Biocatalysis for the Pharmaceutical Industry || Application of Whole-Cell Biotransformation in the Pharmaceutical Industry

10

Application of Whole-CellBiotransformation in thePharmaceutical Industry

Kin Sing Lam

Nereus Pharmaceuticals, Inc., Department of Microbiology and Industrial Fermentation,

San Diego, CA 92121, USA

10.1 Introduction

Biotransformation is the use of biological systems, including whole cells, organs or isolated

enzymes, to catalyze the conversion of organic compounds from synthetics and natural

products. There are many applications of biotransformation in the pharmaceutical industry,

including synthesis of drug metabolites for metabolism studies, lead optimization, chiral

resolution and the creation of libraries of derivatives from highly diverse lead compounds for

structure–activity relationship (SAR) and screening studies. A few of these applications have

been reviewed in this book. Studies in the past decade have witnessed a tremendous growth in

the applications of biotransformation in the pharmaceutical industries [1–3]. These studies

have provided complementary approaches and sometimes powerful alternatives to conven-

tional synthetic chemical techniques, due to the following advantages and traits of biotrans-

formation processes:

. stereoselectivity;

. regioselectivity;

. creating new functional groups at nonactivated sites;

. modification of complex molecules without the need of protection/deprotection steps;

. cloning, overexpression and tailor-made biocatalysts;

. immobilization and reusability of biocatalysts;

Biocatalysis for the Pharmaceutical Industry : Discovery, Development, and Manufacturing edited by J. Tao, G.-Q. Lin, and A. L.

© 2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9

. mild reaction conditions at ambient temperature and atmospheric pressure;

. waste minimization.

10.1.1 Whole-Cell Biotransformation Processes Used in CommercialProduction of Pharmaceuticals

Whole-cell biotransformation processes have been successfully applied for commercial

production of pharmaceuticals, either as the drug substance itself or as an intermediate for

the synthesis of the final drug substance. Some examples of the whole-cell biotransformation

processes used by pharmaceutical industry are shown in Table 10.1. The structures of the

biotransformation products are shown in Figure 10.1.

The examples of thewhole-cell biotransformation processes shown in Table 10.1 illustrate

the aforementioned advantages of the biotransformation reaction. In addition to the

stereoselectivity, mild reaction conditions and waste minimization essentially demonstrated

by all 18 whole-cells processes, the regioselectivity and creation of new functional

groups at nonactivated sites are demonstrated by the hydroxylation production of

11a-hydroxypro-gesterone, b-hydroxy-isobutyric acid, and b-hydroxy-n-butyric acid.

Recombinant microorganisms have been engineered for the production of (R)-ethyl-4,4,

4-trifluoro-3-hydroxybutanoate, (S)-2,2-dimethyl-cyclopropanecarboxamide, L-piperidine-

2-carboxylic acid and L-carnitine. Immobilized-cell technology has been applied for the

production of D-4-hydroxyphenyl glycine, nicotinamide and D-aspartic acid. Modification of

two complex natural products, progesterone and compactin, to the corresponding 11a-hydroxypro-gesterone and pravastatin has been successfully carried out in high yield without

any protection steps. These findings firmly establish that whole-cell biotransformation is an

important complementary tool to organic synthesis in the preparation of drug substances and

useful chiral pharmaceutical intermediates.

10.1.2 Application of Whole-Cell Biotransformation Processin the Synthesis of Chiral Pharmaceutical Intermediates

Fourteen of the 18 whole-cell processes shown in Table 10.1 are used in the preparation of

chiral intermediates for the production of the final drug substances. Whole-cell biotransfor-

mation has been routinely used to generate a wide variety of chiral pharmaceutical

intermediates [2,12,13]. This includes the use of oxido-reductases and aminotransferases

in the whole cells for the synthesis of chiral alcohols, aminoalcohols, amino acids and

amines. Monooxygenases in the whole cells have been used in enantioselective and

regioselective hydroxylation, epoxidation and Baeyer–Villiger reactions. Dioxygenases

have been used in the synthesis of chiral diols. Hydrolytic enzymes in the whole cells have

been applied for the resolution of a variety of racemic compounds and in the asymmetric

synthesis of enantiomerically enriched chiral compounds. Aldolases and decarboxylases

have been effectively used in asymmetric synthesis by aldol and acyloin condensation

reactions. The production of single enantiomers of drug intermediates is increasingly

important in the pharmaceutical industry. Whole-cell biotransformation is expected to play

a significant role in this rapidly growing area of industry.

