Targeted optimization of central carbon metabolism for engineering succinate production in Escherichia coli.
Targeted optimization of central carbon metabolism for engineering succinate production in Escherichia coli.
复制标题
靶向优化大肠杆菌工程琥珀酸生产的中心碳代谢
DOI：
10.1186/s12896-016-0284-7
复制
发表时间：
2016-06-24
影响因子：
3.5
通讯作者：
Zhao GR
中科院分区：
文献类型：
--
作者：
Zhao Y;Wang CS;Li FF;Liu ZN;Zhao GR
琥珀酸盐是一种重要的工业C4平台化学品，用于合成高附加值产品。由于经济和环境的优势，代谢工程和合成生物学在生物基生产琥珀酸盐方面投入了大量的努力。磷酸烯醇丙酮酸（PEP）前体用于葡萄糖的转运和磷酸化，并产生大量副产物，这是阻碍琥珀酸酯产量提高的关键障碍。在本研究中，我们没有删除与乳酸、乙酸和甲酸形成相关的基因，而是通过针对代谢节点PEP优化了工程大肠杆菌的中心碳代谢，以提高琥珀酸盐的产量，减少副产物的积累。结果通过删除ptsG、ppc、pykA、maeA和maeB，构建了琥珀酸产菌初始菌株，琥珀酸产率为0.22 mol/mol葡萄糖，比亲本菌株提高了2.1倍。然后，我们针对还原性TCA臂和PEP羧化，删除了sdh，并共同过表达了pck和ecaA，从而显著提高了1.13 mol/mol葡萄糖的琥珀酸产量。通过抗pykF sRNA调控pykF表达后，乳酸和乙酸的产量分别下降43.48%和38.09%。代谢网络厌氧化学计量模型表明，工程菌株对琥珀酸盐的碳含量显著增加，但对乳酸和醋酸盐的碳含量减少。优化菌株BKS15分批发酵产琥珀酸盐，比产率为5.89 mmol gDCW−1 h−1。结论本研究成功地以中心碳代谢的PEP为靶点，优化了琥珀酸酯的生产。
BackgroundSuccinate is a kind of industrially important C4 platform chemical for synthesis of high value added products. Due to the economical and environmental advantages, considerable efforts on metabolic engineering and synthetic biology have been invested for bio-based production of succinate. Precursor phosphoenolpyruvate (PEP) is consumed for transport and phosphorylation of glucose, and large amounts of byproducts are produced, which are the crucial obstacles preventing the improvement of succinate production. In this study, instead of deleting genes involved in the formation of lactate, acetate and formate, we optimized the central carbon metabolism by targeting at metabolic node PEP to improve succinate production and decrease accumulation of byproducts in engineered E. coli.ResultsBy deleting ptsG, ppc, pykA, maeA and maeB, we constructed the initial succinate-producing strain to achieve succinate yield of 0.22 mol/mol glucose, which was 2.1-fold higher than that of the parent strain. Then, by targeting at both reductive TCA arm and PEP carboxylation, we deleted sdh and co-overexpressed pck and ecaA, which led to a significant improvement in succinate yield of 1.13 mol/mol glucose. After fine-tuning of pykF expression by anti-pykF sRNA, yields of lactate and acetate were decreased by 43.48 and 38.09 %, respectively. The anaerobic stoichiometric model on metabolic network showed that the carbon fraction to succinate of engineered strains was significantly increased at the expense of decreased fluxes to lactate and acetate. In batch fermentation, the optimized strain BKS15 produced succinate with specific productivity of 5.89 mmol gDCW−1 h−1.ConclusionsThis report successfully optimizes succinate production by targeting at PEP of the central carbon metabolism.