产L-苹果酸重组大肠杆菌的构建

Construction of recombinant Escherichia coli for L-malic acid production

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。

Based on the synthesis pathway of succinic acid in E.coli B0013-1050,the synthesize pathway of L-malic acid was constructed by using Red homologus recombination combined with Xer/dif recombinase system to delete the gene fumB,fumC and maeB from chromosome,and a recombinant strain E.coli 2030 was obtained.L-malic acid of 12.5 g/L with yield of 52.1% was achieved in a 15-L bioreactor. The main by-products, including pyruvic acid and succinic acid were analyzed.To increase the yield of L-malic acid, the malate dehydrogenase encoded gene mdh from Aspergillus flavus was integrantly expressed as the recombinant strain and a recombinant strain E.coli 2040 was obtained. The concentration and the yield of L-malic acid increased to 14 g/L and 60.3% in a 15-L bioreactor, respectively.