State Research Institute of Genetics and Selection of Industrial Microorganisms of the National Research Center "Kurchatov Institute", 1-st Dorozhniy pr., 1, 117545, Moscow, Russia.
State Research Institute of Genetics and Selection of Industrial Microorganisms of the National Research Center "Kurchatov Institute", 1-st Dorozhniy pr., 1, 117545, Moscow, Russia.
J Biotechnol. 2019 Mar 10;293:47-55. doi: 10.1016/j.jbiotec.2019.01.013. Epub 2019 Jan 26.
An Escherichia coli K-12 MG1655-derived strain was engineered for respiro-fermentative production of pyruvate from glucose under anoxic conditions, which is preferred for industrial-scale microbial synthesis of valuable chemicals. The pathways of anaerobic pyruvate dissimilation were blocked in the strain by the deletion of the ackA, pta, poxB, ldhA, adhE, and pflB genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was substituted by an alternative ATP-dependent system resulting from the overexpression of galP and glk upon deletion of ptsG. The channelling of pyruvate towards the oxidative branch of the TCA cycle under respiratory conditions was prevented in the strain due to the deletion of aceEF genes, encoding components of pyruvate dehydrogenase, while the operation of the entire reductive branch of the TCA cycle was interrupted by knocking out frdAB and sdhAB. Reoxidation of glycolytic NADH was ensured via anaerobic respiration with nitrate serving as an external electron acceptor. To enforce anaerobic ATP hydrolysis, an ATP-consuming futile cycle of pyruvate-oxaloacetate-malate-pyruvate was established in the strain by expressing the Bacillus subtilis pycA gene, encoding pyruvate carboxylase. In the presence of sufficient amounts of an external electron acceptor and CO source, the engineered strain was able to efficiently utilise glucose and convert it to pyruvate anaerobically with a yield of 1.73 mol/mol, amounting to 87% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient processes of bio-based pyruvate production.
一种源自大肠杆菌 K-12 MG1655 的菌株经过工程改造，可在缺氧条件下将葡萄糖进行(respiro-fermentative)呼吸发酵生产丙酮酸，这对于工业规模的微生物合成有价值的化学品是优选的。该菌株通过缺失 ackA、pta、poxB、ldhA、adhE 和 pflB 基因阻断了厌氧丙酮酸分解代谢途径。通过缺失 ptsG 并过表达 galP 和 glk，葡萄糖运输和磷酸化的磷酸烯醇丙酮酸依赖性磷酸转移酶系统被替代为替代的 ATP 依赖性系统。由于缺失编码丙酮酸脱氢酶组件的 aceEF 基因，在呼吸条件下，丙酮酸被阻止流向 TCA 循环的氧化分支，而整个 TCA 循环的还原分支则通过敲除 frdAB 和 sdhAB 而中断。通过以硝酸盐作为外部电子受体进行厌氧呼吸，确保了糖酵解 NADH 的再氧化。为了强制进行厌氧 ATP 水解，通过表达编码丙酮酸羧化酶的枯草芽孢杆菌 pycA 基因，在菌株中建立了丙酮酸-草酰乙酸-苹果酸-丙酮酸的 ATP 消耗无效循环。在有足够数量的外部电子受体和 CO 源的情况下，该工程菌株能够有效地利用葡萄糖并将其厌氧转化为丙酮酸，产率为 1.73 mol/mol，达到理论最大值的 87%。所实施的策略为开发高效的生物基丙酮酸生产工艺提供了潜力。
