表达细胞色素b562及分子改造L-氨基酸脱氨酶提高全细胞转化法合成丙酮酸效率

Improvement of the Efficiency of Pyruvate Production by Escherichia coli Whole-Cell Biocatalyst through Expression of Cytochrome b562 and Protein Engineering

丙酮酸广泛用于制药、农业化学和化学工业。通过两种策略提高生物转化合成丙酮酸效率。首先,通过表达细胞色素b562提高黄素腺嘌呤二核苷酸(FAD)合成的效率,使反应时间由27 h减少到21 h,生产率提高了28.5%。其次,通过饱和突变技术对L-氨基酸脱氨酶(pm1)进行定向进化提高其催化能力,三突变体E418A/V438I/L278I催化合成丙酮酸产量为25.58 g/L,相比对照菌株提高了44.60%。结果表明,运用饱和突变技术和表达pm1伴侣蛋白(细胞色素b562)分别提高pm1催化能力与FAD合成效率能有效提高全细胞转化法合成丙酮酸的效率。

Pyruvate is widely used in pharmaceutical,agrochemical and chemical industries.Two strategies were used to improve the efficiency of bioconversion to pyruvate.First,the efficiency of adenine dinucleotide(FAD)synthesis was improved by expressing cytochrome b562,reducing the reaction time from 27 h to 21 h and increasing the productivity by 28.5%.Secondly,the directional evolution of L-amino acid deaminase(pm1)was achieved by site-saturation mutagenesis to improve its catalytic ability,and the yield of pyruvate in the triple mutant E418A/V438I/L278I was 25.58 g/L,which was 44.60%higher than that of the control strain.All the results showed that the efficiency of pyruvate produced by E.coli whole-cell biocatalyst could be effectively increased by the improvement of pm1 catalytic ability and FAD synthesis efficiency using site-saturation mutagenesis and expressing pm1 chaperone(cytochrome b562).