Michihiko Kataoka

Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes

Abstract. The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58xa0M (430xa0g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25xa0M (208xa0g/l) (S)-CHBE in an aqueous mono-phase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600xa0mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes

Abstract The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610u2009mM (268u2009mg/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 13u2009500u2009mol/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.

Molecular Cloning and Overexpression of the Gene Encoding an NADPH-Dependent Carbonyl Reductase from Candida magnoliae, Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate

An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.

Stereoselective reduction of alkyl 3-oxobutanoate by carbonyl reductase from Candida magnoliae

Abstract The enantioselective reduction of alkyl 3-oxobutanoates by carbonyl reductase (S1) from Candida magnoliae was investigated. S1 reduced alkyl 4-halo-3-oxobutanoates to the corresponding enantiomerically pure ( S )-3-hydroxy esters. Escherichia coli HB101 transformant co-overproducing the S1 and glucose dehydrogenase from Bacillus megaterium , produced optically pure alkyl 4-substituted-3-hydroxybutanoates in a two-phase water/organic solvent system.

Diversity of microbial threonine aldolases and their application

Threonine aldolase catalyzes the reversible interconversion of certain β-hydroxy-α-amino acids and glycine plus the corresponding aldehydes. Various microbial threonine aldolases with different stereospecificities were found on extensive screening, and the genes encoding the proteins were cloned and heterogeneously overexpressed in Escherichia coli. By using recombinant threonine aldolases, an enzymatic resolution process was established for the production of optically pure β-hydroxy-α-amino acids. In addition, the threonine aldolase-catalyzed direct synthesis of β-hydroxy-α-amino acid from aldehyde and glycine is discussed.

Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug.

Abstract The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18u2009U/mg (purified enzyme 38.6u2009U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinsons disease.

Chiral alcohol synthesis with yeast carbonyl reductases

Abstract Synthesis of chiral alcohols, ( R )- and ( S )-4-chloro-3-hydroxybutanoate ethyl esters (CHBE), was performed through the enzymatic asymmetric reduction of 4-chloroacetoacetate ethyl ester (CAAE). The enzymes reducing CAAE to ( R )- and ( S )-CHBE were found to be produced by Sporobolomyces salmonicolor and Candida magnoliae , respectively. The enzyme of S. salmonicolor is a novel NADPH-dependent aldehyde reductase (AR) belonging to the aldo-keto reductase superfamily. The C. magnoliae enzyme also seems to be a novel NADPH-dependent carbonyl reductase. When AR-overproducing Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent–water two-phase system, 300 or 90 mg/ml of CAAE was almost stoichiometrically converted to ( R )- or ( S )-CHBE (>92% e.e.), respectively.

Purification and characterization of an aldehyde reductase from Candida magnoliae

Abstract An NADPH-dependent aldehyde reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue-Sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 33,000 on high performance gel-permeation chromatography and 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The substrate specificity of the enzyme was broad and resembled those of other aldo–keto reductases. The partial amino acid sequences of the enzyme showed that it belongs to the aldo–keto reductase superfamily. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding ( R )-alcohol, with a 100% enantiomeric excess. The enzyme was inhibited by 1 mM quercetin, CuSO 4 , ZnSO 4 and HgCl 2 . The thermostability of the enzyme was inferior to that of the ( S )-CHBE-producing enzyme from the same strain.

Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae

Multiple ethyl 4-chloro-3-oxobutanoate (COBE)-reducing enzymes were isolated from a cell-free extract of Candida magnoliae. A NADPH-dependent COBE-reducing enzyme, distinct from the carbonyl reductase and aldehyde reductase previously reported, was purified to homogeneity using five steps, including polyethylene glycol treatment. The relative molecular mass of the enzyme was estimated to be 86,000 on high performance gel-permeation chromatography and 29,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme catalyzed the reduction of COBE to the corresponding (S)-alcohol with a 51% enantiomeric excess. The substrate specificity of the enzyme was different from those of the other COBE-reducing enzymes of the same strain. The partial amino acid sequences of the enzyme showed that it belongs to the short chain alcohol dehydrogenase/reductase (SDR) family. This is the first report of multiple COBE-reducing enzymes with various stereoselectivities occurring in the same strain but belonging to different (super)families.

Lactone-ring-cleaving enzymes of microorganisms: their diversity and applications.

Microbial lactonohydrolases (lactone-ring-cleaving enzymes) with unique characteristics were found. The Fusarium oxysporum enzyme catalyzes the reversible and stereospecific hydrolysis of aldonate lactones and D-pantolactone (D-PL), and is useful for the optical resolution of racemic PL. The Agrobacterium tumefaciens enzyme hydrolyzes several aromatic lactones, and catalyzes the stereospecific hydrolysis of PL like the Fusarium enzyme, but its selectivity is opposite. The Acinetobacter calcoaceticus enzyme catalyzing the specific hydrolysis of dihydrocoumarin belongs to serine-enzyme family, and is useful for enantioselective hydrolysis of methyl DL-beta-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate. This enzyme also catalyzes the bromination of monochlorodimedon when incubated with H(2)O(2) and dihydrocoumarin.