以野生型大肠杆菌Escherichia coli W为出发菌株,利用Red同源重组系统分别敲除了乳酸脱氢酶基因(ldhA)、乙醇脱氢酶基因(adhE)、丙酮酸甲酸裂解酶基因(pflB)、丙酮酸氧化酶基因(poxB)和乙酸激酶基因(ackA),再通过无氧生长进化筛选过程,构建得到在厌氧条件下能有效生长,并以琥珀酸为主要发酵产物的重组大肠杆菌WS100(△ldhA,△adhE,△pflB,△poxB,△ackA)。利用15 L发酵罐进行厌氧发酵测定显示,经72 h发酵,菌体密度OD600最大值可提高至6.48,琥珀酸产量达到70.13 g/L,琥珀酸的生产强度为0.98 g/(L.h),葡萄糖-琥珀酸转化率为76%。发酵液中副产物含量低,乙酸含量为5.34 g/L,乳酸产量仅为0.15 g/L,未检测到甲酸和乙醇生成。结果表明,厌氧条件下,该工程菌可有效利用低营养成分的无机盐培养基,在不表达任何外源基因的条件下可稳定高产琥珀酸,具有极大的工业化开发前景。

In this study,metabolic pathway engineering using Red recombinase system was performed on Escherichia coli W,a wild type strain,for the production of succinic acid.In order to eliminate the by-product formation of E.coli fermentation,five genes leading for production of lactate(ldhA),formate(pflB) ethanol(adhE),and acetate(poxB and ackA) were deleted from the chromosome.The resulting mutant was then further selected through adaptive(metabolic) evolution for improved anaerobic growth.The evolved strain WS100 could produce succinate as the major fermentation product.In a 15 L fermentation under anaerobic condition using mineral salt medium,WS100 produced 70.13 g/L succinate from 100 g/L glucose in 72 h,with a volumetric productivity of 0.98 g/(L·h) and a yield of 76% based on sugar metabolism.Although small amount of acetate and lactate were still produced as the minor by-products,there was no detectable production of formate and ethanol.This result demonstrated the great potential of the engineered E.coli WS100 for fermentative production of succinic acid at large scale.