PART 2, SECTION 3 Screening of Microbial Diversity for ...shodhganga.inflibnet.ac.in/bitstream/10603/29814/16/17_section 3.pdf · The application of these microorganisms and their

PART 2, SECTION 3

Screening of Microbial Diversity for Preparation of Ethyl (S)-4-chloro-3-hydroxybutanoate, A High V aloe

Intermediate for Statio Drugs

Part 2, Section 3, Introduction

2.3.1 Introduction

Oxido-reductases have many applications in chemical synthesis, especially in

asymmetric synthesis of chiral compounds. The asymmetric reduction of carbonyl

compounds with microorganisms or enzymes is a useful method for the synthesis of

optically active alcohols. Optically active (R)- and (S)-ethyl or methyl 4-chloro .. 3-

hydroxybutanoate are very useful chiral building blocks in the synthesis of

pharmaceutical target compounds. e.g. the (R)-enantiomer is an important chiral

building block for the synthesis of(-)-macrolactin A (111), L-carnitine (112) and (R)

y-amino-~-hydroxybutyric acid (GABOB) (113), or can be converted into (+)

negamycin (114) or to a chiral2,5-cyclohexadienone synthon.191-195 On the other hand

the (S)-enantiomer is a key chiral intermediate in the enantioselective synthesis of

slagenins B (115) and C (116) and in the total synthesis of a class of HMG-CoA

reductase inhibitors atorvastatin (117), fluvastatin (118) and rosuvastatin (119) or can

be converted into a 1 ,4-dihydropyridine-type ~-blocker. 196-198

111

112 113

157

0 i

114


F

OH

F

117 118 119

A racemic mixture of such compounds, which have been chemically

synthesized, can no longer be used, especially for pharmaceuticals; thus, the

troublesome optical resolution of the racemic mixture using a conventional organic

synthetic process for such optically active substances is unavoidable. To overcome the

disadvantage of using a conventional organic synthetic process, microbial

transformation with enzymes possessing high stereospecificities has been applied to

the asymmetric synthesis of optically active substances.

Several microorganisms have been found to catalyze the reduction of ethy] 4-

chloroacetoacetate (COBE) to 4-chloro-3-hydroxybutanoate (CHBE). Enzymes

reducing CAAE to (R)- and (S)-CHBE were found to be produced by Sporobolomyces

salmonicolor and Candida magnoliae, respectively, and were characterized in

detail. 199-201 The enzyme from S. salmonicolor was a novel NADPH-dependent

aldehyde reductase and that from C. magnoliae also seemed to be a novel carbonyl

reductase. The application of these microorganisms and their enzymes to the practical

synthesis of chiral CHBE was investigated. 202-208 Efficient conversion of CAAE to

(R)- and (S)-CHBE with a satisfactory enantiomeric excess (e.e.) was attained in an

organic solvent-water diphasic system without further treatment of the cells or

fractionation of the enzymes (Scheme 39).

Scheme 39

0

CI~COOEt ~DP~NADP+ H'-..+'~OH _..;;:::--='"~·~ CI~COOEt

158

(R)

H'·~OH CI~COOEt

(S)


Of the two enantiomers, the (S)-enantiomer is the more common and more

easily available, whereas (R)-enantiomer is in greater demand yet less readily

available.209 Several synthetic routes have been developed to obtain both enantiomers,

comprising asymmetric synthesis and biocatalytic reduction, as well as a lipase

catalyzed kinetic resolution through ammonolysis of racemic 4-chloro-3-

hydroxybutanoate.192•193•210-212 Biocatalytic reduction appears to have attracted most

attention, several enzymatic systems having been studied with very good results,

particularly for the (S)-enantiomer.213

Of these, baker's yeast continues to play an important role in the attempts to

synthesize chiral compounds by biocatalysis because it is inexpensive, readily

available, and very easy to handle, and also because, in recent years, some examples

have been reported in which both enantiomers can be obtained from yeast reduction

of the same substrate by adopting appropriate reduction conditions such as the use of

organic solvents, the use of additives and the application of particular cell culture

conditions.214-216 The presence of multiple CAAE-reducing enzymes that show

opposite stereoselectivities in one microorganism has been reported in bakers' yeast.

It has been reported that when allyl bromide214 and allyl alcohol,217 two

commonly available chemicals, are added to baker's yeast reduction of 4-

halogenated-3-oxobutanoates, they interact with the enzymatic system of yeast by

changing the relative activity of L- and D-enzymes, thereby affording one isomer or

the enantiomer in good enantiomeric excess by reducing the substrate in suitable

conditions with the additive. By reducing methyl 4-chloro-4,4-difluoro-3-

oxobutanoate with baker's yeast in the presence of allyl bromide or allyl alcohol,

efficient stereochemical control of both (R)- and (S)-hydroxy derivatives was

obtained.

Ethyl (S)-4-chloro-3-hydroxybutyric acid ethyl ester can be produced by the

asymmetric reduction of ethyl 4-chloro-3-oxobutyrate with bakers' yeast. In such

systems, the optical purity of the product and the reaction yield are in general the two

indexes of reaction performance.218-219 The change of the medium composition in the

cultivation of yeast may enhance the reaction efficiency of the asymmetric

bioreduction. An effective approach that used the desirability function to optimize this

dual-response system, as well as the Taguchi's orthogonal array method and the

steepest ascent method have been employed crosswise to determine the optimal

conditions.220'221 This study investigated the feasibility of improving the

159


stereospecificity of yeast by the adjustment of the culture medium composition and

the reaction conditions. The investigation was performed systematically, using an

approach that integrates the Taguchi' s array method and the steepest ascent method.

A desirability function was applied to combine these two indexes as a single objective

function. The removal of peptone and malt extract from the YM medium increased

the yeast's stereoselectivity, without reducing the production of biomass. The medium

composition and the reaction conditions were then simultaneously optimized. The

resulting optimal conditions were 30 g/L glucose for cultivation, 12 g/L yeast extract,

a cultivation time of 12 h, 15 g/L glucose for reaction, 150 g/L yeast for reaction, a

reaction buffer concentration of 0.2 M and a buffer pH of 8.5. Compared to the one

before this study, the product's e.e. was improved from 82.1 to 92.3%, and the

reaction yield was enhanced from 77.3 to 82.3%.222

It has appeared in literature that Candida magnolia AKU4643 cells reduced

ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96%

enantiomeric excess (e.e.).223 As this yeast has at least three different stereoselective

reductases, the (S)-CHBE produced by this yeast was not optically pure.201•224

•225

From among these three enzymes, an NADPH-dependent carbonyl reductase,

designated as S 1, was purified and characterized in some detail. 201 The gene encoding

S 1 was cloned and sequenced and over-expressed in Escherichia coli cells. This E.

coli transformant reduced COBE to optically pure (S)-CHBE in the presence of

glucose, NADP+, and commercially available glucose dehydrogenase (GDH) as a

cofactor generator.226 Later, the construction of three E. coli transformants

coexpressing the S 1 from C. magnolia and GDH from Bacillus megaterium genes was

described and the reduction of COBE catalyzed by these strains was analyzed.

Previous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical

purity of 92% e. e. recommended an organic-solvent two phase system reaction for an

enzymatic or microbial reduction, because the substrate (COBE) is unstable in an

aqueous solvent and inactivates enzymes.227'228 The reductions of COBE to optically

pure (S)-CHBE by E. coli transformants in a water monophase system reaction has

been described and the possible use of this type of reaction system in industrial

applications discussed. From an industrial viewpoint, the results of this study make it

very difficult to determine which solvent system is best suited for effecting an

enzymatic COBE reduction. A two-phase system is an attractive procedure because it

is not necessary to control the COBE concentration in the reaction mixture. On the

160


other hand, a water mono-phase system is also attractive because it is possible to

accumulate a high concentration of CHBE without the need for special facilities to

handle a large quantity of organic solvent.

Several synthetic procedures have recently been developed to obtain (R)

CHBE. For example, asymmetric reduction of COBE by a microbial aldehyde

reductase in an organic solvent-water diphasic system, or by Escherichia coli strains,

which coexpress both the aldehyde reductase gene and the glucose dehydrogenase

gene.227-230 Although all of these procedures are successful in raising the yield and e.e.

of (R)-CHBE, typically also result in higher costs in much effort directed towards

screening different microorganisms. The baker's yeast, Saccharomyces cerevisiae, is

also generally used to reduce dicarbonyl compounds (in particular a and ~-diketones

and keto esters) to chiral alcohols with high e.e. However, products are formed at a

low rate.231'232 In one of the examples, the use of baker's yeast in stereoselective

reduction in which the introduction of allyl bromide to baker's yeast and interact with

the enzymatic system of yeast shifts the stereoselectivity of the reduction of COBE

toward the (R)-CHBE in 97% e. e., but only had 3 mmol L-1 of COBE was transformed

with a conversion of 98% to the corresponding (R)-CHBE has appeared in the

literature.233 Reduction of ethyl or methyl 4-chloro-3-oxobutanoate with baker's yeast

and in the presence of the additive allyl bromide or allyl alcohol were performed,

which yielded both (R)- and (S)-4-chloro-3-hydroxybutanoate in nearly optically pure

form and with total conversion ofthe substrate (Scheme 40).

Scheme 40

0

Cl~COOEt yeast ""-···''OH H~ .. ~OH allyl bromide ~r Cl~COOEt or Cl~COOEt

allyl alcohol (R) (S)

The technique is apparently not suitable for the large-scale production required

by industry. In another report, the results of microbial reduction of COBE to the

corresponding (R)-alcohol using permeabilization of fresh brewer's yeast cells by

CTAB in the presence of allyl bromide and using the ADH/2-propanol and

G6PDH/glucose-6-phosphate system for NAD(P)H-regeneration has appeared

(Figure 9). Ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) is obtained by

161


cetyltrimetylammonium bromide (CTAB) permeabilized fresh brewer's yeast whole

cells bioconversion of ethyl 4-chloro-3-oxobutanoate (COBE) in the presence of allyl

bromide. The results showed that the activities of alcohol dehydrogenase (ADH) and

glucose-6-phosphate dehydrogenase (G6PDH) in CTAB permeabilized brewer's yeast

cells increased 525 and 7.9-fold, respectively, compared with that in the non

permeabilized cells and had high enantioselectivity to convert COBE to (R)-CHBE ..

As one of co-substrates, glucose-6-phosphate was prepared using glucose

phosphorylation by hexokinase-catalyzed of CTAB permeabilized brewer's yeast

cells. In a two phase reaction system with n-butyl acetate as organic solvent and with

2-propanol and glucose-6-phosphate as co-substrates, the highest (R)-CHBE

concentration of 44 7 mM was obtained with 110-130 g/L of the CT AB permeabilized

cells at optimized pH, temperature, feeding rate and the shake speed of 125r/min. The

yield and enantiomeric excess (e.e.) of (R)-CHBE reached 99.5 and 99%,

respectively, within 6 h. 234

0

CI~COOEt~~~~~ H,_,,OH CI~COOEt

(R)

2-propanol

glucose-6-phosphate

Figure 9: Bioconversion of ethyl 4-chloro-3-oxobutanoate to ethyl 4-chloro-3-hydroxybutanoate by CTAB permeabilized fresh brewer's yeast cells in the presence of allyl bromide and co-factor regeneration using ADH/2-propanol and G6PD HI glucose-6-phosphate

Kataoka et.al. reported that a red yeast, Sporobolomyces salmonicolor,

produced an NADPH-dependent aldehyde reductase (AR), and that the enzyme can

catalyze the stereospecific reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to

ethyl (R)-4-chloro-3-hydroxybutanoate.235'236 Using AR isolated from S.

salmonicolor, a practical procedure for the production of (R)-CHBE was

developed.237'238 Furthermore, since the gene encoding AR was cloned from S.

162


salmonicolor cells and AR could be overproduced in Escherichia coli cells, the direct

use of E. coli cells expressing the AR gene (E. coli JM109 harboring pKAR) as a

catalyst for the efficient production of (R)-CHBE was shown to be more advantageous

than the use of AR isolated from S. salmonicolor.239'240 In these two systems,

commercially available glucose dehydrogenase (GDH) should be added to the

reaction mixture together with glucose and NADP+ as a cofactor regenerator, because

AR requires NADPH for COBE reduction. Then, it was shown that E. coli cells

overexpressing the GDH gene (E. coli JM109 harboring pGDA2) could be used as an

NADPH regenerator instead of commercially available GDH, in the production

system for (R)-CHBE involving E. coli JM 109 cells harboring pKAR. 241 The

asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-

chloro-3-hydroxybutanoate [(R)-CHBE] using this transformant as a catalyst has been

investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the

organic phase amounted to 1610 mM (268 mg/mL ), with a molar yield of 94.1% and

an optical purity of 91.7% enantiomeric excess. The calculated turnover number of

NADP+ to CHBE formed was 13500 mol/mol. Since the use of E. coli JM109 cells

harboring pKAR and pACGD as a catalyst is simple, and does not require the addition

of GDH or the isolation of the enzymes, it is highly advantageous for the practical

synthesis of (R)-CHBE. 242 The calculated turnover of NADP+, based on the amounts

NADP+ added and CHBE formed, was about 5100 mol L-1• When glucose is oxidized

to glucolactone by glucose dehydrogenase (GDH, E.C.1.1.1.47), NADP+ is reduced to

NADPH and thus could be coupled to the reduction of COBE to (R)-CHBE. NADPH

or GDH, or both of them together should be added into the biotransformation system

when a recombinant E. coli harboring the ALR gene is applied. 240'243 Recombinant E.

coli strains with two plasmids harboring ALR or GDH gene respectively, or with a

single plasmid harboring both ALR and GDH genes, have been constructed.242 In the

above cases, the results were encouraging with high productivity but the large size of

plasmid with two target genes or the co-existence of two different plasmids might

increase plasmid instability. Also the co-expression of ALR and GDH might be a

problem in bioprocess development. It is reported that the A TP regeneration system in

one strain can be used as the energy supplier to assist the biosynthesis of glutathione

in another different strain.244'245 Taking a lead from this, two plasmids were

constructed for ALR gene from Sporobolomyces salmonicolor ZJU0307 and GDH

163

Part 2, Section 3, Results and discussion

gene from Bacillus megaterium ZJU031 0, and then transformed into E. coli strains to

obtain E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), respectively.

The two recombinant strains were cultivated separately at optimized conditions and

then mixed up to asymmetrically catalyze the reduction of COBE to (R)-CHBE. The

former strain acted as catalyst and the latter functioned in NADPH regeneration. The

biotransformation was completed effectively without any addition of glucose

dehydrogenase or NADP+/NADPH. An optical purity of 99% (e.e.) was obtained and

the product yield reached 90.5% from 28.5 mM substrate.246 The principle of the two

strain system is similar to the A TP regeneration system between two microorganisms

(Figure 1 0).245 The permeability of cell membrane was further improved with the

addition of organic substrate (COBE) in the system. One GDH-expressing strain, E.

coli M 15 (pQE30-gdh031 0), was employed to recycle NADPH with exogenous

addition of glucose, and the intracellular NADPH in this strain would be secreted into

the medium; another ALR expressing strain, M15 (pQE30-alr0307), completed the

bioreduction reaction by utilizing intracellular NADPH in that strain, most of which

was from the trans-membrane transportation ofNADPH in the medium.

H ~OH

CI~COOEt

E. Coli MIS pQE30-alr0307

0

CI~COOEt

NADP Glucose

E.ColiMlS pQE30-gdh0310

gluconolactone

Figure 10: Biotransformation with two co-existing recombinant E. Coli strains

2.3.2 Present work: Results and discussion

Statin drugs are an important class of cholesterol-lowering drugs. About half

of statin drugs come from microbial sources, but others such as atorvastatin (117),

fluvastatin (118) and rosuvastatin (119) are synthesized. These drugs command a

multibillion dollar global market; atrovastatin, the active ingredient of Pfizer's

Lipitor, alone has sales value of over$ 12 billion per year.

164


Statin drugs have a (3R,5S)-dihydroxyhexanoate (120) side chain, which

accounts for above 25% of the compounds molecular weight, that should make it

worth at least $ 3 billion on prorated basis. The annual requirement for intermediate

120 exceeds 220 tons.

OH OH

X~COOEt

120 (X=CN, OBn, Cl etc)

OH

Cl~COOEt

121

No wonder that a number of fine chemical compames are competing to

develop economically viable routes for 120. Making side chain 120 is challenging

because it has two chiral centres and greater than 99.5% e.e., and 99% d.e. are

required. In the past, there has been some debate about whether chemocatalytic

processes are more cost competitive than biocatalytic routes for the side chain, but

presently biocatalytic processes for 120 appear economically and environmentally

more viable. A summary of the processes being developed is given in Figure 11.

Two major strategies are being followed, making either a shorter-chain with

only one chiral center or longer chain in which both chiral centres are targeted.

Reduction of ethyl 4-chloro-3-oxobutanoate is particularly interesting because of easy

availability of starting material at reasonably low price. Thus, ethyl (S)-4-chloro-3-

hydroxybutanoate (121) has become an important intermediate that commands a

market sales value of over $ 1 billion per year. Available biocatalysts for the

reduction of ethyl4-chloro-3-oxobutanoate have been discussed in Section 2.3.1.

A key issue in the biocatalyzed reduction of ethyl 4-chloro-3-oxobutanoate is

the requirement for NADPH-recycle system, which tends to not only increase the

cost but also complicates the process development. Whole cell systems offer

advantage as co-factor recycling can be avoided. But, so far, e.e. and/or productivity

of known microorganisms is less than optimal. Since different microorganisms

contain different enzymes, screening of microorganisms is desired to find a

microorganism having optimal properties. India is endowed with a rich microbial

diversity and screening of microorganisms isolated from various niches in India is

being currently pursued in our laboratory under a wider C.S.I.R. programme

"Exploration and Exploitation of Microbial Diversity of India".

165


Daicel 0 ADH or CR and OH

Cl~COOEt GDH or FDH )lr Cl~COOEt

Dowpharma 247

NC~CN

DSM248 0 0

Cl~+~

Bristol-Myers Squibb 249

OH Nitrilase)lr NC~COOH

OH OH

DERA )lr Cl~CHO

0 0 0 OH OH 0

BnO~~ ADH)Ir Bn~0~ ADH = alcohol dehydrogenase, CR = carbonyl reductase, FDH = formate dehydrogenase,

GDH = glucose dehydrogenase, DERA = deoxyribose-5-phosphate aldolase, Bn = benzyl

Figure 11: Routes targeted by various companies for side chain intermediate for statin drugs

2.3.3 Screening of microorganisms for enantioselective reduction of ethyl 4-

chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (121)

In view of the importance of ethyl (S)-4-chloro-3-hydroxybutanoate (121) as

intermediate of side-chain of statins, we screened a large number of microorganisms

for enantioselective reduction of ethyl 4-chloro-3-oxobutanoate. During the

screening, each organism was also tested for its ability to oxidize cinnamyl alcohol

and/or cinnamaldehyde with the aim of developing a cofactor regeneration system.

Each microorganism was also tested for reduction of ethyl 3-oxobutanoate and

cinnamic acid to get preliminary data on the nature of dehydrogenase activity of

various microorganisms. The results of the screening effort are summarized in Table

2.

166


Table 2: Screening of microbial strains for oxidoreductase activity

Strain* Ethyl-3- Ethyl 4-chloro-

Cinnamaldehyde Cinnamic Cinnamyl

oxobutanoate 3-oxobutanoate Acid Alcohol

BIMT 9012 - - - - -

BIMT 9013 + + - - -

BIMT 9014 - - - - -

BIMT 9044 - - - - -

BIMT9002 - - - - -

BIMT 9003 - + - - -

BIMT 9005 - - - - -

BIMT 9011 - - - - -

BIMT 9033 - - - - -

BIMT 9034 ++ + - - -

BIMT 9032 + - - - -

BIMT 9035 - - - - -

BIMT 9001 ++ ++ - - -

BIMT 9010 - - - - -BIMT 9012 - - - - -BIMT 9043 - - - - -

BIMT 9036 - - - - -

BIMT9037 - - - - -

BIMT9038 - - - - -

BIMT 9019 - - + - -

BIMT 9050 - - - - -

BIMT 9018 - - - - -

BIMT9055 + + - - -

BIMT 9030 - - - - -

BIMT 9075 - - + - -BIMT 9066 - - - - -BIMT 9058 + + - - -

BIMT9059 - - - - -

167





BIMT9073 ++ + - - -

FIMT 9046 + ++ - - -

FIMT 9047 - + - + -

FIMT 9017 + + - - -

FIMT 9024 + + - - -

FIMT 9029 + + - - -

FIMT 9028 - - - - -

FIMT 9005 + + - - -

FIMT 9021 + + - - -

FIMT 9025 + + - - -

FIMT 9061 + + - - -

NIO 069N - - - - -

NIO 066N - - - - -

NIO 058N - - - - -

NIO 053N - - - - -

NIO 055N + ++ + - -

NIO 072N ++ +++ - - -

NI0075N +++ +++ + - -

NI0059N + +++ - + -

706 - - - - -

334 + + + + -340 + +++ + + -

716 + +++ + - -

715 - - - + -343 + - - - -

709 + + - - -BIMT 9093 ++ ++ - - -BIMT 9092 ++ +++ - - -

168





BIMT 9086 ++ ++ + + -

BIMT9090 ++ +++ - - -

BIMT 9023 + + + - -

BIMT 9082 +++ +++ + - -

BIMT 9083 - - - - -

BIMT 9006 - - - - +

BIMT 9049 + ++ - + -

BIMT9094 ++ ++ - + -

BIMT 9053 - - - + -

BIMT 9005 - - - - -BIMT 9080 + + - + -

BIMT9079 ++ +++ - + -

BIMT 9152 + + - - -

BIMT 9140 + ++ - ++ -

BIMT 9163 + ++ - - -

BIMT 9128 + + - - -

BIMT 9120 - + - ++ -BIMT 9151 + ++ - ++ -

BIMT 9160 + ++ - ++ -

BIMT 9139 + + - ++ -

BIMT 9167 - + - ++ -

BIMT 9119 +++ ++ - + -

BIMT 9143 + ++ - - -BIMT 9144 + + - + -

BIMT 9141 - - - - -

BIMT 9136 - - - - -

BIMT 9108 - - - - -

BIMT 9124 - - - - -

BIMT9102 - + - + -

169





BIMT 9154 - - - + -

BirMT 9159 - - - + -

BIMT 9166 - + - + +

BIMT 9134 - - - - -

BIMT 9132 - - - - -

BIMT 9130 - - - - -

BIMT 9142 + - - + -

BIMT 9135 + + - + -

BIMT 9146 - - - + -

BIMT 9147 + + - + -

BIMT 9162 - - - + +

BIMT 9155 - - - - +

BIMT 9158 - - - - ++

BIMT 9168 - - + - ++

BIMT 9117 - - - + -

BIMT 9114 - - - + -

BIMT 9154 - - - - -

BIMT 9165 - - - - -

BIMT 9133 - - - - -

BIMT 9145 + - - - +

BIMT 9161 - + - - -

BIMT 9169 ++ + - - -BIMT 9113 + - - - -

BIMT 9119 - ++ - - -

BIMT 9100 - ++ - - -BIMT 9139 + - - - -BIMT 9112 - - - - -

BIMT 9136 - - - - -

BIMT 9137 - - - - -

170





BIMT 9125 - - - - -

BIMT 9116 - - - - -

BIMT 9117 - - - - -

BIMT 9105 - - - - -

BIMT 9110 - - - - -

BIMT 9138 - - - - -

BIMT 9115 ++ - - - -

BIMT 9119 + - - - -

BIMT 9027 - - - - -

BIMT 9062 - - - - -

BIMT 9400 - - - ++ -

BIMT 9064 ++ +++ - ++ -

BIMT 9900 + + - - -

BIMT 9067 + + - + -

BIMT 9074 - - - - -

BIMT 9054 + - - ++ -

101 - + - ++ -

148 + ++ - - -

49 - - - + -

137 + + - + -

82 - - - - -

125 + ++ + + -

77 + + - + -

71 + - - + -

123 - - - + -

139 + + - + -+++. htgh, ++. medmm, +.low, and -.no conversiOn

*Strain designation according to MTCC, IMTECH, Chandigarh

171


A number of bacterial and fungal strains were able to reduce both ethyl 4-

chloro-3-oxobutanoate and ethyl 3-oxobutanoate with low to high conversion rate.

The conversion rate for most organisms was similar for both substrates except for few

which showed some preference. For example, BIMT 9034, 9073, 9119, 9169 showed

some preference for ethyl 3-oxobutanoate and FIMT 9046, NIO 072, 059, 340, 716,

9090, 9092, 9049 and 9079 showed varying degree of preference for ethyl 4-chloro-3-

oxobutanoate. A few microorganisms were able to reduce aldehyde group, but with

low conversion rates. Some strains reduced cinnamic acid to cinnamyl alcohol, but

with low to moderate conversion rate. Oxidation of alcohol to acid or aldehyde turned

out to be much more difficult with very few strains showing the capability. But the

conversion rates were low to moderate making them unsuitable for cofactor

regeneration.

Enantiomeric excess of the alcohol formed from ethyl 4-chloro-3-

oxobutanoate and ethyl 3-oxobutanoate was tested for all positive strains, irrespective

of the level of conversion. Either none or low e.e. (10-45%) was obtained for ethyl 3-

hydroxybutanoate. However, the e.e. of ethyl 4-chloro-3-hydroxybutanoate (121) was

typically higher and ranged from 48-91%. Both (R)- and (S)-selectivity was observed.

The e.e. was determined by chiral HPLC using 250 x 4.6 mm chiralcel OB-H (Diacel,

Japan) column. Elution was done with isopropanol/hexane (4:96) as mobile phase at

flow rate 0.5 mL/min and detection was done at A.217 . The retention times for (R)- and

(S)- isomers were 31.6 min and 34.4 min respectively.

Unfortunately, the maximum e.e. obtained for the desired molecule, ethyl (S)-

4-chloro-3-hydroxybutanoate (121) was only about 91%. An organism, Penicillium

funiculosum, MTCC 5246 has already been described from our laboratory, which

gave 100% conversion and >97% e.e.184 Therefore, further efforts were directed

towards cofactor regeneration and reaction conditions optimizations using this

organism, increase in productivity being the main objective.

2.3.4 Enantioselectivity in Penicillium funiculosum whole-cell catalyzed

reduction of P-ketoesters

It has been reported previously, that Penicillium funiculosum reduced ethyl 3-

oxobutanoate (EOB) to give ethyl 3-hydroxybutanoate with high conversion rate, but

with poor e.e. of <1 0%. 184 In contrast, ethyl 4-chloro-3-oxobutanoate (ECOB) was

reduced at lower rate but e.e. of the product (S)-121 was >97%. This suggested the

172


presence of multiple dehydrogenases in Penicillium funiculosum with opposite

selectivity. In all likelyhood, EOB is a substrate for multiple dehydrogenases with

opposite selectivity, but ECOB is a substrate for a single dehydrogenase or more than

one dehydrogenase with similar selectivities. Activity staining studies were performed

to confirm these possibilities. Thus, cell-free extract prepared as described184 was

cooled to 4 °C and brought to 20% ammonium sulphate saturation. The precipitate

was discarded and supernatant brought to 75% saturation, maintaining the

temperature and pH at 4 oc and 7.0 respectively. The precipitated proteins, which

contained all the dehydrogenase activity was desalted using PD-1 0 column. Native

PAGE was performed as described in the experimental section.250 Gel was stained for

NADP-dependent dehydrogenase activity as described.251 Briefly, gel was washed

with Tris-Cl buffer, pH 8.0 and then immersed in a solution containing phenazine

methosulfate (0.05 mg/mL), nitroblue tetrazolium (0.3 mg/mL), NADP (0.5 mM),

glucose (20 mM), glucose dehydrogenase (15 units) and EOB (20 mM) in Tris-Cl

buffer, pH 8.0. The contents were incubated overnight when four white bands

appeared on purple background (Figure 12a). When the procedure was repeated with

ECOB as substrate, only one band appeared on purple background (Figure 13a). This

confirms that (a) EOB is a substrate for 4 different dehydrogenases and (b) ECOB is a

substrate for only one dehydrogense.

(a) (b)

Figure 12: Dehydrogenase activity staining with ethyl 3-oxobutanoate (20 mM) as substrate in presence (a) of glucose-glucose dehydrogenase (b) glutamic acid as cosubstrate

173


(a) (b)

Figure 13: Dehydrogenase activity staining with ethyl 4-chloro-3-oxobutanoate (20 mM) as substrate in presence of (a) glucose-glucose dehydrogenase (b) glutamic acid as co-substrate

2.3.5 Presence of ethanol, isopropanol or glucose as co-substrate has no effect

on Penicillium funiculosum whole cell catalysis

It has been reported that ethanol or isopropanol can be used as co-substrate for

cofactor regeneration. But the presence of 2-5% ethanol or isopropanol as co-substrate

in the Penicillium funiculosum catalyzed reduction of ECOB resulted in neither the

increase in consumption of ECOB nor the increase in the rate of the reaction (Entry 1,

2; Table 3). Similar results were obtained, when the reaction was done in the presence

of 2% glucose in the reaction medium (Entry 3; Table 3). In all cases, 0.2 mL of

ECOB (added in fractions of 50 J.!L each) was consumed in about 6 h. No further

reaction occurred when the time period was increased, indicating irreversible

inhibition of enzyme with ECOB.

2.3.6 Demonstration of the presence of glutamate-dehydrogenase in Penicillium

funiculosum

In order to find a co-substrate suitable for efficient cofactor regeneration in

whole cell catalysis by Penicillium funiculosum, presence of glutamate dehydrogenase

was tested using activity staining method. Native-PAGE was run and stained as

described above in Section 2.3.4 except that glucose-glucose dehydrogenase was

replaced with 20 mM glutamic acid in the staining solution with EOB as substrate and

glutamate as co-substrate. Four white bands appeared on purple background (Figure

174


12b ), which corresponded with the bands that appeared in the presence of glucose

glucose dehydrogenase. In addition, a faint purple band appeared just below major

white band (not apparent in Figure 12b). When ECOB was used as substrate and

glutamate as co-substrate, a single white band appeared on purple background (Figure

13b), which corresponded with the band that appeared in the presence of glucose

glucose dehydrogenase These results clearly show the presence of a glutamate

dehydrogenase in Penicillium funiculosum.

Table 3: Effect of different co-substrates and 2-phase system on the productivity in reduction of ECOB catalyzed by Penicillium funiculosum

Productivity

ECOB1'2

Time1 ECOB

Entry Co-substrate Reaction volume consumed consumed

mmolh-1L-1

1 Isopropanol

100 mL 0.2 mL 6h 2 to 5 mL 2.46

Ethanol 2 100mL 0.2mL 6h

2 to 5 mL 2.46

3 Glucose 2-3% 100mL 0.2 mL 5h 2.95

4 None 100mL 0.2mL 6h 2.46

Glutamic acid 5 100 mL 1.0 mL 10 h

2 equivalents 7.36

6 Glycine

100mL l.OmL 12 h 2 equivalents 6.21

7 None but 2-phase 50 mL (buffer-

buffer/n-butyl ether3 l.OmL 6h

solvent, 4:1) 24.53

Glycine + 2-phase 50 mL 8

buffer/n-butyl ether3 3.5 mL 12 h

(buffer-solvent, 4:1) 43.90 ..

1. No further reactwn occurred by addition of more ECOB or mcrease m time. 2. ECOB was added m portions of 50 flL 3. A solution of ECOB in n-butyl ether was slowly added to cell suspension in buffer.

2.3. 7 The effect of glutamic acid and glycine on the productivity of reaction

Penicillium funiculosum catalyzed reduction of ECOB was performed in the

presence of 2-equivalent of glutamic acid. Approximately 3-fold increase in the

productivity occurred (Entry 5: Table 3). We were pleased to note that a similar 2.5-

175

Part 2, Section 3, Conclusions

fold increase in the productivity occurred when glycine was used in place of glutamic

acid (Entry 6: Table 3).

2.3.8 Application of 2-phase system increased the productivity of reaction

Several solvents systems, such as hexane, ethyl acetate, n-butyl acetate and

n-butyl ether were tested for studying the reaction in 2-phase system. No reaction

occurred in the presence of ethyl acetate and n-butyl acetate. Enhancement in the

productivity occurred with hexane, but the solubility of substrate and product was

very low in this solvent. n-Butyl ether gave the best results. Thus, when a solution of

ECOB inn-butyl ether was slowly added to a suspension of cells in buffer (Entry 7:

Table 3), a 1 0-fold increase in the productivity occurred compared to monophasic

aqueous system. When the reaction was performed in the presence of 2 equivalents of

glycine as co-substrate, a substantial increase in the rate and amount of consumption

of ECOB occurred (Entry 8: Table 3). Thus, by using glycine as co-substrate and

applying 2-phase solvent system, nearly 18-fold increase in the productivity was

obtained.

2.3.9 Conclusions

Screening of a large number of microorganisms failed to improve e.e. of (S)

ethyl 4-chloro-3-hydroxybutanoate (121) as compared to a strain of fungi, Penicillium

funiculosum already described from our laboratory. By rational modification of the

conditions, the productivity was increased from 2.46 mmolh-1r 1 to 43.90 mmolh-1r 1.

An important feature to note is that no extraneous NAD(P) or cofactor regeneration

system has been added, instead, glycine was used as cosubstrate, thereby significantly

reducing the cost of production of 121. This became possible with the identification of

the presence of an amino acid oxidase in Penicillium funiculosum, which was used for

recycle of cofactor, NADPH, already present within the cells.

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