State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, China.
Appl Microbiol Biotechnol. 2012 May;94(4):959-68. doi: 10.1007/s00253-012-3896-4.
In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce byproduct formation and increase succinate accumulation,pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates,such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus,the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l−1 with a yield of1.02 g g−1 total sugars during anaerobic fermentation.
在大肠杆菌 K12 中，琥珀酸不是木糖的主要发酵产物。为了减少副产物的形成和增加琥珀酸的积累，失活了编码丙酮酸甲酸裂解酶和乳酸脱氢酶的 pflB 和 ldhA 基因。然而，这些突变消除了细胞生长和木糖的利用。在细菌的厌氧生长过程中，有机中间产物，如丙酮酸，作为电子受体来维持整体氧化还原平衡。在这些条件下，细胞生长所需的 ATP 来自底物水平磷酸化。在大肠杆菌 K12 中，木糖转化为丙酮酸在厌氧发酵过程中仅产生 0.67 个净 ATP 每木糖。然而，大肠杆菌从两个丙酮酸中产生等量的乙酸盐和乙醇，这些反应产生一个额外的 ATP。将木糖转化为乙酸盐和乙醇将净 ATP 产量从每木糖 0.67 增加到 1.5，这可以满足木糖代谢所需的 ATP。pflB 缺失菌株不能将丙酮酸转化为乙酰辅酶 A，这是乙酸盐和乙醇生产的前体，也不能产生额外的 ATP。因此，双重突变消除了细胞生长和木糖的利用。为了提供足够的 ATP，在 ldhA、pflB 和 ppc 缺失菌株中过表达来自枯草芽孢杆菌 168 的磷酸烯醇丙酮酸羧激酶可显著增加细胞质量和琥珀酸的产量。此外，发酵含有高比例木糖和葡萄糖的玉米秸秆水解物在厌氧发酵过程中最终产生 11.13 g l−1 的琥珀酸浓度，总糖得率为 1.02 g g−1。
