Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial microbiology. A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include antibiotics and pigments such as resins and terpenes etc. Some antibiotics use primary metabolites as precursors, such as actinomycin which is created from the primary metabolite, tryptophan.
Examples of primary metabolites produced by industrial microbiology:
Glutamic acid, Aspartic acid
5' guanylic acid
Acetic acid, Lactic acid
The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions.
Fermentation products of primary metabolism such as ethanol, acetic acid, and lactic acid were the first commercial products of the fermentation industry. These industrial revelations were soon followed by citric acid production along with other products of fungal origin. Due to the high product yield and the low reproducibility costs, major interest has been shown in the respective markets. Production of cell constituents i.e. lipids, vitamins, polysaccharides as well as intermediates in the synthesis of cell constituents such as amino acids and nucleotides are also of great economic importance in present-day industry. The effectiveness of yeasts along with other microorganisms as sources of the B-group vitamins has been recognized for more than 50 years and like products of catabolic primary metabolism e.g. ethanol, citric acid etc. are of great commercial importance.
Citric acid is an organic acid that is of major economic use in today’s industry. It is a very important commercial product and is widely used in the food and beverage
industries as a food additive. A simple diagram outlining citric acid production is given below in Figure 1. The present annual global requirement is nearly 4 lakh MTons that is produced almost entirely by fermentation of molasses sugar with selected strains of Aspergillus niger. (www.fgsc.net/asilommar/citric.html) In addition to the beverage and food industry, citric acid is used in effervescent powders as well as being used in boiler
and metal cleaning. Citric acid was Pfizer’s most popular product for decades. The process was vastly improved in 1917 when a Pfizer employee, Dr. James Currie began
fermentation experiments in order to develop the sugar under conversion in acid citrate (SUCIAC) process. This began to outperform conventional citric acid production
methods. Deep tank fermentation using molasses was also developed and aspects of this process subsequently contributed to the design of penicillin production.
Factors effecting citric acid production vary considerable and depend predominantly on the strain of A. niger used. Other factors that effect citric acid production include the type of raw material fermented, the amount of methyl alcohol present, the substrate’s initial moisture content as well as the fermentation time and temperature. Much research has been conducted over the years in order to increase the yield of citric acid production.
Much of the research carried out is in the area of improving the strains of A. niger used in 4 citric acid production using mutations. One such mutant of strain 72/4 was produced by X-ray and ultraviolet irradiation, which clearly gave better yields than that of the parent
. Recently, research carried out in the Institute of Food and Radiation Biology in Bangladesh has found several high citric acid producing strains of Aspergillus niger.
Using molasses as its substrate, these strains were found to yield 3-5 times more citric acid than that of its parent strain (CA16), thus being more resourceful than the parent form
Figure 1. A basic outline of citric acid cycle
Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. This promises to save half the costs, reduce waste mand energy requirements, and use renewable resources like sugar or plant oil.
Vitamin B12 is found in practically all animal tissues and originates either from organisms within the animal’s own digestive tract of from ingestion of animal food.
Human beings are completely dependent on dietary vitamins as they are unable to utilize any of the intestinally synthesized vitamins. It therefore seems apparent that the only primary source of the vitamin B12 in nature is the metabolic activity of microorganisms.
It is synthesized by a wide range of bacteria and streptomycetes, however not so much by yeasts and fungi. Three microorganisms are currently in use for industrial riboflavin production, which naturally overproduces the vitamin: the hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, yeast. Riboflavin production is also possible using the gram-positive Bacillus subtilis, however the deregulation of purine synthesis is required.
Riboflavin production is identifiable among all three organisms by the growth of yellow coloured colonies. This is of great importance during the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii,
tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analogue of riboflavin used for B. subtilis, have been applied successfully for mutant selections. These mutations greatly improve the production yield of the vitamin riboflavin, in comparison to the parent strain.
Nucleotides are used in the preparation of poly and oligonucleotides as well as being of potential nutritional and medical interest. However the greatest interest in nucleotides lies in the fact that they have the ability to enhance the flavour of foods. Yeast extract is extensively used as a flavouring agent in the food industry and is widely available either in powder or paste form. After autolysis and partial hydrolysis of RNA, ribonucleotides
such as 5’-monophpsphate (GMP) and inosine 5’ monophosphate (IMP) may be extracted from the biomass.(Ref) Flavour enhancement is a property of these purine ribonucleosides as well as the ribonucleoside, xanthylic acid (XMP). These food enhancers are responsible for meaty flavours found in foods and are available on the market worldwide. These products are of major importance in the food industry and currently international trade surpasses US $1.1 billion per year.
Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs.
Most of the secondary metabolites of interest to humankind fit into categories which classify secondary metabolites based on their biosynthetic origin. Since secondary metabolites are often created by modified primary metabolite synthases, or "borrow" substrates of primary metabolite origin, these categories should not be interpreted as saying that all molecules in the category are secondary metabolites (for example the steroid category), but rather that there are secondary metabolites in these categories.
Small "small molecules"
- Alkaloids (usually a small, heavily derivatized amino acid):
- Hyoscyamine, present in Datura stramonium
- Atropine, present in Atropa belladonna, Deadly nightshade
- Cocaine, present in Erythroxylon coca the Coca plant
- Scopolamine, present in the Solanaceae (nightshade) plant family
- Codeine and Morphine, present in Papaver somniferum, the opium poppy
- Tetrodotoxin, a microbial product in Fugu and some salamanders
- Vincristine & Vinblastine, mitotic inhibitors found in the Rosy Periwinkle
- Terpenoids (come from semiterpene oligomerization):
- Azadirachtin, (Neem tree)
- Artemisinin, present in Artemisia annua Chinese wormwood
- tetrahydrocannabinol, present in Cannabis
- Steroids (Terpenes with a particular ring structure)
- Saponins (plant steroids, often glycosylated)
- Glycosides (heavily modified sugar molecules):
- Natural phenols:
- Phenazine-1-carboxylic acid (and derivatives)
- Big "small molecules", produced by large, modular, "molecular factories"
- Fatty acid synthase products :
- Nonribosomal peptides:
- Hybrids of the above three:
Antibiotics were first defined as a chemical compound produced by a microorganism, which has the capacity to inhibit the growth of and even destroy bacteria and
microorganisms in dilute solutions (A. Waksman 1942).
