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"$ Modern Industrial Microbiology and Biotechnology

(a) Isolation from field cultivated ergot (or parasitic production): This method is widely used in

Europe. Inoculation of the rye plants with conidiospores of Claviceps and other fungi

takes place two to three weeks before flowering begins and may continue during

flowering. Harvesting of ergotized ears of rye takes place about two months later. This

method has numerous disadvantages. Firstly, only one crop a year can be obtained.

Secondly, the yield of alkaloid in terms of quality (type) and quantity is highly

unpredictable. Thirdly, the vicissitudes of the weather attendant on all field operations

make the operation highly unstandardizable.

(b) Fermentation production: Due to the above problems, efforts have been put in over the

years to devise fermentation methods. A good measure of success has been achieved

especially for the clavine alkaloids and simple lysergic acid derivates. Ergot alkaloids

can be produced in submerged fermentation by Claviceps or Penicillium species, which are

used for their industrial production. Initial work in Japan showed that submerged

cultures did not produce the typical alkaloids associated with the sclerotium but instead

produced a series of new non-peptide bases (clavines) which did not possess any

significant pharmacological action. Attempts were made by many workers to influence

alkaloid production by modification of the culture medium and the fungus strain. The

first pure ergot alkaloid, ergotamine, was obtained by Stoll in 1920. Subsequently, others

reported the discovery of the “water soluble uterotonic principle of ergot” which was

subsequently determined to be ergonovine (also called ergometrine). As a result of further

successful experiments the commercial manufacture of simple lysergic acid derivatives

by fermentative growth of a strain of Claviceps paspali became feasible

(i) Production of clavine alkaloids: Different species of Claviceps parasitizing a variety of

grasses have been isolated and grown in liquid medium and the different alkaloids

assayed. Clavine alkaloids were obtained from Cl. litoralis, Cl. microcephals. One

strain Cl. purpurea produced a clavine and the other a pepetide alkaloid. The

medium used contained mannitol (5%) ammonium citrate (0.7%), KH

PO4 (0.1%)

and MgSO4 (0.03%). The pH was 5.2. The use of 10% sucrose instead of mannitol

gave higher yields.

The fermentation lasted from 30 days to 40 days and by classical strain

improvement methods yields of up to 1.0-1.5 gm/liter were obtained.

Improvements have since been obtained by other workers who found that a

mixture of mannitol (6.5%) and glucose (1%) gave up to 1 gm/liter yield in about

14 days. A high carbon to nitrogen in the medium increased yield.

(ii) Production of simple lysergic acid derivates: The production of simple lysergic acid

derivates was achieved in 1961 using Claviceps paspali isolated from an infected

Paspalum digitatum.

Higher yields have since been attained by slight modifications of the original

mannitol-succinic acid-mineral salts medium. Fumaric acid gave higher yields

when it was used in place of succinic acid. The fermentation lasted nine days. The

addition of hydrophilic non-ionic surfactants had the greatest effects on yield.

Aeration was vigorous.

(iii) Peptide alkaloids: The physiology of ergotamine formation has been studied using

Claviceps purpurea, C. litoralis, Elymus mollis. Improved ergotoxine yields were

Production of Ergot Alkaloids "$

obtained by the mutation and selection of a strain of Claviceps purpurea. This strain

on a mannitol-ammonium-succinate medium produced up to 40 gm/liter.

25.4 PHYSIOLOGY OF ALKALOID PRODUCTION

(i) Induction: Tryptophan is the central precursor of all ergot alkaloids and it is

therefore required in the medium. In addition to being a direct precursor of ergot

alkaloids, tryptophan is also a factor in the induction and derepression of enzymes

necessary for alkaloid synthesis. If the amino acid is not added to the medium the

organism manufactures it. When it is added the organism accumulates it in its

hyphae.

