关键词: 氯霉胺, 短链脱氢酶, 分子改造, 定点突变, 甲酸脱氢酶, 双酶共表达

Abstract:

2-amino-1-（4-nitrophenyl）-1,3-propanediol, commonly known as chloromycetin（ANP）, has a wide range of applications due to its two chiral centers and the good coordination ability of the O and N atoms in its structure. For responding to the many drawbacks of chemical synthesis such as high production cost, low atomic economy and high environmental pressure, here we aim to construct a new pathway for the synthesis of（1R）-ANP using p-nitro-α-acetamido-β-hydroxypropiophenone（p-NAH）as a substrate by combining chemical hydrolysis with biocatalysis. Firstly, 1-（4-nitrophenyl）-2-amino-3-hydroxypropiophenone（AHNA）was prepared by chemical hydrolysis of p-NAH, and a carbonyl reductase with catalytic activity for the hydrolysis product was screened. The catalytic activity of the enzyme was improved by molecular modification, and the enzymatic properties of a mutant mut-V112Y were investigated. Then, a dual enzyme co-expression or fusion expression recombinant strain containing mut-V112Y and formic acid dehydrogenase was constructed, and the catalytic efficiencies of the recombinant strains were verified. Finally, the catalytic reaction conditions were optimized and the preparative reactions were performed. The results showed that the chemical method can be used to hydrolyze p-NAH to AHNA, the screened short-chain dehydrogenase Lvchun calatyzed AHNA to（1R）-ANP, and a mutant mut-V112Y with 3.47-fold increase in catalytic efficiency was obtained by targeted mutation of carbonyl reductase, which presented good temperature and pH stability with an optimal temperature of 30℃ and an optimal pH of 7.5. A dual enzyme co-expression and fusion expression recombinant strain was successfully constructed, and the highest catalytic efficiency was found in strain mut-V112Y-CbFDH. The catalytic reaction conditions were optimized, 14.56 mmol/L（1R）-ANP was produced from 50 mmol/L AHNA in 30 min under optimal conditions, with a yield of 29.12%. The combination of chemical hydrolysis and biocatalysis can effectively catalyze the synthesis of（1R）-ANP from p-NAH, which provides a new way to synthesize optically pure ANP.

Key words: chloromycetin, short-chain dehydrogenase, molecular modification, site-specific mutagenesis, formic acid dehydrogenase, dual enzyme co-expression