Primary metabolism, also referred to as trophophase, is characterized by balanced growth of microorganisms. It occurs when all the nutrients needed by the organisms are provided in the medium. Primary metabolism is essential for the very existence and reproduction of cells. In the trophophase, the cells possess optimal concentrations of almost all the macromolecules (proteins, DNA, RNA etc.).
It is during the period of trophophase, an exponential growth of microorganisms occurs. Several metabolic products, collectively referred to as primary metabolites, are produced in trophophase (i.e., during the period of growth).
The primary metabolites are divided into two groups:
1. Primary essential metabolites:
These are the compounds produced in adequate quantizes to sustain cell growth e.g. vitamins, amino acids, nucleosides. The native microorganisms usually do not overproduce essential primary metabolites, since it is a wasteful exercise. However, for industrial overproduction, the regulatory mechanisms are suitably manipulated.
2. Primary metabolic end products:
These are the normal and traditional end products of fermentation process of primary metabolism. The end products may or may not have any significant function to perform in the microorganisms, although they have many other industrial applications e.g. ethanol, acetone, lactic acid. Carbon dioxide is a metabolic end product of Saccharomyces cerevisiae. This CO2 is essential for leavening of dough in baking industry.
Limitations in growth:
Due to insufficient/ limited supply of any nutrient (substrate or even O2), the growth rate of microorganisms slows down. However, the metabolism does not stop. It continues as long as the cell lives, but the formation of products differs.
Overproduction of primary metabolites:
Excessive production of primary metabolites is very important for their large scale use for a variety of purposes.
Overproduction of several metabolites has been successfully accomplished by eliminating the feedback inhibition as briefly described below:
1. By using auxotrophic mutants with a block in one of the steps in the biosynthetic pathway concerned with the formation of primary metabolite (this should be an intermediate and not the final end product). In this manner, the end product (E) formation is blocked, hence no feedback inhibition. But overproduction of the required metabolite (C) occurs as illustrated below.
In the above example, an un-branched pathway is shown. This type of manipulation for overproduction of metabolites can be done for branched metabolic pathways also.
2. Mutant microorganisms with antimetabolite resistance which exhibit a defective metabolic regulation can also overproduce primary metabolites.
As the exponential growth of the microorganisms ceases (i.e. as the trophophase ends), they enter idiophase. Idiophase is characterized by secondary metabolism wherein the formation of certain metabolites, referred to as secondary metabolites (idiolites) occurs.
These metabolites, although not required by the microorganisms, are produced in abundance. The secondary metabolites however, are industrially very important, and are the most exploited in biotechnology e.g., antibiotics, steroids, alkaloids, gibberellins, toxins.
Characteristics of secondary metabolites:
1. Secondary metabolites are specifically produced by selected few microorganisms.
2. They are not essential for the growth and reproduction of organisms from which they are produced.
3. Environmental factors influence the production of secondary metabolites.
4. Some microorganisms produce secondary metabolites as a group of compounds (usually structurally related) instead of a single one e.g. about 35 anthracyclines are produced by a single strain of Streptomyces.
5. The biosynthetic pathways for most secondary metabolites are not clearly established.
6. The regulation of the formation of secondary metabolites is more complex and differs from that of primary metabolites.
Functions of secondary metabolites:
Secondary metabolites are not essential for growth and multiplication of cells. Their occurrence and structures vary widely. Several hypotheses have been put forth to explain the role of secondary metabolites, two of them are given below.
1. The secondary metabolites may perform certain (unknown) functions that are beneficial for the cells to survive.
2. The secondary metabolites have absolutely no function. Their production alone is important for the cell, whatever may be the product (which is considered to be useless).
Overproduction of secondary metabolites:
As already stated, the production of secondary metabolites is more complex than primary metabolites. However, the regulatory manipulations employed for excess production of primary metabolites can also be used for the secondary metabolites as well.
Several genes are involved in the production of secondary metabolites. Thus, around 300 genes participate in the biosynthesis of chlortetracycline while 2000 genes are directly or indirectly involved in the production of neomycin. With such complex systems, the metabolic regulation is equally complex to achieve overproduction of secondary metabolites. Some regulatory mechanisms are briefly discussed hereunder.
Addition of methionine induces certain enzymes and enhances the production of cephalosporin. Tryptophan regulates ergot alkaloid biosynthesis.
End product regulation:
Some of the secondary metabolites inhibit their own biosynthesis, a phenomenon referred to as end product regulation e.g. penicillin, streptomycin, puromycin, chloramphenicol. It is possible to isolate mutants that are less sensitive to end product inhibition, and in this manner the secondary metabolite production can be increased.
In this regulation process, a key enzyme involved in a catabolic pathway is inactivated, inhibited or repressed by adding a commonly used substrate. Catabolic repression can be achieved by using carbon or nitrogen sources. The mechanism of action of catabolite regulation is not very clearly understood.
The most commonly used carbon source is glucose. It is found to inhibit the production of several antibiotics e.g. penicillin, streptomycin, bacitracin, chloramphenicol, puromycin. The nitrogen sources such as ammonia also act as catabolite regulators (i.e. inhibitors) for the overproduction of certain antibiotics.
Inorganic phosphate (Pi) is required for the growth and multiplication of prokaryotes and eukaryotes. Increasing Pi concentration (up to 1 mM) is associated with an increased production of secondary metabolites e.g. antibiotics (streptomycin, tetracycline), alkaloids, gibberellins. However, very high Pi concentration is inhibitory, the mechanism of action is not very clear.
In some microorganisms (particularly actinomycetes), there occurs a self regulation for the production of secondary metabolites. A compound designated as factor A which is analogous to a hormone is believed to be closely involved in auto regulation for the production streptomycin by Streptomyces griseus. More such factors from other organisms have also been identified.
Microorganisms are also used for chemical transformation of unusual substrates to desired products. This process, also referred to as biotransformation, is very important in producing several compounds e.g. conversion of ethanol to acetic acid (in vinegar), sorbitol to sorbose, synthesis of steroid hormones and certain amino acids.
In bioconversion, microorganisms convert a compound to a structurally related product in one or a few enzymatic reactions. The bioconversions can be carried out with resting cells, spores or even killed cells. Non-growing cells are preferred for bioconversions, since high substrate concentration can be used, besides washing the cells easily (to make them free from contamination).
Sometimes, mixed cultures are used for bioconversions to carry out different reactions. In recent years, the yield of bioconversion is increased by using immobilized cells at a lower cost.