(ii) Feedback regulation: Feedback regulation studies have been hampered by the cell

wall as studies using protoplasts of Claviceps sp. have unambiguously

demonstrated. It was shown that the addition of elymoclavine inhibited (not

repressed) the first enzyme in the synthesis of the alkaloid, namely dimethylollyl

tryptophane synthase (DMAT synthase). This was demonstrated by supplying to

washed stationary phase cultures of the producing organism, basal culture

medium or basal culture medium supplemented with elymoclavine. Cultures with

fresh medium synthesized the alkaloid at a slower rate than when supplemented.

Both of them however reached the same final alkaloid concentration.

(iii) Phosphate repression: Like many secondary metabolites ergot alkaloid formation is

inhibited by increasing the level of phosphate. In this case the unfavorable limit

was 1.1. gm/liter. The addition of tryptophan helped to nullify the effect of

phosphate, showing that phosphate inhibition was mediated through tryptophan

probably by preventing its accumulation from exceeding the amount required for

alkaloid synthesis induction. In support of this explanation, it is noted that 5-

methyltryptophan also overcomes the inhibition of alkaloid synthesis by

tryptophan. Phosphate inhibited culture had very low levels of the first enzyme in

the synthetic chain, namely DMAT synthase; tryptophan caused more than 10-

fold increase in the activity of the enzyme.

(iv) Catabolite regulation: High levels of glucose (5%) greatly inhibited enzyme

production. The addition of a small amount of camp restored alkaloid synthesis to

a little extent.

(v) Alkaloid formation and morphological structures: It is interesting to note that alkaloids

seem to form in one structure and accumulated in another. Thus, in the plant genus

Ipomea, alkaloids are formed in the leaves and accumulated in the seeds, which do

not produce alkaloids at all. In ergotoxine alkaloid fermentation, alkaloids are not

elaborated until specific morphological structures, pellets, are formed in the

medium. The medium composition appears to influence the formation of these

pellets. Sucrose appears to encourage their formation while they are poorly formed

in malt. The peptide alkaloids are found in these structures, while clavines and

simpler derivates of lysergic acid are found in the medium.

(vi) Biosynthesis: Work on biosynthesis of alkaloids has been greatly facilitated by the

use of protoplasts. Being secondary products, alkaloids are produced by pathways

different from those of general metabolism. Furthermore, synthesis initiates with

carbon limitation. The ergot skeleton is derived from tryptophan, mevalonic acid,

"$ Modern Industrial Microbiology and Biotechnology

and methionine. The central precursor of the ergoline skeleton is tryptophan,

which is initially dealkylated. The five-carbon unit is obtained from mevalonic

acid. Although a component of the ergoline unit, it is neither limiting nor does it

participate in the regulation of alkaloid synthesis induction. Mevalonic acid may

be formed from the malonyl COA pathway with biotin and decarboxylation.

Clavines are simpler in terms of biosynthesis than lysergic acid derivates.

Lysergic acid itself is a key substrate in ergot alkaloid synthesis and the simpler

amide as well as the peptide derivates come from it (see Fig. 25.5).

Surprisingly not many enzymes appear to be involved in the synthesis of

alkaloids. Two of them have so far been isolated: Dimethyl tryptophan synthase

(DMTPS) and chanoclavine synthase. DMTPS catalyses the first specific reaction

of the formation of 8-ergoline and introduces the isoprene residue to the C

position

of tryptophan. Analogues of tryptophan increase the activity of the enzyme. The

compound produced, dimethyl tryptophan (4-isoprenyltryptophan) is 5-10 times

more effective as a precursor of the clavines than tryptophan.

Chanoclavine cyclase catalyses the cyclization of chanoclavine – 1 to the four-

ring 8-ergoline i.e. to elgmoclavine and agroclavine, both individually and

simultaneously. Some biogenetic relationships among alkaloids are shown in Fig.

25.5

(vii) Extraction of the alkaloid: Alkaloids are readily isolated at an alkaline pH by various

organic solvents such as ether, chloroform, and ethyl acetate.

Fig. 25.5 Synthetic Routes for Ergot Alkaloids

Production of Ergot Alkaloids "$!

The earliest procedures used for extraction were designed to obtain alkaloids

from sclerotia. In general the sclerotia would be dried, powdered, alkalinized, and

extracted with tartaric acid. For further purification, the tartaric acid solution was

made alkaline and extracted with chloroform. This chloroform layer would then

contain all the alkaloids. This has led to the terms water-soluble alkaloids which

include the simple amide lysergic acid derivatives and the clavines, whereas the

water-insoluble alkaloids now include the peptide-type. Nowadays the method

adopted for both water-soluble and water-insoluble peptides is to extract the

powdered ergot with chloroform to which a small amount of methanolic ammonia

has been added. The chloroform extract is concentrated to a small volume, diluted

with ether and extrated with concentrated H

. On neutralization with

ammonia the water-soluble alkaloids can be extracted with water and the water-

insoluble ones with carbon-tetrachloride.

SUGGESTED READINGS

Boichenko, L.V., Boichenko, D.M., Vinokurova, N.G., Reshetilova, T.A., Arinbasarov, M.U. 2001.

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the

Polymerase Chain Reaction, Microbiology, 70: 306-307.

Dongen, van P.W.J., de Groot, A.N.J.A. 1995. History of ergot alkaloids from ergotism to

ergometrine, European Journal of Obstetrics & Gynaecology and Reproductive Biology, 60:

109-116.

Evans, W.C. 1996. Pharmacognosy. 14

ed, W B Saunders Company Ltd, London, UK.

Groot, N.J.A. de Akosua, van Dongen, Pieter W.J, Vree, Tom, B., Hekster, Yechiel A., van

Roosmalen, Jos. 1998. Ergot Alkaloids - Current Status and Review of Clinical Pharmacology

and Therapeutic Use Compared with Other Oxytoxics in Obstretrics and Gynaecology, Drugs,

56: 525-8.

Hardman, J.G., Limbird, L.E. (eds) 1996. The Pharmacological Basis of Therapeutics, 9

ed,

McGraw-Hill

Kobel, H., Kobel, J. 1986. Ergot alkaloids. In: Biotechnology, H.J., Rehm, G. Reed, (eds) Vol 4,. 2nd

Ed. VCH, Weinheim, Germany, pp. 569-609.

Komarova, E.L., Tolkachev, O.N. 2001. The Chemistry of Peptide Ergot Alkaloids,

Pharmaceutical Chemistry Journal, 35: 504-506.

Lange, Klaus W. 1998. Clinical Pharmacology of Dopamine Agonists in Parkinson’s Disease,

Drugs & Aging, 13: 385-386.

Langley, D. 1998. Exploiting the Fungi: Novel Leads to New Medicines, Mycologist, 11: 165-166.

Menge, J.M.M. 2000. Progress and Prospects of Ergot Alkaloid Research. Advances in

Biochemical Engineering/Biotechnology. 68, 1-20.

Thompson, F., Muir, A., Stirton, J., Macphee, G., Hudson, S. 2001. Parkinson’s Disease, The

Pharmaceutical Journal, 267: 600-612.

Votruba, V., Flieger, M. 2000. Separation of Ergot Alkaloids by Adsorption on Silicates,

Biotechnology Letters, 22: 1281-1282.

Rehacek, Z., Sajdl, P. 1990. Ergot Alkaloids: Chemistry, Biological Effects, Biotechnology.

Academia Praha: Czech Republic.

464 Modern Industrial Microbiology and Biotechnology

26.1 NATURE AND USE OF STEROIDS AND STEROLS

Steroids are a large group of organic compounds with the perhydro- 1, 2-cyclopentano –

phenanthrene nucleus, which consists of four fused rings (Fig. 26.1).

Sterols are hydroxylated steroids – that is, they are alcohols derived from steroids. The

hydroxyl (OH) group of sterols is usually substituted at position C

. Unsaturation is

usually at C

and often as C

and C

. The term sterol comes from the Greek (Steros = solid)

because the earliest members studied were solid alcohols resulting from the

unsaponifiable (i.e. could not be broken down by NaOH) fractions of fats of plants and

animals. As the variety of known structures increased the general term steroid came into

use about 1935. In higher animals the principal sterol is cholesterol but a wider variety

exists in lower animals and in plants (Fig. 26.1).

Steroids and sterols are widely distributed in nature and are present in bile salts,

adrenal-cortical and sex hormones, insect molting hormones, sapogenins, alkaloids and

some antibiotics.

Steroids and sterols differ from each other in two ways: (a) the number, type, and

position of the substituents; (b) the number and position of the double bonds in the ring.

Steroid molecules are usually flat. However, the substituents at each of the junctions of

Rings A and B, Rings B and C, and Rings C and D may be either above or below the plane

of the ring. When the substituent group lies above the plane (denoted by a solid line) of the

molecule the substituent is denoted by > ; when it is below (denoted by a broken line) it is

denoted by =. When as is the case in many steroid hormones a double bond exists

between C

and C

the situation is denoted ,

. The individual compounds are named

systematically as derivatives of steroidal hydrocarbons the more important of which are

gonane, estrane, androstane, pregnane, cholane and cholestane. Thus cortisone which is

a derivative of pregnane is ,

- pregnene – 17=, 24 – diol – 3 11, 20 – trione.

The steroid hormones of the mammalian body have profound effects on the body

function and even the behavior of the animal. Thus, the male hormones, androgens

secreted by the testes are responsible for the development of the male reproductive organs

and the secondary sexual characters such as hairiness among several other functions.

Microbial Transformation of

Steroids and Sterols

CHAPTER

Microbial Transformation of Steroids and Sterols 465

The female sexual hormones include estrogens and progesterone. Estrogens are

produced by the ovary – they stimulate the development of the female reproductive

organs and secondary sexual characteristics such as enlarged breasts, etc.

Progesterone is produced by the corpus luteum, a body formed by the mature egg in the

female ovary. In association with the oestrogens, progesterone prepares the uterus for the

implanation of the fertilized egg in the uterine wall. Corticosteroid hormones are

produced by the cortex surrounding the adrenal glands, which are themselves located

just above the kidneys. The main steroid hormones produced by the glands are

corticosterone, cortisol, and aldosterone. Aldosterone is involved with mineral

metabolism, mainly of sodium ions and hence indirectly of the blood pressure. Cortisol

Fig. 26.1 Structures of some Steroids and Sterols

466 Modern Industrial Microbiology and Biotechnology

and corticosterone help the body handle physiological stress including extreme cold. It is

important to underscore, even at the risk of sweeping generalization, the significance of

hormones as a background towards appreciating the impetus for the transformation of

steroids.

Among insects steroid hormones are also very important in post-embryonic

development: juvenile hormones control larval growth; ecdysone controls

metarmorphosis of larval-larval larval-pupal and pupal-adult moulting processes; a

third hormone affects the brain and controls the production and release of the moulting

hormone. These hormones and their laboratory synthesized analogues (pheromones) are

used for controlling insects. Bile salts, sterols, oestrogens, progesterone, androgens

cortisone and cortisol and other steroids from animal and plants were isolated and

studied from 1903.

26.2 USES OF STEROIDS AND STEROLS

The world sale of steroids runs into billions of dollars (see Table 26.1)

26.2.1 Sex Hormones

As will be seen below, many steroids and sterols are manufactured through microbial

action. The largest economic impact of synthetic estrogen and progestin production has

been for use as contraceptive agents and for treatment and prevention of osteoporosis.

Contraceptive steroid mixtures have also been used to treat a variety of related abnormal

states including endometriosis, dysmenorrhea, hirsutism, polycystic ovarian disease,

dysfunctional uterine bleeding, benign breast disease, and ovarian cyst suppression.

Estrogens are routinely prescribed to post-menopausal women to prevent the

development and exacerbation of osteoporosis because it can increase bone density and

reduce fractures.

Table 26.1 Total worldwide sales of systemic sex hormones and corticosteroids

Sales, $x 10

Steroid class 1990 1994

Systemic sex hormones 3,582 5,436

Corticosteroids

Topical 1,558 1,891

Systemic 903 1,181

Respiratory 988 2,170

Nasal 382 665

Inhalants, systemic 606 1,505

Steroids for sensory organs 396 507

Total 7,427 11,185

Testosterone, alkylated testosterone, or testosterone esters are the primary anabolic–

androgenic steroid drugs. Most of these synthetic testosterone derivatives were in failed

attempts to separate the hormones’ masculinizing (androgenic) and skeletal muscle-

building (anabolic) effects. The medicinal uses for these drugs include treatment of

Microbial Transformation of Steroids and Sterols 467

certain types of anemias, hereditary angioedema, certain gynecological conditions,

protein anabolism, certain allergic reactions, and use in replacement therapy in gonadal

failure states.

Anabolic–androgenic steroids are best known for their nonmedical, and illegal, use to

aid in body-building or to increase skeletal muscle size, strength, and endurance by

athletes.

26.2.2 Corticosteroids

The greatest portion of steroid drug production is aimed at the synthesis of

glucocorticoids which are highly effective agents for the treatment of chronic

inflammation. Glucocorticoids exert their effects by binding to the cytoplasmic

glucocorticoid receptor within the target cell and thus either increase or decrease

transcription of a number of genes involved in the inflammatory process. Specifically,

glucocorticoids down-regulate potential mediators of inflammation such as cytokines

(Chapter 28). Typical oral glucocorticoids used to treat rheumatoid arthritis are

prednisone and 6 =-methylprednisolone. Corticosteroids are the most efficacious

treatment available for the long-term treatment of asthma, and inhaled corticosteroids are

considered to be a first-line therapy for asthma. They are also used to treat rhinitis, or

nasal congestion and inflammations of the skin.

26.2.3 Saponins

These are used for their hypocholesterolemic (cholesterol lowering) activity. Synthetic

steroids that are structurally related to saponins have been shown to lower plasma

cholesterol in a variety of different species

26.2.4 Heterocyclic Steroids

Dihydrogentesterone (DHT) is a more potent androgen than testerone. Elevated levels of

DHT lead to enlarged prostate (benign hyperplasia), sometimes prostate cancer, and

male baldness and the enzyme antagonistic to DHT steroid 5 =-reductase is being

developed as treatment for these ailments.

26.3 MANUFACTURE OF STEROIDS

In 1937, the first microbial transformation of steroids was carried out. Testerone was

produced from dehydroepiandrosterone using Corynebacterium sp. Subsequently,

cholesterol was produced from 4-dehydroeticholanic acid and 7-hydroxycholesterol

using Nocardia spp. These developments were virtually unexploited until 1949, when the

dramatic curative effect of cortisone on rheumatoid arthritis, a disease in which painful

swellings occur at the joints of the body, was announced. The cortisone used in this work

had been prepared by complex and tedious chemical synthesis beginning with

deoxycholic acid, a bile acid. So tedious was this that it took 32 chemical steps and two

years to produce only 11 gm of cortisone acetate. Additionally, it was difficult to find

enough of the starting materials. To meet the demand for cortisone to treat the large

number of people suffering from rheumatoid arthritis, a great burst of activity along the

four following lines ensured:

468 Modern Industrial Microbiology and Biotechnology

(i) The improvements of the original chemical method along lines suitable for

commercial production;

(ii) The development of methods of chemical synthesis using other more available and

more abundant starting materials including steroids and steroid-containing

compounds of plant origin. One of such compounds was diosgenin (a glycoside

formed from a steroid and glucose) obtained from a species of yams, Dioscorea

composita and the South African ‘elephant foot’ Testudinaria sylvatica. The other

was a plant sterol, stigmasterol obtained from soy bean, Glycine max.

(iii) Total chemical synthesis of cortisone and cortisol from relatively simple materials;

(iv) The use of biological agents to transform readily available steroids by introducing

oxygen at carbon C

, a process which took 12 steps in chemical synthesis. The use

of biological agents originally consisted of the use of ground or homogenized

adrenal tissues and fungi.

The first of the two methods mentioned above had moderate successes and for some

time provided steroids for clinical use. The third method remained an academic exercise.

The fourth method however gave dramatic results of which microbial transformations

eventually became more important. Two of the earliest such microbial transformations

were the conversion of progesterone to 11 – a hydroxy progesterone by the introduction of

– OH at the position 11 using Rhizopus nigricans and the conversion of cortisol to

prednisolone by Corynebacterium simplex (Fig. 26.2).

Fig. 26.2 Some Steroid Transformations Brought about by Microorganisms

Microbial Transformation of Steroids and Sterols 469

This latter transformation was notable because the new product was more active than

the starting one. Since then a large number of steroid analogues have since been

produced using a wide variety of microorganisms. Indeed virtually every steroid is

transformable in some way by some microorganism or the other.

From the beginning of 1960, intensive research interest shifted from rheumatoid

steroids to the area of sex hormones, especially the progresterone-based drug principally

used for birth control pills. This shift of interest was as a result of concern for rising world

population. The disclosure about 1965 of the steroidal nature of insect hormones

stimulated interest in them as a means of controlling insect pests of agriculture and food

and vectors of disease.

A large number of steroids have since been produced and tested for a variety of

purposes. Several of them have been found useful as anti-inflammatory, anti-tumor and

anti-allergy drugs; as birth control pills; for treatment in heart disease and a vast array of

medical and veterinary uses.

The use of microorganisms to transform steroids revolutionized the steroid

transformation industry. For example the price of cortisone, a widely used anti-

inflammatory drug, fell from US $200 per gm in 1949 to less then US $1.0 per gm in 1979

as a result of this development.

The microbial transformation of steroids differs from the ‘traditional’ fermentations

such as that of penicillin thus:

(i) In many cases steroid transformations are one-step-processes which bring about

relatively minor structural changes in the substrate, i.e. the steroid molecule. This

differs from the synthesis of penicillin and many other fermentation products in

which the product is synthesized entirely from the substrate offered in the medium.

(ii) Whereas in many industrial fermentations, the process of production is completed

in the fermentor, in the case of steroid transformations, readily available steroids

are micro-biologically transformed into important intermediates which are then

converted chemically to the final product. Alternately, the chemical syntheses are

first performed and the products transformed microbiologically later.

26.3.1 Types of Microbial Transformations in

Steroids and Sterols

Transformations by microorganisms affecting various positions in a wide range of

steroids and sterols have been carried out. Although steroid hormones have been most

widely studied, the transformation of bile acids, plant and animal sterols, steroid

alkaloids have also occurred. The transformation reaction include: hydroxylation,

dehydrogenation, reduction, side chain degradation, lactone formation, aromatization,

isomerization, epoxidation, hydrolysis, esterification, halogenation, and cleavage of the

steroid skeleton.

All of these have been carried out on steroid hormones, but only some of them have

been done on the other natural steroids and sterols. Two examples of these reactions have

already been described: Progesterone is converted to 11= - hydroxyl progesterone by

hydroxylation or the introduction of an OH group at position 11. Similarly, cortisol is

converted to prednisolone by dehydrogenation at position 1. Some other examples are

given in Fig. 26.2.