Okafor N. Modern Industrial Microbiology and Biotechnology

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' Modern Industrial Microbiology and Biotechnology

Fig. 5.7 b-oxidation of Fatty Acids

(ii) the assembly of these intermediates in an unusual way, by means of a combination

of standard general mechanisms with a selection from a relatively small number of

special mechanism;

(iii) these special mechanisms while being peculiar to secondary metabolism are not

unrelated to general or primary mechanism;

(iv) the synthetic activity of secondary metabolism appears in response to conditions

favorable for cell multiplication.

From the above, it becomes clear that although secondary metabolites are diverse in

their intrinsic chemical nature as well as in the organism which produce them, they use

only a few biosynthetic pathways which are related to, and use the intermediates of, the

primary metabolic pathways. Based on the broad flow of carbon through primary

metabolites to secondary metabolites, (depicted in Fig. 5.8) the secondary metabolites

may then be classified according to the following six metabolic pathways.

(i) Secondary products derived from the intact glucose skeleton: The carbon skeleton of

glucose is incorporated unaltered in many antibiotics and other secondary

metabolites. The entire basic structure of the secondary product may be derived

from glucose as in streptomycin or it may form the glycoside molecule to be

combined with a non-sugar (aglycone portion) from another biosynthetic route.

Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products '

Fig. 5.8 Biosynthetic Routes Between Primary and Secondary Metabolites

Main carbon routes

Primary metabolic routes

Secondary metabolic routes

Compounds in heavy type = secondary metabolites.

' Modern Industrial Microbiology and Biotechnology

The incorporation of the intact glucose molecule is more common among the

actinomycetes than among the fungi.

(ii) Secondary products related to nucleosides: The pentose phosphate pathway

provides ribose (5 carbon) for nucleoside biosynthesis. Many secondary

metabolites in this group are antibiotics and are produced mainly by

actinomycetes and fungi. Examples are nucleoside antibiotics such as bleomycin.

(Chapter 21).

(iii) Secondary products derived through the Shikimate-Chorismate Pathway:

Shikimic acid (C

) is formed by the condensation of erythrose-4- phosphate (C

)

obtained from the PP pathway with phosphoenolypyruvate (C

) from the EMP

pathway. It is converted to chorismic acid which is a key intermediate in the

formation of numerous products including aromatic aminoacids, such as

phynylalamine, tryrosine and tryptophan. Chorismic acid is also a precursor for a

number of secondary metabolites including chloramphenicol, p-amino benzoic

acid, phenazines and pyocyanin which all have anticrobial properties (Fig. 5.9).

The metabolic route leading to the formation of these compounds is therefore

Fig. 5.9 Metabolites in the Shikimic-Chorismate Pathway

Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products '!

referred to as the shikimate pathway. In view of this central role of chorismic acid,

however, the route is more widely known as the shikimate-chorismate route. The

shikimate-chorismate route is an important route for the formation of aromatic

secondary products in the bacteria and actinomycetes. Examples of such

secondary products include chloramphenicol and novobiocin. The route is less

used in fungi, where the polyketide pathway is more common for the synthesis of

aromatic secondary products.

(iv) The polyketide pathway: polyketide biosynthesis is highly characteristic of the

fungi, where more secondary metabolites are produced by it than by any other.

Indeed most of the known polyketide-derived natural products have been obtained

from the fungi, a much smaller number being obtained from bacteria and higher

plants. The triose (C

) derived from glucose in the EMP pathway is converted via

pyruvic acid to acetate, which occupies a central position in both primary and

secondary synthesis. The addition of CO

to an acetate group gives a malonate

group. The synthesis of polyketides is very similar to that of fatty acids. In the

synthesis of both groups of compounds acetate reacts with malonate with the loss

of CO

. By successive further linear reactions between the resulting compound and

malonate, the chain of the final compound (fatty acid or polyketide) can be

successively lengthened.

However, in the case of fatty acid the addition of each malonate molecule is

followed by decarboxylation and reduction whereas in polyketides these latter

reactions do occur. Due to this a chain of ketones or a b-polyketomethylene (hence

the name polyketide) is formed (Fig. 5.10). The polyketide (b - poly-ketomethylene)

Fig. 5.10 Formation of Polyketides

chain made up of repeating C-CH

or ‘C

units’, is a reactive protein-bound

intermediate which can undergo a number of reactions, notably formation into

rings. Polyketides are classified as triketides, tetraketides, pentaketides, etc.,

depending on the number of ‘C

units’. Thus, orsellenic acid which is derived from

the straight chain compound in Fig. 5.11 with four ‘C

-units’ is a tetraketide.

Although the polyketide route is not common in actinomycetes, a modified

polyketide route is used in the synthesis of tetracyclines by Streptomyces griseus.

(v) Terpenes and steroids: The second important biosynthetic route from acetate is that

leading via mevalonic acid to the terpenes and steroids. Microorganisms

'" Modern Industrial Microbiology and Biotechnology

Fig. 5.12 Isoprene Derivatives

especially fungi and bacteria synthesize a large number of terpenes, steroids,

carotenoids and other products following the ‘isoprene rule’. The central point of

this rule is that these compounds are all derivatives of isoprene, the five-carbon

compound.

