Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli
Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli
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DOI：
10.1016/j.ymben.2014.05.003
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发表时间：
2014-07-01
影响因子：
8.4
通讯作者：
Zhang, Xueli
中科院分区：
文献类型：
--
作者：
Zhu, Xinna;Tan, Zaigao;Zhang, Xueli
还原当量是有效合成目标产物的重要辅助因素。在提高大肠杆菌丁二酸酯产量的代谢进化过程中，两条还原等效保守途径被激活以提高丁二酸酯产量。丙酮酸脱氢酶对NADH抑制的敏感性被lpdA基因的三个核苷酸突变消除。在厌氧条件下，丙酮酸脱氢酶活性增加，提供了额外的NADH。转酮醇酶和可溶性转氢酶SthA的活性增加，激活了磷酸戊糖途径和转氢酶。这些数据表明，更多的碳通量通过磷酸戊糖途径，从而导致更多以NADPH形式存在的还原当量的产生，NADPH然后通过可溶性转氢酶转化为NADH来生产琥珀酸。在一株未经代谢进化的亲本菌株中进一步进行反向代谢工程，以验证激活这两条还原等价保守途径对提高琥珀酸产率的效果。激活丙酮酸脱氢酶可使琥珀酸产率从1.12增加到1.31，而激活磷酸戊糖途径和转氢酶则使琥珀酸产率从1.12增加到1.33。同时激活这两个途径可使琥珀酸产率达到1.5m o l/m o l(理论最大值的88%)，表明它们在提高丁二酸酯产率方面表现出协同效应。(C)2014年国际代谢工程学会。爱思唯尔公司出版。版权所有，
Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31 mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33 mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5 mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved,