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A synthetic gene encoding a Streptomyces l -proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l -proline to cis-3-hydroxy- l -proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy- l -proline.

Previously we have demonstrated the reduction of ethyl and t-butyl diketoesters 1 to the corresponding syn-(3R,5S)-dihydroxy esters 2a by Acinetobacter sp. 13874. The syn-(3R,5S)-dihydroxy ester 2a was obtained with an enantiomeric excess (e.e.) of 99% and a diastereomeric excess (de) of 63%. In this report, we identified a gene encoding desired ketoreductase III which catalyzed the diastereoselective reduction of diketoesters 1 to syn-(3R,5S)-dihydroxy esters 2a and describe cloning and expression of ketoreductase III into Escherichia coli. Cells or extracts of recombinant E. coli efficiently reduced the diketoester 1 to the corresponding syn-(3R,5S)-dihydroxy ester 2a in 99.3% yield, 100% e.e., and 99.8% de.

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP+-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD+-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.

A novel enantioselective N-carbobenzyloxy (Cbz)-cleaving enzyme was purified to homogeneity from Sphingomonas paucimobilis SC 16113. The gene encoding the protein has been identified and its complete DNA sequence was determined. The Cbz-cleaving enzyme was expressed in Escherichia coli.

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates and fine chemicals are in high demand both from pharmaceutical and agrochemical industries for the preparation bulk drug substances and agricultural products. In this review article, microbial/enzymatic processes have been described for the synthesis of chiral intermediates for anticancer drugs, antiviral agents, β3-receptor agonists, antihypertensive drugs, melatonin receptor agonists, anticholesterol drugs, and anti-Alzheimer’s drugs.

Three different biochemical approaches were used for the synthesis of ethyl 5-(S)-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2. In the first approach, ethyl 5-oxo-hexanoate 3 and 5-oxo-hexanenitrile 4 were reduced by Pichia methanolica (SC 16116) to the corresponding (S)-alcohols, ethyl (S)-5-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2, with an 80–90% yield and >95% enantiomeric excess (e.e). In the second approach, racemic 5-hydroxyhexanenitrile 5 was resolved by enzymatic succinylation, leading to the formation of (R)-5-hydroxyhexanenitrile hemisuccinate and leaving the desired alcohol 5-(S)-hydroxyhexanenitrile 2 with a yield of 34% (50% maximum yield) and >99% e.e. In the third approach, enzymatic hydrolysis of racemic 5-acetoxy hexanenitrile 6 resulted in the hydrolysis of the R-isomer to provide 5-(R)-hydroxyhexanenitrile, leaving 5-(S)-acetoxyhexanenitrile 7 with a 42% yield (50% maximum yield) and >99% e.e.

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine ϵ-aminotransferase. This enzyme catalyzed the oxidation of the ϵ-amino group of lysine in the dipeptide dimer N2-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required α-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to α-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine ϵ-amino transferase and glutamate oxidase, respectively. L-lysine ϵ-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine ϵ-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine ϵ-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine ϵ-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65–70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine ϵ-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine ϵ-aminotransferase. N-α-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine ϵ-aminotransferase.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l−1 and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD+, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l−1. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.

A key chiral intermediate, lactol 2[3aS(3aα, 4α, 7α, 7aα)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one, and the corresponding chiral lactone 3 were made in high optical purity by stereoselective enzymatic and microbial oxidation of the parent diol 1 . Horse liver alcohol dehydrogenase (HLADH) in the presence of nicotinamide adenine dinucleotide (NAD + ) and riboflavin oxidized diol 1 (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding lactol 2 and lactone 3 . Regeneration of NAD + required for the oxidation of diol 1 was carried out by the NADH-dependent alanine dehydrogenase and leucine dehydrogenase in the presence of alanine and leucine, respectively. Since expensive enzyme (HLADH) and cofactors (NAD + ) were required for the oxidation reaction, various microorganisms were screened for the ability to catalyze the stereoselective oxidation of diol 1 . Two organisms, Nocardia globerula ATCC 21505 and Rhodococcus sp. ATCC 15592, catalyzed the efficient oxidation of diol 1 to the corresponding chiral lactol 2 and lactone 3 . The reaction yield of 70% and optical purity of 96% was obtained for lactone 3 prepared from the oxidation of diol 1 by cell suspensions (10% w/v, wet cells) of N. globerula ATCC 21505. Substrate was used at 5 gl −1 concentration. A 10% (w/v, wet cells) cell suspension of Rhodococcus sp. ATCC 15592, after 120 h reaction period, produced lactol 2 in 12% yield and lactone 3 in 34% yield. An overall 46% reaction yield was obtained based on diol 1 oxidation. Substrate was used at 5 gl −1 concentration. Optical purity of 96.7% and 98.4% was obtained for lactol 2 and lactone 3 , respectively. Lactone 3 purified by slilica gel chromatography gave a specific rotation of +117° and enantiomeric excess of >99%.

A key chiral intermediate S (−)-4-chloro-3-hydroxybutanoic acid, methyl ester 2 was made in high optical purity by the stereoselective reduction of 4-chloro-3-oxobutanoic acid methyl ester 1 by cell suspensions of Geotrichum candidum SC 5469. A reaction yield of 95% and optical purity of 96% was obtained for 2 by glucose-, acetate-, or glycerol-grown cells (10% w/v) of G. candidum SC 5469. Substrate was used at 10 mg ml −1 concentration. The optical purity of 2 was increased to 99% by heat treatment of cell suspensions (55°C for 30 min) prior to conducting bioreduction of 1 at 28°C. Glucose-grown cells of G. candidum SC 5469 have also catalyzed the stereoselective reduction of ethyl-, isopropyl-, and tertiary-butyl esters of 4-chloro-3-oxobutanoic acid and methyl- and ethyl esters of 4-bromo-3-oxobutanoic acid. A reaction yield of > 85% and optical purity of > 94% were obtained. NADP-dependent oxidoreductase responsible for the stereoselective reduction of β-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of the purified enzyme is 950,000. The purified oxidoreductase was immobilized on Eupergit C and used to catalyze the reduction of 1 to 2. The cofactor NADP required for the reduction reaction was regenerated by glucose dehydrogenase.

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.