Simply put the isoprene rules consist of the following (Fig. 5.12):

(i) Synthesis of mevalonate from acetate or leucine

(ii) Dehydratopm and decarboxylation to give isoprene followed by

condensation to give isoprenes of various lengths.

(iii) Cyclization (ring formation) e.g., to give steroids (Chapter 26)

Fig. 5.11 Formation of the Triketide, Orsellenic Acid

Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products '#

(iv) Further modification of the cyclised structure. The route leads to the

formation of essential steroid hormones of mammals and to a variety of

secondary metabolites in fungi and plants. it is not used to any extent in the

actinomycetes.

(vi) Compounds derived from amino acids: The amino acids are derived from various

products in the catabolism of glucose. Serine (C

N) and glycine (C

N) are derived

from the triose (C

) formed glucose; valine (C

N) is derived from acetate (C

);

aspartatic acid (C

N) is derived from oxeloacetic acid (C

) while glutamic acid

N) is derived from oxoglutamic acid (C

). The biosynthetic pathways for the

formation of amino acids are shown in Fig. 5.13 from which it will be seen that

aromatic amino acids are derived via the shikimic pathway.

Secondary products may be formed from one, two or more amino acids. an example of

the first group (with one amino acid group) is hadacidin which inhibits plant tumors and

Fig. 5.13 Synthetic Routes of the Amino Acids

'$ Modern Industrial Microbiology and Biotechnology

is produced from glycine and produced by Penicillium frequentants according to the

formula shown below

NCH

H ® HN(OH) CH

H ® OHCN(OH) CH

Glycine Hadacidin

Other examples are the insecticidal compound, ibotenic acid (Amanita factor C)

produced by the mushroom Amanita muscaria and psilocybin, a drug which causes

hallucinations and produced by the fungus Psiolocybe (Fig. 5.14), the ergot alkaloids

(Chapter 25) produced by Clavicepts purpureae also belong in this group as does the

antibiotic cycloserine.

Among the secondary products derived from two amino acids are gliotoxin which is

produced by members of the Fungi Imperfecti, especially Trichoderma and which is a

highly active anti-fungal and antibacterial (Fig. 5.14) and Arantoin, an antiviral drug

also belongs to this group.

Top: Ibotenic acid (from one amino acid)

Middle: Indole (from one amino acid)

Bottom: Gliotoxin (from two amino acids)

Fig. 5.14 Secondary Metabolites Formed from Amino Acids

Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products '%

Top: Malformin A, a secondary metabolite formed from three amino acids

Bottom: Amanitin, a secondary metabolite formed from two amino acids

Fig. 5.15 Secondary Metabolites from Amino Acids

The secondary products derived from more than two amino acids include many

which are of immense importance to man. These include many toxins from mushrooms

e.g the Aminita toxins (Fig. 5.15) (phalloidin, amanitin) peptide antibiotics from Bacillus

app and a host of other compounds.

An example of a secondary metabolite produced from three amino acids is malformin

A (Fig. 5.15) which is formed by Aspergillus spp. It induces curvatures of beam shoots and

maize seedlings. It is formed from L-leucine, D-leucine, and cysteine.

'& Modern Industrial Microbiology and Biotechnology

SUGGESTED READINGS

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. 2

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.

Overproduction of Metabolites of Industrial Microorganisms 99

The complexity of the activities which go on within a cell was mentioned at the beginning

of Chapter 5 when we discussed the metabolism of a yeast cell introduced into an

aqueous solution of glucose and ammonium salts. The yeast cell must first permit the

entry into itself of the glucose and ammonium salts. Under suitable environmental

conditions such as pH and temperature it will grow by budding within about half an

hour. For these buds to occur, hundreds of activities will have gone on within the cell.

New proteins to be incorporated into enzymes and other structures will have been

synthesized; nucleic acids for the chromosomes and carbohydrates for the cell walls will

all have been synthesized. Hundreds of different enzymes will have participated in these

synthetic activities. The organism must synthesize each of the compounds at the right

time and in the appropriate quantities. If along side ammonium salts, amino acids were

supplied, the yeast cells would stop absorbing the ammonium salt and instead utilize the

supplied ‘readymade’ substrate.

A few yeasts can utilize starch. If our yeast belonged to this group and was supplied

nothing but starch and ammonium salts, it would secrete extracellular enzyme(s) to

breakdown the starch to sugars. These sugars would then be absorbed and would be

used with ammonium salts, for the synthetic activities we described earlier.

Clearly therefore, while the organism’s genetic apparatus determines in broad terms

the organism’s overall synthetic potentialities, what is actually synthesized depends on

what is available in the environment. Most importantly, the organism is not only able to

‘decide’ when to manufacture and secrete certain enzymes to enable it to utilize materials

in the environment, but it is able to decide to stop the synthesis of certain compounds if

they are supplied to it. These sensing mechanisms for the switching on and off of the

synthetic processes enable the organism to avoid the overproduction of any particular

compound. If it did not have these regulatory mechanisms it would waste energy and

resources (which are usually scarce in natural environments) in making materials it did

not require.

An efficient or ’stringent’ organism which does not waste its resources in producing

materials it does not require will survive well in natural environments where competition

Overproduction of

Metabolites of Industrial

Microorganisms

CHAPTER