214 Biocatalysis for the Pharmaceutical Industry

Table

10.1

Commercial

whole-cellbiotransform

ationprocesses

forthepreparationofpharmaceuticalsandpharmaceuticalinterm

ediates

Biotransform

ationProduct

Final

product

Indication

Reactiontype

Microorganism

Process

Ref.

11a-H

ydroxypro-gesterone1

Cortisone

Anti-inflam

mation

Hydroxylation

Rhizopusarrhius

Single-stageferm

entation

[4]

b-H

ydroxy-isobutyricacid

2Captopril

Treatmentfor

hypertension

Hydroxylation

Candidarugosa

Single-stageferm

entation

[4]

b-H

ydroxy-n-butyricacid

3Carbapenem

Antibacterial

Hydroxylation

Candidarugosa

Single-stageferm

entation

[4]

Pravastatin4

Pravastatin

Anti-cholesterol

Hydroxylation

Streptomyces

carbophilus

Single-stageferm

entation

[5]

5-M

ethylpyrazine-2-

carboxylicacid

5

Acipim

ox

Anti-lipolytic

Oxidation

Pseudomonasputida

Single-stageferm

entation

[6]

5-M

ethylpyrazine-2-

carboxylicacid

5

Glipicide

Anti-diabetic

Oxidation

Pseudomonasputida

Single-stageferm

entation

[6]

Pyridyl-3-aceticacid

6Risedronate

Treatmentfor

osteoporosis

Oxidation

Pseudomonasoleovorans

Single-stageferm

entation

[7]

(4S,6S)-Hydroxy-sulfone7

Trusopt

Treatmentfor

glaucoma

Reduction

Neurospora

crassa

Single-stageferm

entation

[6]

(R)-Ethyl-4,4-4-trifluoro-3-

hydroxybutanoate8

Belfoxatone

Anti-depressant

Reduction

Recombinant

Escherichia

coli

Two-phasewater–butyl

acetate

[7]

D-4-H

ydroxyphenylglycine9

Ampicillin/

amoxycillin

Antibacterial

Hydrolysis

Bacillusbrevis

Immobilized

cells

[8]

Nicotinam

ide10

Nicotinam

ide

Treatmentofpellagra

Hydrolysis

Rhodococcusrhodochrous

Immobilized

cells

[7]

(S)-Pipecolicacid

11

Incel

Anticancer

Hydrolysis

Pseudomonasfluorescens

Single-stageferm

entation

[7]

CBZ-D-proline12

Eletriptan

Treatmentofmigraine

Hydrolysis

Arthrobacter

sp.

Single-stageferm

entation

[7]

(S)-2,2-D

imethyl-cyclopro-

panecarbox-amide13

Cilastatin

Dehydropeptide

inhibitor

Hydrolysis

Comononasacidivoransand

recombinantE.coli

One-pot,two-stepprocesses

[7]

D-Carnitine14

L-Carnitine

Thyroid

inhibitor

Multi-enzymatic

pathway

Agrobacterium

sp.

Single-stageferm

entationof

fourprocesses

[7]

Phenylacetylcarbinol15

Ephedrine

Treatmentforasthma

Lyase

Saccharomyces

cerevisiae

Single-stageferm

entation

[9]

D-A

sparticacid

16

Apoxycillin

Antibacterial

Decarboxylation

Pseudomonasdacunhae

Immobilized

cells

[10]

3,4-D

ihydroxy- L-

phenylalanind( L-D

OPA)17

L-D

OPA

Treatmentof

Parkinsonism

Lyase

Erw

inia

herbicola

Single-stageferm

entation

[11]

Application of Whole-Cell Biotransformation in the Pharmaceutical Industry 215

O

HO

O

HOCOOH COOH

OH

NaOOCHO

OH

HO

HO

H

O

6

4321

N

NOH

O

N

OH

O S S

OH

O O

F3C

CO2Et

OH

8765

HO

H2NCOOH

N

CONH2

NH

COOH

N COOH

OO

9 12 11 10

OH

OCOOHN

OH

HO

O

HOOCCOOH

NH2

13 16 15 14

HO

HO

COOH

NH2

17

Figure 10.1 Structures of biotransformation products from Table 10.1


10.2 Disadvantages of Whole-Cell Process Comparedwith the Isolated Enzyme Process

Although whole-cell biotransformation has been established as a valuable tool for the synthesis

of pharmaceuticals and pharmaceutical intermediates, there are several inherent problems

associated with this process that have hindered the advancement of this technology in the

pharmaceutical industry.Attempts to clear some of these obstacles have recently allowed the use

of isolated enzyme in the biotransformation process to garner greater attention than whole-cell

biotransformation, since the reactionmechanism and kinetics of a single biocatalyst are simpler

than the complex biochemical synthetic methods which usewhole cells. In comparison with the

whole-cell processes, fewer side-products are formed in enzymatic transformations, complex

expensive fermentors are not required, aeration, agitation and sterility need not necessarily be

maintained and the substrate is not diverted into the formation of a de novo cellular biomass. In

this and the next section, comparison of application of whole cells and isolated enzymes in

biotransformation processes is made. The following are the disadvantages of the whole-cell

biotransformation process compared with the isolated enzyme biotransformation process.

10.2.1 Substrate Availability and Recovery of Productsin Low Concentrations

Whole-cell biotransformation usually occurs in a water medium with substrate concentration

rarely exceeding 10 gL�1. The substrate concentration is also affected by the mass transfer

limitations imposed by the cellular membrane, which further reduces the production yield, an

issue not seen with the use of isolated enzymes. The low substrate availability in the reaction

leads to the formation of a large volume of aqueous solution containing a relatively low

concentration of product, which imposes technical difficulty in the downstream process and

adds to themanufacturing cost of the product. The ability to effectively overcome the problems

of substrate availability, effective space–time yield production and product recovery is very

important in achieving cost-effective production with this technology. Without much im-

provement of the above technical issues, the application of whole-cell biotransformation is

limited to high-priced chiral pharmaceutical intermediates or high value-added chemicals.

10.2.2 Undesirable Side Reactions

Whole-cell biotransformations frequently showed insufficient stereoselectivities and/or unde-

sired side reactions because of competing enzymatic activities present in the cells. These side

reactions canmodify the substrates and/or products. Furthermore,whole-cell biotransformations

are limited due to the intrinsic need to growbiomass,which generates its ownmetabolites that are

not related to the biotransformation reactions and, therefore,whichneed to be removedduring the

downstream process. Both the cells themselves and the unrelated metabolites produced are

impurities that need to be removed after the biotransformation reaction. With isolated enzymes,

there are no organism and unrelatedmetabolites to remove after the biotransformation processes.

10.2.3 Toxicity of Substrate and Product

The biochemical system of a microorganism is more complicated than an isolated enzyme

and possesses dynamic and regulatory properties. Both substrate and product of the


biotransformation process can exert inhibition or toxicity to the microorganism, resulting in

reduced production [14].

10.3 Advantages of Whole-Cell Process Compared with the IsolatedEnzyme Process

In this section, the advantages of the use of whole cells over the use of isolated enzymes in

biotransformation processes are presented.

10.3.1 More Stable Sources than Isolated Enzymes

Compared with isolated enzymes, enzymes used in whole-cell biotransformations are often

more stable due to the presence of their natural environment inside the cell. This is especially

true for the enzymes involved in the oxidation and hydroxylation reactions that are labile once

isolated from the cells. They are a convenient and stable source of enzymes that are often

synthesized by cells in response to the presence of the substrate.

10.3.2 Regeneration of Cofactors and Multi-Enzyme Reactions

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleo-

tide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations

are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive

edge over the isolated enzymes in complex conversions involving multiple enzymatic

reactions [14].

10.3.3 Diversity and Availability

One can easily access a collection of microbial biocatalysts with broader diversity and higher

activities and selectivities than the commercially available enzymes. Most academic and

industrial institutions possess microbial culture collections that can be utilized for whole-cell

biotransformation processes. Furthermore, one can rapidly assemble a relatively small number

of nonpathogenic, often food-grade microorganisms (such as baker�s yeast and bacteria), for

whole-cell biotransformation applications. Themicrobial biocatalysts are inexpensive, readily

available and renewable. The selection of the microorganisms from the culture collection for a

specific biotransformation reaction is based on the internal experience of the research group

and on published information. Screening of the microbial biocatalysts takes advantage of

microbial diversity as much as possible by evaluating a broad range of microbial species that

have been isolated from very diverse environments.

10.3.4 Reactions with Non-Commercially Available Isolated Enzymesfor Preparative-Scale Synthesis

10.3.4.1 Cytochrome P450 Monooxygenases

Cytochrome P450 monooxygenases (P450s) have significant potential in biotransformation

applications because their ability to insert molecular oxygen regiospecifically and


stereoselectively into allylic positions or into unactivated C�H bonds under mild conditions

has no equivalent in organic synthesis [16]. The membrane-bound nature of the majority of

these enzymes and typically low activities [17], together with their functional dependence on

the presence of cofactors and related electron transport proteins, has ensured that preparative

biocatalytic hydroxylations are most usefully performed with whole-cell catalysts [16,17].

10.3.4.2 Aromatic Hydrocarbon Dioxygenases

Aromatic hydrocarbon dioxygenases catalyze the oxidation of the aromatic-ring hydrocar-

bons, compounds containing only carbon and hydrogen. Representatives of this class of

enzymes are toluene dioxygenase (TDO) and naphthalene dioxygenase (NDO) [18,19]. Like

P450s, several properties associated with aromatic-ring-hydroxylating dioxygenases pose

challenges for their application to the targeted preparation of oxidation products. These include

their cofactor requirements for reduced nicotinamide cofactor(s) NAD(P)H, the multicompo-

nent nature of the iron–sulfur-containing subunit, low specific activity and instability.

Therefore, applications based on aromatic-ring-hydroxylating dioxygenases have been based

largely on whole-cell biotransformations with enzymes typically overexpressed in recombi-

nant hosts such as Escherichia coli or Pseudomonas sp. [20,21]. An excellent example of the

application of aromatic hydrocarbon dioxygenase in whole-cell biotransformation is the

production of indigo to levels exceeding 18 g L�1 by overexpressed NDO from Pseudomonas

putida in a recombinant Escherichia coli strain [21].

10.3.4.3 Enoate Reductase in Reduction of Triply Substituted Double Bonds

Enoate reductase reduces a,b-unsaturated carboxylate ions in an NADPH-dependent reactionto saturated carboxylated anions. Useful chiral synthons can be conveniently prepared by the

asymmetric reduction of a triply substituted C�Cbond by the action of enoate reductase, when

the double bond is activated with strongly polarizing groups [22]. Enoate reductases are not

commercially available as isolated enzymes; therefore, microorganisms such as baker�s yeastor Clostridium sp. containing enoate reductase are used to carry out the reduction reaction.

10.3.4.4 D-Aminoacylase for Production of D-Amino Acid

Since there is no commercially available D-aminoacylase, the production process of D-amino

acids involves cloning of the D-aminoacylase and thewhole cells containing the recombinant D-

aminoacylase are used in biotransformation of N-acetyl-D-amino acid. D-Amino acids can be

generated in large quantities at low cost using whole-cell biotransformation [23].

10.3.5 Cost Effectiveness and Ease of Operation

Compared with isolated enzymes, application of whole cells as biocatalysts is usually more

economical since there is no protein purification process involved. Whole cells can be used

directly in chemical processes, thereby greatly minimizing formulation costs. Whole cells are

cheap to produce and no prior knowledge of genetic details is required. Microorganisms have

adapted to the natural environment and produce both simple and complex metabolic products

from their nutrient sources through complex, integrated pathways.


10.4 Approaches to Address the Disadvantages of Whole-CellBiotransformation

During the last decade, significant advancements in biochemistry, molecular cloning, and

random and site-directed mutagenesis, directed evolution of biocatalysts, metabolic engineer-

ing and fermentation technology have led us to devisemethods to circumvent the disadvantages

of whole-cell biotransformation discussed in Section 10.2. The applications of these methods

are summarized in this section.

10.4.1 Control of Substrate and Product Concentrationby Absorbing Resins

Absorbing resins have been extensively applied for enhancing the production and the recovery

of natural products frommicrobial fermentations [24]. Resins act as ‘stabilization agents’ when

added to the fermentation broths during the production phase by capturing the products and

preventing product degradation and repression of their production. Addition of resin to the

fermentation broths significantly reduces the volume of the fermentation broth for downstream

processing, since resin can be effectively separated from the microbial cells and the liquid

medium [24]. This technique has been applied to thewhole-cell biotransformation processes to

circumvent the problems associated with substrate availability and recovery of products in low

concentrations. Listed below are a few examples of the use of resins in the whole-cell

biotransformation processes.

10.4.1.1 Production of (S)-4-(3, 4-Methylene-Dioxyphenyl)-2-Propanol

Compound LY 300 164, a 2,3-benzodiazepin, is a drug candidate being evaluated clinically for

treatment of epilepsy and Parkinson�s disease. It was prepared from (S)-4-(3,4-methylene-

dioxyphenyl)-2-propanol, which in turn was derived from 3,4-methylenedioxyphenylacetone

by a dehydrogenase from the yeast Zygosaccharomyces rouxii via the whole-cell biotransfor-

mation process [25]. The volumetric productivity of this biotransformation process is limited

by the toxicity of the substrate to the yeast cells at a concentration of 6 g L�1. By binding 80 g of

the substrate to 1 L of Amberlite XAD-7 resin, the substrate concentration in thewater medium

was kept around 2 g L�1, significantly lower than the toxicity level. A sevenfold greater

substrate concentration (40 g L�1) could be reached with 96% conversion and >99.9%

enantiomeric excess (ee) by simple addition of a polymeric absorbing resin to the reaction

mixture.

10.4.1.2 Reduction of Bromocinnamaldehyde

2-(R)-Benzylmorpholine is a potent appetite suppressant drug. The synthesis of 2-(R)-benzyl-

morpholine began with the reduction of the unsaturated bromocinnamaldehyde to the corre-

sponding saturated (S)-bromo-alcohol by baker�s yeast, with a very low ee of 63%. However, an

efficient transformation can be achieved by controlling the substrate concentration with the

addition of hydrophobic resin Amberlite XAD-1180 [26]. With a resin-to-substrate ratio of one

and an initial substrate concentration of 5 gL�1, the saturated (S)-bromo-alcohol was recovered

at nearly quantitative yield and 98.6% ee.


10.4.1.3 Production of Vanillin

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is widely used in foods, beverages, perfumes

and the pharmaceuticals industries. Biotransformation of isoeugenol from essential oil to

vanillin represents an economic route for the supply of vanillin, which has a limited supply due

to the availability of vanilli pod plants. The conversion yield of isoeugenol to vanillin by the

whole-cell biotransformation process of Bacillus fusiformis was low due to the product

inhibition effect. Adding resin HD-8 to the whole-cell biotransformation eliminated the

product inhibition effect, yielding 8 g L�1 of vanillin in the final reaction mixture [27]. The

resin HD-8 also facilitated the separation of vanillin from the used substrate. The recovered

isoeugenol can be used for the subsequent biotransformation reaction.

10.4.2 Immobilized-Cell Technology

In order to extend the biocatalytic activities of the biotransformation processes and reduce the

frequency of producing cell mass and undesirable side products, immobilized-cell technology

has been successfully applied to the whole-cell biotransformation processes. In addition to the

three commercial immobilized whole-cell biotransformation processes shown in Table 10.1,

examples of immobilization of three different microorganisms for whole-cell biotransforma-

tions are shown below to demonstrate the broad application of the immobilized whole-cell

biotransformation processes.

10.4.2.1 Production of Acrylamide

The significance of the application of immobilized-cell technology in the production of

industrially important chemicals is exemplified by the production of acrylamide by immo-

bilized Escherichia coli cells containing nitrile hydratase. The immobilized Escherichia coli

cells convert acrylonitrile to acrylamide, yielding 6000 tons of acrylamide per year by this

process [28].

10.4.2.2 Production of (5R)-Hydroxyhexane-2-One by Immobilized Cells

Tan et al. [29] demonstrated the use of a plug flow reactor of immobilized Lactobacillus kefiri

cells for the synthesis of the intermediate (5R)-hydroxyhexane-2-one. This immobilized-cell

reactor operated at a maximum conversion yield of 100% and a selectivity of 95%. The

production of (5R)-hydroxyhexane-2-one was extended to an operation time of 6 days. During

this time (91 residence times), a space–time yield of 87 g L�1 day�1 and a productivity of

0:7 g gwet cell weight�1 were obtained.

10.4.2.3 Side-Chain Cleavage of b-Sitosterol

b-Sitosterol is an abundant and low-cost raw material for the production of pharmaceutical

steroids. 4-Androstene-3,17-dione, the precursor for the synthesis of corticosteroid hormones,

canbederived fromthe side-chaincleavageofb-sitosterol. Immobilizedcells ofMycobacterium

sp. NRRLB-3805 onCelitematrix (80–120mesh) was found to be effective in cleaving the side

chain of b-sitosterol (5 gL�1) with a molar conversion yield of 70% in 50 h [30].


10.4.3 Aqueous–Organic Two-Phase System

The issues of autotoxicity and product recovery associated with the whole-cell biotransfor-

mation processes can be addressed with the aqueous–organic two-phase system. Similar to the

effect of absorbing resins, aqueous–organic two-phase biotransformations offer the advantage

of controlling the concentrations of both substrate and product in the biotransformation

reaction, allowing higher productivities and product concentrations [14]. The two-phase

systems enable substrate transfer from the organic phase to the aqueous biocatalyst phase

in a long-term regeneration and equilibrium manner to avoid substrate toxicity and inhibi-

tion [14,31]. Similarly, the product preferentially and continually partitions into the organic

phase and away from the aqueous biocatalyst phase, thereby preventing product toxicity and

inhibition. Furthermore, as the product accumulates in the organic solvent phase, downstream

processing has already been partially implemented during the biotransformation process and,

therefore, simplified the in situ product isolation and recovery [14,32].

One of themain obstacles for whole-cell microbial transformation in an organic solvent is its

biocompatibility, which has led to screening for organic-solvent-tolerant microorganisms.

Numerous organic-solvent-tolerant microorganisms have been found and their tolerance

mechanisms have been reviewed [14,33,34]. Two-phase biotransformation systems have been

successfully implemented for the production of pharmaceutically relevant metabolites.

10.4.3.1 Production of Ethyl-(S )-4-Chloro-3-Hydroxybutanoate

The asymmetric reduction of ethyl-4-chloro-3-oxobutanoate to ethyl-(S)-4-chloro-3-hydroxy-

butanoate is efficiently carried out by Escherichia coli cells (containing carbonyl reductase)

growing in a water–n-butyl acetate two-phase system. n-Butyl acetate was selected as the

organic solvent to avoid substrate degradation and enzyme inhibition. Glucose and glucose

dehydrogenase are used for cofactor regeneration [35].

10.4.3.2 Production of a-Terpineol

A recombinantEscherichia coli strain containing the cloned limonene hydratase genewas able

to grow in a water–limonene two-phase system and converted limonene to a-terpineol [36].Limonene, a cost-effective and readily available monoterpene, served both as the substrate and

the neat solvent for the production of a-terpineol.

10.4.3.3 Production of (S )-2-Octonol

(S)-2-Octonol is an intermediate for the production of several optically active pharmaceuticals,

such as steroids and vitamins. The asymmetric reduction of 2-octanone to (S)-2-octonol by

baker�s yeast was inhibited severely by substrate and product concentration of 10mM and 6mM

respectively. Whole-cell biotransformation of 2-octanone in a water–n-dodecane biphasic

system yielded a high product concentration of 106mM with 89% ee in 96 h [37].

10.4.3.4 Production of (S )-2-Ethylhexan-1-ol

(S)-2-Ethylhexan-1-ol is a useful building block for the pharmaceutical, food, cosmetic and

chemical industries. The reduction of 2-ethylhex-2-enal to (S)-2-ethylhexan-1-ol by baker�s


yeast can be effectively carried out in a biphasic system of water–ether and resulted in high

yield (91%) and high ee value of >99% [38].

10.4.4 Genetic Engineering Approaches

By developing recombinantmicroorganisms, whole-cell biocatalysts have been created that can

compete against isolated enzymes. The overexpression of oxidoreductases and cofactor

regenerating enzymes leads to sufficient specific activities of these enzymes, so that side

reactions are negligible. Incomplete stereochemical purity of the products can often be corrected

by inhibiting or improving the synthesis of a specific enzyme inside the cells. The permeability

of the substrates into the cells can be overcome by expressing the required enzymes on the cell

surface. Listed below are a few examples to demonstrate the application of genetic engineering

techniques in improving the whole-cell biotransformation processes.

10.4.4.1 Increased Stereoselectivity of Reductase in Saccharomyces cerevisiae

To address the lack of stereoselectivity of Saccharomyces cerevisiae in conducting the

reduction of ketones to alcohols, the parent yeast strain was modified through genetic

engineering to selectively increase the amount of the desirable reductase inside the yeast

cells [39].Biotransformationwith the geneticallymodified strains using glucose or galactose as

carbon source and various b-keto esters as substrates showed that the ee of the products was

increased significantly. Another successful example to overcome the limitations of wild-type

Saccharomyces cerevisiae strain for stereoselective reductionb-keto esters is the application ofa recombinant Saccharomyces cerevisiae strain that overexpressed a stereospecific carbonyl

reductase and a cofactor regeneration enzyme [40].

10.4.4.2 Increased Efficiency of Aromatic-Ring Hydroxylating Dioxygenases

Owing to the cofactor requirements and increased efforts associated with the use of purified

enzyme components, biocatalytic applications employing aromatic-ring-hydroxylating dioxy-

genases (TDO and NDO) have been predominantly developed using whole-cell biotransfor-

mations [41,42]. These whole-cell biotransformations are typically facilitated by the inducible

overexpression of multicomponent dioxygenases in recombinant host strains, such as Escher-

ichia coli or Pseudomonas sp., which can be grown to high cell densities. These approaches

have been employed for the application of oxygenases in the production of multi-kilogram

quantities of chiral metabolites for the preparation of natural products, polyfunctionalized

metabolites, and pharmaceutical intermediates [15]. Two representative examples from the

above applications are the production of (�)-cis-(1S,2R)-indandiol by recombinant Pseudo-

monas putida strain overexpressing TDO [41] and the production of indigo by recombinant

Escherichia coli strain overexpressing NDO [42]. (�)-cis-(1S,2R)-Indandiol is a key interme-

diate for the production of HIV-1 protease inhibitor Crixivan. Indigo is an important

commodity chemical.

10.4.4.3 Developing a New Whole-Cell Biotransformation Process

for the Synthesis of Simvastatin

Simvastatin is an important cholesterol-lowering drug and is currently synthesized from the

natural product lovastatin via a tedious multistep chemical synthesis. A one-step, whole-cell


biotransformation process for the synthesis of gram quantity simvastatin with >90% conver-

sion yield and >98% purity using a recombinant Escherichia coli strain overexpressing

acyltransferase LovD has been reported recently [43]. Further genetic modification of the

Escherichia coli strain was performed by inactivating an undesirable enzyme that reduced both

the substrate conversion and recovery yields by hydrolyzing the substrate [44]. The improved

Escherichia coli strain has a lower substrate requirement and a faster rate of simvastatin

synthesis comparedwith the parent recombinant strain. In the improved strain, 99% conversion

of 6 g L�1 of simvastatinwith higher purity (devoid of hydrolyzed product)was achieved in less

than 12 h. While the economic feasibility of this process still requires further in-depth

evaluation, this example nevertheless demonstrates the significance of genetic approaches in

enhancing the efficiency of whole-cell biotransformation processes.

10.4.4.4 High Cell-Density Cultivation of Escherichia coliwith Surface-Anchored Transglucosidase

One of the disadvantages of whole-cell biotransformation is the mass transfer limitations

imposed by the cellmembrane,which leads to lower productivities. Development ofwhole-cell

biocatalysts displaying the target enzyme directly on the cell surface is one of the methods of

overcoming the substrate permeation barrier and to improve product yields. An application of

this technique was demonstrated by the production of arbutin by recombinant Escherichia coli

cells anchoring surface-displayed transglucosidase in a fed-batch process [45]. Arbutin is a

hydroquinone glucoside that inhibits tyrosinase activity in melanin biosynthesis and has

market potential in the cosmetics industry. The whole-cell biocatalysts showed a specific

activity of 501 nkat/g cell and produced 21 g L�1 of arbutin. Other examples include using

Pseudomonas syringae ice-nucleation protein as a carrier protein, and levansucrase [46],

carboxymethylcellulase [47] and organophosphorus hydrolase [48] have been successfully

displayed on Escherichia coli surface for use as whole-cell biocatalysts in the related

applications.

10.4.4.5 Designed Biocatalysts

Gr€oger et al. [49] demonstrated the use of ‘designed biocatalysts’ for efficient asymmetric

reduction of ketones (>90% yield and>99% ee) at high substrate concentrations and with no

external addition of cofactor. The designed biocatalyst was prepared by cloning alcohol

dehydrogenase (Lactobacillus kefir or Rhodococcus erythropolis) with high specific activity

(1000Umg�1) and glucose dehydrogenase (Thermoplasma acidophilum or Bacillus subtilis)

with high cofactor regenerating activity into Escherichia coli DSM 1445 host strain. This

designed biocatalyst demonstrated 95% conversion of 4-chloro-acetophenone at a concentra-

tion of 78 g L�1. This is an excellent example of a tailor-mademicroorganism for carrying out a

specific biotransformation with extremely high efficiency.

10.5 Conclusions

It is clear from the discussion in Section 10.4 that the advancements in biochemistry, molecular

cloning, random and site-directed mutagenesis, directed evolution of biocatalysts, metabolic

engineering and fermentation technology can circumvent the major technical problems


associated with thewhole-cell biotransformation processes. The genetic engineering approach

can be used in combinationwith other approaches to address the problems encounteredwith the

whole-cell processes. The number of tailor-made microorganisms created to address specific

problems of the biotransformation processes will be increased rapidly in the next few years.

There are many microorganisms, such as Clostridium spp. and cyanobacteria, which show

tremendous potential in biotransformation applications [50,51]. The special growth require-

ments of these microorganisms slow their development as useful microbial biocatalysts.

However, any experienced fermentation scientists should have no problem in developing these

microbial biocatalysts for whole-cell biotransformation applications. Furthermore, new

microbial biocatalysts will continuously be isolated from different environments due to the

tremendous microbial diversity in nature [52]. The discovery and development of new

microbial biocatalysts would lead to new whole-cell biotransformation applications. There-

fore, whole-cell biotransformations will likely play an increasingly important role in the

synthesis of pharmaceuticals and pharmaceutical intermediates.

One advantage of whole-cell biotransformation that has not been addressed adequately in

this chapter is the ability to modify compounds with complex structure, such as natural

products. Natural products are ideal substrates for biotransformation reactions since they are

synthesized in a series of enzymatic reactions by the whole cells. The modification of natural

products by biotransformation has been reviewed recently byAzerad [13] and amajority of the

modifications were carried out by whole-cell biotransformations. Additional examples of

modification of natural products by whole-cell biotransformations can also be found in the

review article by Patel [2]. Natural products are an important source of new drugs and new drug

leads [53]. The use of biotransformation, especially whole-cell biotransformation, in modifi-

cation of natural products for lead optimization and generating libraries of derivatives for SAR

and screening studies is important for the pharmaceutical industry.

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Biocatalysis for the Pharmaceutical Industry || Application of Whole-Cell Biotransformation in the Pharmaceutical Industry