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.
Yasohara
Y
Y
Fine Chemical Research Laboratories, Kaneka Corporation, Takasago, Japan.
Kizaki
N
N
Hasegawa
J
J
Wada
M
M
Kataoka
M
M
Shimizu
S
S
D000090:Acetoacetates / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D000090:Acetoacetates; D004271:DNA, Fungal; C111982:ethyl 4-chloro-3-oxobutanoate; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C000614209:AKR7A5 protein, mouse