Antibiotic production includes 160 different products and has a total annual worldwide market of $23 billion.
Sir Alexander Fleming first discovered the antibiotic properties of the mould Penicillin notatum in 1929 at St. Mary’s hospital in London, when he noticed that Penicillin notatum destroyed a staphylococcus bacterium in culture. Penicillin is bactericidal to a number of gram-positive bacteria and acts by inhibiting transpeptidation thus preventing new cells from forming walls. It belongs to the beta-lactam family of antibiotics.
A team at Oxford University further proved penicillin’s value as a drug by developing methods of growth, extraction and purification. During World war two research was moved to the USA where large-scale growth of the mould began. Firstly penicillin moulds were grown in small shallow containers on nutrient broth. Methods of growth were improved by using deep fermentation tanks with continuous sterile air supply and corn steep liquor as a source of nutrients. In 1943 a cantaloupe mould, P. Chysogenum was found to produce twice the amount of penicillin than P. notatum. Since then researchers continued to find higher yielding penicillin moulds and have also improved yields further by exposing moulds to x-rays and UV light. The first type of penicillin produced was Penicillin G, which had to be administered to patients parenterally because it is broken down by stomach acid. Penicillin V was later formulated so that it could be
taken orally, unfortunately it was less active than Penicillin G.
The enhancement of antibiotic industrial yield has been achieved through traditional strain improvement programs based on random mutation and screening. Recombinant
DNA techniques have existed since the 1970’s and involve the introduction of DNA fragments into host cells using a vector (a plasmid or phage) that contains a selection marker. The DNA fragments are integrated into the host genome or autonomously replicated as a plasmid. Transformants are then screened for improved characteristics.
The pharmaceutical company Eli Lilly was responsible for the first recombinant DNA improvement of an antibiotic producing microorganism. Transformation of C.
acremonium 394-4 caused an increase in the amount of antibiotic cephalosporin excreted by the organism. Cephalosporins are beta-lactam compounds that are
structurally and pharmacoligically related to penicillins. Cephalosporins resist hydrolysis by enzymes referred to as penecillinases, which are secreted by a number of bacteria.
They are now one of the most widely prescribed antibiotics and are very effective for the treatment of hospital-acquired infections. Actinomycetes are aerobic spore forming bacteria that originate from soil. A large
number of antibiotics are produced by actinomycetes and in particular Streptomyces.
They resemble a fungal mycelium in form, but have thinner filaments. These filaments are formed when cells divide to form long chains of up to 50 cells.
Actinomyces griseus was first isolated from soil in the Andes, this bacterium produced a substance that killed many bacteria unaffected by penicillin, including Tuburculosis bacillus. The antibiotic was named streptomycin. However tubercle bacilli soon became
resistant to streptomycin and it has since been replaced by para-amino-salicylic acid (PAS). Stretomycetes are still very important bacterial producers of antibiotics and
cytostatics. Due to the emerging resistance of bacteria to common antibiotics, new technologies such as combitatorial biosynthesis are being used for the production of novel metabolites using streptomycetes. This technology involves the use of a combination of genes from different biosynthetic pathways to produce modified metabolites.
Ordinarily Actinomycetes use the EMP pathway to metabolise glucose because this pathway is a more efficient one than the ED pathway. However, one actinomycete,Nonomureae in Mycobacterium smegmatis, has been found to preferentially use the ED pathway. This bacterium is now used to produce a novel commercial antibiotic for the treatment of multiresistant gram-positive bacteria.
The secondary metabolites of the fungi including Drechslera, Trichoderma, Aspergillus and Curvularia have the ability to produce green dyes / anthraquinones.. These dyes are from natural sources and do not cause the pollution to the environment associated with chemical dyes.
The production of penicillin G or V usually begins with lyophilized spores of P. chrosogenum. Using these spores, a sterile agar slant is inoculated, sporulation and growth occur under controlled conditions. Spores are harvested from mature slants and a spore suspension is used to inoculate a shake flask containing vegetative medium. A
typical vegetative medium will contain an organic nitrogen source (corn-steep liquor) and a sufficient concentration of fermentable carbohydrate (2% sucrose or glucose). Log phase growth is required in this stage and a minimum doubling time of six hours is usually required. The purpose of this inoculum development stage is to increase the concentration of the fungal mycelium necessary for a high volumetric rate of penicillin synthesis. Addition of lactose in combination with limited nitrogen availability stimulates a greater accumulation of penicillin because of the slower hydrolysis rate of the
disaccharide. Slow continuous feeding of culture with glucose also produces the same result. In order to produce particular penicillin a specific precursor can be added to the medium, for example phenyl acetic acid is added to maximize the production of penicillin G. The pH is kept neutral by adding sterile alkali, which stabilizes the newly synthesized penicillin. The fermentation usually takes 6-7 days; the fungal mycelium is then separated from the broth and processed by adsorption, precipitation and crystallization to yield the final product.
The fermentation of penicillin G or V is carried out in a liquid culture in volumes of10,000 to 200,000 litres. This aerobic process has a volumetric oxygen uptake of 0.4 to