical Journal, 293, 607–611. Martin, J. F. (1998).Newaspects of genes and enzymes for β-lactam antibiotic biosynthesis.
Applied Microbiology and Biotechnology, 50, 1–15.
Elander, R. P. (2003). Industrial production of β-lactam antibiotics. Applied Microbiology and Biotechnology, 61, 385–392.
Thykaer, J., and Nielsen, J. (2003). Metabolic engineering of β-lactam production. Metabolic Engineering, 5, 56–69.
Nestrovich, E. M.,Danelon, C.,Winterhalter, M., andBezrukov, S. M. (2002).Designed to penetrate: time-resolved interaction of single antibiotic molecules with bacterial
pores. Proceedings of the National Academy of Sciences U.S.A., 99, 9789–9794.
Tondi, D., Morandi, F., Bonnet, R., Costi, M. P., and Shoichet, B. K. (2005). Structurebased optimization of a non-β-lactam lead results in inhibitors that do not upregulate
 Alexander N.Glazer et al Hiroshi Nikaido, September 1981.
Demain, A. L. 1999. Metabolites, primary and secondary. In Encyclopedia of bioprocess technology: Fermentation, biocatalysis, and bioseparation, 1713–32. New York: John
Wiley & Sons, Inc.
Demain, A. L. 2000. Pharmaceutically active secondary metabolites of microorganisms.
Appl. Microbiol. Biotechnol. 52:455–63.
King, L. A., and Possee, R. D. 1992. The Baculovirus expression system. New York:
Chapman & Hall. Lancini, G.; and Demain, A. L. 1999. Secondary
metabolism in bacteria: Antibiotic pathways, regulation, and function. In Biology of the prokaryotes, 627–51. New York: Thieme.
Stevenson, R. 1994. Extremozymes. Am. Biotechnol. Lab. 12(9):5–8.
Strohl,W. R. 1997. Biotechnology of antibiotics. New York: Marcel Dekker, Inc
 . Faber, K. (2004). Biotransformation in Organic Chemistry, Berlin: Springer-Verlag Schmidt, E., and Blaser, H.-U. (2003). Asymmetric Synthesis on Industrial Scale: Challenges,
Approaches, and Solutions, New York:Wiley-VCH.
Mattlack, A. S. (2001). Biocatalysis and biodiversity. In Introduction to Green Chemistry, pp. 241–289. New York and Basel:Marcel Dekker. Organization for Economic Co-operation and Development. (2001). The Application of Biotechnology to Industrial Sustainability, Paris: OECD.
Liese, A., Seelbach, K., and Wandrey, C. (2000). Industrial Biotransformations – A
Straathof, A. J. J., Adlercreutz, P. (eds.) (2000). Applied Biocatalysis, 2nd Edition,
Amsterdam: Harwood Scientific Publishers
 microbiology an introduction 10th edition , Gerard J. Tortora, Berdell R. Funke et al Christine L. Case, © 2010
food fermentation and micro organisms,charles w. bamforth,2005)
Berry, D.R., ed. (1988) Physiology of Industrial Fungi. Oxford: Blackwell.
Branen, A.L. & Davidson, P.M., eds (1983) Antimicrobials in Foods. New York:
Brown, C.M., Campbell, I. & Priest, F.G. (1987) Introduction to Biotechnology.
Oxford: Blackwell Publishing.
Caldwell, D.R. (1995) Microbial Physiology and Metabolism. Oxford: William C.
Dawes, I.W. & Sutherland, I.W. (1992) Microbial Physiology, 2nd edition. Oxford:
Demain, A.L., Davies, J.E. & Atlas, R.M. (1999) Manual of Industrial Microbiology
and Biotechnology. Washington, DC: American Society for Microbiology.
Frankel, E.N. (1998) Lipid Oxidation. Dundee: Oily Press.
Griffin, D.H. (1994) Fungal Physiology, 2nd edn. New York: Wiley-Liss.
Jennings, D.M. (1995) The Physiology of Fungal Nutrition. Cambridge: Cambridge
Lengeler, J.W., Drews, G.&Schlegel, H.G. (1999) Biology of the Prokaryotes. Oxford:
McNeil, B. & Harvey, L.M. (1990) Fermentation: A Practical Approach. Oxford: IRL.
O’Brien, J., Nursten, H.E., Crabbe, M.J.C. & Ames, J.M., eds (1998) Maillard
Reaction in Foods and Medicine. Cambridge: Royal Society of Chemistry.
Pirt, S.J. (1975) The Principles of Microbe and Cell Cultivation. Oxford: Blackwell
Salminen, S. & Von Wright, A., eds (1998) Lactic Acid Bacteria: Microbiology and
Functional Aspects. New York: Marcel Dekker.
Stanbury, P.F., Whitaker, A. & Hall, S.J. (1995) Principles of Fermentation
Technology, 2nd edn. Oxford: Butterworth-Heinemann (Pergamon).
Tucker, G.A. & Woods, L.F.J. (1995) Enzymes in Food Processing. London: Blackie.
Waites, M.J., Morgan, N.L., Rockey, J.S. & Higton, G. (2001). Industrial
Microbiology: An Introduction. Oxford: Blackwell Publishing.
Walker, G.M. (1998) Yeast Physiology and Biotechnology. Chichester: Wiley.
Ward, O.P. (1989) Fermentation Biotechnology: Principles, Processes and Products.
UK: Open University Press.
Wood, B.J.B. and Holzapfel, W.H. (1996) The Genera of Lactic Acid Bacteria. London: