Microbial Transformation

05.04.21
 Introduction
 The microorganisms or their enzymes are used to carry out
the conversion of substrates to desired products and such
reactions or processes are popularly known as microbial
transformation or biotransformation.
 Microbial transformations have many advantages e.g. a)
reaction-specific processes
b) higher stereo-and region-specificity
c) milder reaction condition and are therefore
preferred over chemical transformation in industries
usually involved in preparative organic chemistry.
 Microbial transformation reactions are mainly
categorized into oxidation/ reduction, hydrolysis,
condensation and isomerization reactions
In biotransformation reactions, microbial cultures
are grown in a suitable medium and then used as a
source of enzymes.
The poorly soluble substrates are either used in low
concentration or mixed with emulsifiers.
Lipophilic substrates are transformed in biphasic or
polyphasic system, in which enzyme is present in
aqueous phase and substrate is in the solvent phase.
 Steroids and sterol transformation

 Steroids are natural

hormones secreted by
adrenal gland. Steroids share
a basic structure called
cyclopentanoperhydrophena
nthrene
 Steroids and its derivatives
are of commercial use
because of their therapeutic
(glucocorticoids) and
contraceptive properties (e.g.
progesterone and estrogen).

Basic structural unit

(cyclopentanoperhydrophenanthrene) of
steroid
 These are also used as sedative and antitumor agents.
Cortisone is of commercial importance because of its anti-
inflammatory action against rheumatoid arthritis and can
be transformed to prednisolone (by the introduction of
double bond in ring A), which is more effective steroid as
compared to its precursor
 Chemical steroid synthesis involves 31 reaction steps to
yield 1g cortisone from 615 g of deoxycholic acid (purified
from beef bile).
 Transformation of simple natural precursor molecule to
product using microbes (Rhizopus arrhizus and Aspergillus
niger) reduced the cost of manufacturing cortisone from
200$ (1949) to 1$ (1979) and shortened 31 chemical steps to
11 steps. The introduction of oxygen atom in C-11 is crucial
for anti-inflammatory activity
 The microbial steroid transformations were made cost
effective by substituting expensive precursor by cheaper
plant based sterol, which is modified by mycobacteria.
 Plant sterols like stigmasterol, sitosterol (byproducts of
soya bean oil production), campesterol (byproduct of paper
industry) and diosgenin (Maxican yam roots- Dioscorea
composite or Testudinaria sylvatica) or animal sterol
(cholesterol) are the precursors used in steroid
transformation reactions catalyzed by microbes.
 Presently all steroids can be easily hydroxylated at specific
position using microbial monooxygenases (e.g. 11α- or 11β-
hydroxylase, 17α- hydroxylase and 21α- hydroxylase). These
microbial transformations are accomplished at 37ºC in
aqueous medium at atmospheric pressure. The market
demand for four major steroids (cortisone, aldosterone,
prednisone and prednisolone) is about 700,000 kg/year
Production of some steroids using microorganisms
DESIGN OF BIOTRANSFORMATION PROCESSES

 (a) oxidation ; (b) reduction ; (c) hydrolysis ; (d)

condensation ; (e) isomerization ; (f) formation of newer C–
C bonds ; and (h) introduction of hetero functional moieties
Biotransformation Reactions of Different Types
 Transformation of Steviol
(aglucon) into Stevioside
(glucoside) :
The transformation of Steviol
(i.e., hydroxydehydrostevic
acid) by the cells of Stevia
rebaudiana (Bert.) Hemsl.
(Eupatorium rebaudianum
Bert.) Compositae, also called
yerba dulce (Habitat :
Paraguay),
into a glucoside known as
stevioside which is proved to
be 300 times sweeter than
sucrose, and hence used as a
sweetner.
 Hydroxylation of β-Methyl digitoxin
to Digoxin :
 It has been observed that the most
significant biotransformation process
of pharmaceutical importance is the
12-hydroxylation of β-methyl digitoxin
to digoxin by the aid of cell cultures of
Digitalis lanata.
 In fact, the two cardiotonic
compounds digitoxin and digoxin are
duly isolated from the leaves of
Digitalis.
 It is a well-known fact that the
cardiotonic substance digoxin cannot
be produced either by microbial
biotransformation (bioconversion) or
chemically.
 Selection of Microorganisms

 The selection of strains either from its natural sources or

from the various available cultures which are solely
responsible for catalyzing the desired biotransformation
reaction(s) is not only vital and critical but also of great
importance.
 In steroid its rather difficult problem due to the lack of
selective methods so as to identify the colonies precisely which
usually perform the marked specific activity.
 Plate Assay

 The ‘plate assay’ may be successfully employed to select

organisms which may aromatize several steroidal entities, for
instance : 19-nor steroids; 19-substituted steroids; and sterols
(e.g., β-sitosterol, ergosterol etc.) into equilin and related
oestrogen.
These ring A aromatic oestrogenic products I and II above
usually react particularly with the reagent p-nitrobenzene
diazonium fluoborate to give rise to the production of an
intense red colouration.
Therefore, the development of such colonies in a solid
medium containing an appropriate steroidal substrate, are
duly replicated before the reagent is sprayed ; and thus, a
red-ring gets developed all around the active colonies.
 Modified Enrichment Method

In this specific instance, a steroid substrate is normally

incorporated as the sole C-source exclusively in a ‘minimal
medium’ seeded adequately with the soil dilutions.
The cells that causes the degradation of the substrate will
ultimately grow ; and are, therefore, subsequently transferred
to the same medium but particularly enriched with another C-
source, for instance : glucose.
However, the mutants may be present which are strategically
blocked at different stages in the process of degradation of the
steroid substrate, but may consume glucose as the C-source.
 Filtration Enrichment Method

 After mutagenesis the spores of filamentous organisms e.g.,

actinomycetes, fungi, are made to develop in a liquid minimal
medium. The ensuing microcolonies of prototroph's thus
developed are meticulously separated by filtration, whereby the
spores of auxotrophs that were unable to grow left behind in the
filtrate.
 The filtrate obtained in this manner in subsequently plated and
the resulting colonies are adequately checked for auxotrophic
characteristics
 Agar Plug Method

 The agar plug method is regarded to be one of the most reliable

and precise techniques wherein the agar cylinders having' single-
colonies’ are transferred to test plates after due incubation
preferably in a moist chamber.
 Biotransformation of Steroids

 Animal source — corticosteroids, oestrogens, sex-

hormones, cholesterol etc.
 Plant source — ergosterol, cardiac glycosides, β-sitosterol
 Glucocorticoids : Cortisol ; Cortisone ; Corticosterone ;
Dexamethasone
 Mineralocorticoids : Aldosterone ; Fludrocortisone ;
 Androgens : Testosterone ; Oxandrolone ;
 Oestrogens : Estrone ; Estriol ; Estradiol ;
 Gestogens : Progesterone
 (a) Total Synthesis :
The ‘total synthesis’ of cortisone (yield = 1g) obtained from deoxycholic acid (615 g) via
the original chemical process essentially involved as many as 31 different individual
reaction steps which eventually held the cost of the product abnormally high.
Hence, this kind of research is only limited to academic interest and very little
commercial interest.
 (b) Site-specific and Stereospecific Manipulation :
Intensive and extensive research with regard to microbial introduction of an O-atom
strategically into the steroid nucleus particularly in a site-specific and sterospecific
fashion encouraged the 11∝-hydroxylation of progesterone, and that too without any
prior activation whatsoever. Interestingly, these biochemical processes proceeded quite
effectively and efficiently, which ultimately paved the way for a viable, feasible and cost-
effective production of cortisone. Cost of 1g of cortisone stood at USD 200 per g in 1949.
 (c) Microbial Process :
The entirely microbial-based process discovered in 1979 having the same prime objective
to cause an effective 11α-hydroxylation of progesterone helped in the historical drastic
decreased in the cost of pure cortisone to less than even USD 1 per g.
 Production of Cortisone, Prednisolone, and
Prednisone from Diosgenin :

 Diosgenin is a sapogenin obtained from Dioscorea tokoro

Makino, belonging to the natural order Dioscoreaceae.
 The classical example based on the combined chemical-microbial
synthesis may be vividly represented by the production of
cortisone and prednisone (i.e., its 1-dehydro derivative) starting
from diosgenin via Reichstein’s Substance S (i.e., 11-
deoxycortisol) as illustrated under :
 Step-I : Diosgenin undergoes several chemical-microbial
transformations to give rise to the formation of Reichstein’s
Substance S which may also be called as 11-deoxycortisol.
 Step-II : The resulting Reichstein’s Substance S undergoes
11β-hydroxylation in the presence of the microorganism
Curvularia lunata to yield cortisol i.e., hydrocortisone.
 Step-III : Cortisol obtained from the previous step-III affords
dehydration at C-1 position in the presence of the organism
Corynebacterium simplex to produce prednisolone.
 Step-IV : It is essentially a non-microbial step and gives rise to
cortisone via chemical oxidation whereby the 11-hydroxy
moiety is converted to a 11-ketonic function conveniently.
 Step-V : Once again the organism Corynebacterium
Simplex (see step-III) has been successfully utilized to
cause dehydration at C-1 to yield the desired product
prednisone
 Biotransformation of Steroids

Steroids are the complex chemical substances

containing tetra cyclic carbon ring.
They are important therapeutic agents as steroid
hormones regulate various metabolic aspects in man
(including animals) and the human sexuality.
The chemical synthesis of steroids is very complex
and expensive and, therefore, use of microorganisms
such as micro fungi and bacteria has become very
important in their production (biotransformation) in
pharmaceutical industry.
Biotransformation of steroids differ from
conventional fermentation processes in that the
products are not synthesized from the ingredients
added in the medium; steroid precursors are added
into the culture medium towards the end of the
growth phase and these are minor modified by
microorganisms resulting in the steroids.
In a typical biotransformation process of steroid, a
high biomass of the microorganism is obtained in a
fermentation tank using an appropriate growth
medium and incubation conditions.
After this, the steroid to be bio transformed is added
in the fermentation with solvent, purified
chromatographically, and recovered by
crystallization.
Cortisone is a steroid hormone used to relieve the
pain associated with rheumatic arthritis in
humans.
This steroid is chemically synthesized from
deoxycholic acid; the chemical
synthesis completes in 37 different steps, many of
which require extreme conditions of temperature
and pressure for completion and the resulting
product is very expensive.
The most difficult aspect of the chemical synthesis
of cortisone is the introduction of an oxygen atom
at the number 11 position of the steroid ring of
progesterone, an intermediate.
This difficulty is easily solved by microorganisms.
When progesterone is added to a fermentation
tank containing Rhizopus nigricans growing for
approximately a day, the progesterone is
hydroxylated at the number 11 position of its
steroid ring to from 11-a-hydroxyprogesterone.
The product is recovered by extraction with
methylene chloride or various other solvents, purified
chromatographically, and recovered by crystallization.
11-a-hydroxyprogesterone is now subjected to
chemical synthesis steps and, finally, cortisone is
obtained.
The use of biotransformation process to obtain
cortisone has made the whole process easy and has
lowered the original cost 400-fold especially in U.S.
pharmaceutical market.
Steroid Bioconversion via Fermentative Procedures

 Generally, the bioconversion of steroids is invariably

accomplished by employing submerged aeration
technique carried out in SS tanks charged with minimal
nutritional quantities so as to permit the maximum ease
and conversion with regard to the extraction and
purification of the ultimate desired transformation.
 In usual practice, the fermentative procedure is adopted
by either of the two following phases:
Phase I
 It represents predominantly the growth phase whereby the
maximum extent of the maximum extent of the culture growth.
Both optimized temperature and aeration is required. The
duration of incubation solely depends upon the type of culture
being employed such as : bacteria :12-24 hrs; fungi 24-72 hrs.
Phase II
When the growth phase almost comes to an end, the careful
addition of the respectively steroid commences. Importantly ,the
quantum of steroid need to be added solely depends upon three
vital components namely:
a) Transforming capacity of culture
b) Potential toxicity of substrate
c) Nature of product
It is essential to take care of problem of toxicity in the culture
medium by either continuous or periodic incorporation of
substrate of into the fermentation vessel
 The organic solvents that are utilized profusely are
acetone, ethanol and methanol which essentially
contribute two main advantages factors, which are:
a) afford relatively low toxicity for the conversion enzymes
b) cause appreciable solubility of steroids
DMF does possess an exceptional solubility characteristic
feature for steroids, whereas its non-toxic level stands at
below 2% conc. in the culture medium.
The bioconversion capacity actually is based upon the load
of the prevailing substrate, and the ability of the
microorganism
 General scheme for steroid drug production

Steroid drugs are synthesized by chemical or

microbial routes, both of which involve conversion of
steroid precursors to drug intermediates and final
conversion of intermediates
Microbial transformations cleave the complex side
chains of precursor steroids in one single step and
incorporate desirable modifications in steroid
nucleus.
Microorganism involvements in steroid production.
 Potential microbial transformations of steroids
with cortical side chain
 The bioconversion of 3β-acetoxypregna-5, 16-diene-20-one
(commercially known as 16-DPA) by a mixed culture of Pseudomonas
diminuta and Comamonas acidovorans has been reported
 Biotransformation of hydrocortisone, a corti-costeroid by Neurospora
crassa FGSC 4335 was investigated
 The studies with growing cells of Pseudomo-nas putida MTCC 1259
were carried for bio-transformation of a steroid with cortical side chain,
namely, 11β,17α-dihydroxy-4-pregnene-3,20-dione-21-O-succinate
 Alternative Bioconversion Methods

Steroids are insoluble in water and uniform dispersal

of steroids in bioconversion medium is the major
problem associated with steroid bioconversions that
limits the yield of such processes

The yield of products is restricted by upper limit of

substrate concentration as the enzymes involved in
bioconversions are inhibited by high concentration
of their substrates.
 Alternative methods like use of immobilized cells, emulsification of
substrate, use of organic solvents in biphasic systems and aqueous
biphasic systems for in-creasing the solubility and dispersal of
steroids have been attempted by many researchers.
 The use of immobilization of microbial cells provides the ability to
minimize the deactivation of biocatalyst and controls reaction times,
reuse of cells for many reaction cycles and lowers the total
production cost of cell mediated reactions.
 Cells can be used for many reaction cycles and lowers the total
production cost of cell mediated reactions.
 The 11β-hydroxylation and 1-dehydrogenation on a cortical side chain
containing precursor by using immobilized spores of Cunninghamella
elegans was studied and maximum production of cortisol and predni-
solone were obtained after 72 h transformation period using
immobilized spores of C. elegans of concentration 2 x 107 spores/ml for
both entrapment and adsorption