中文摘要：

为了高效生产L-苹果酸，首先在大肠杆菌w3110中敲除ldhA、poxB、pflB和pta-ackA基因积累丙酮酸，为L-苹果酸合成提供前体，并且通过苹果酸酶的引入构建L-苹果酸一步合成路径，将丙酮酸转化为L-苹果酸. 在此基础上，敲除frdBC、fumB和fumAC阻断L-苹果酸代谢路径，并结合pos5基因的表达对胞内辅因子路径进行优化. 结果表明：（1）ldhA、poxB、pflB和pta-ackA基因的敲除能有效地提高丙酮酸产量到20.9 g/L；（2）苹果酸酶突变及过量表达使得L-苹果酸和琥珀酸产量分别提高了87.2%和31.6%，达到1.46 g/L和3.25 g/L；（3）通过敲除frdBC、fumB和fumAC，L-苹果酸产量增加到3.42 g/L；（4）pos5基因的表达降低了胞内NADH/NAD＋比率，增加了NADPH含量，最终突变菌株Escherichia coli F0921的L-苹果酸产量达到9.34 g/L. 因此，通过苹果酸酶构建L-苹果酸生物合成路径提高L-苹果酸的生产是可行的，结果可为代谢工程改造大肠杆菌生产L-苹果酸提供了新的研究思路.

英文摘要：

The aim of our study was to engineer Escherichia coli to produce L-malate. The pool of pyruvate was first increased by knocking out the genes ldhA, poxB, pflB, and pta-ackA in E. coli w3110, and the one-step L-malate synthesis pathway was constructed via malic enzyme. Furthermore, the L-malate consumption pathway was disrupted by knocking out frdBC, fumB, and fumAC, and the cofactor level was optimized by overexpressing pos5. The pyruvate production was increased to 20.9 g/L with knockout of ldhA, poxB, pflB, and pta-ackA, and the titers of L-malate and succinate were respectively increased to 87.2% and 31.6% with overexpression of C490S. The titer of l-malate was further improved to 9.34 g/L in E. coli F0921 with knockout of frdBC, fumB, and fumAC, and optimization of the cofactor level. In summary, the one-step L-malate synthesis pathway was successfully constructed in E. coli to produce L-malate by malic enzyme, providing a new method to produce L-malate.