College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
Metab Eng. 2019 May;53:1-13. doi: 10.1016/j.ymben.2019.01.006. Epub 2019 Jan 24.
Medium-chain (C-C) chemicals are important components of fuels, commodities and fine chemicals. Numerous exciting achievements have proven reversed β-oxidation cycle as a promising platform to synthesize these chemicals. However, under native central carbon metabolism, energetic and redox constraints limit the efficient operation of reversed β-oxidation cycle. Current fermentative platform has to use different chemically and energetically inefficient ways for acetyl-CoA and NADH biosynthesis, respectively. The characteristics such as supplementation of additional acetate and formate or high ATP requirement makes this platform incompatible with large-scale production. Here, an artificial micro-aerobic metabolism for energy and carbon-efficient conversion of glycerol to MCFAs was constructed to present solutions towards these barriers. After evaluating numerous bacteria pathways under micro-aerobic conditions, one synthetic metabolic step enabling biosynthesis of acetyl-CoA and NADH simultaneously, without any energy cost and additional carbon requirement, and reducing loss of carbon to carbon dioxide-emitting reactions, was conceived and successfully constructed. The pyruvate dehydrogenase from Enterococcus faecalis was identified and biochemically characterized, demonstrating the most suitable characteristics. Furthermore, the carbon and energy metabolism in Escherichia coli was rewired by the clustered regularly interspaced short palindromic repeats interference system, inhibiting native fermentation pathways outcompeting this synthetic step. The present engineered strain exhibited a 15.7-fold increase in MCFA titer compared with that of the initial strain, and produced 15.67 g/L MCFAs from the biodiesel byproduct glycerol in 3-L bioreactor without exogenous feed of acetate or formate, representing the highest MCFA titer reported to date. This work demonstrates this artificial micro-aerobic metabolism has the potential to enable the cost-effective, large-scale production of fatty acids and other value-added reduced chemicals.
中链（C-C）化学品是燃料、大宗商品和精细化学品的重要组成部分。大量令人兴奋的成果证明，反β-氧化循环是合成这些化学品的有前途的平台。然而，在天然中心碳代谢下，能量和氧化还原限制限制了反β-氧化循环的有效运行。目前的发酵平台分别需要使用化学和能量效率低下的方法来合成乙酰辅酶 A 和 NADH。需要补充额外的乙酸盐和甲酸盐或高 ATP 的特性使得该平台与大规模生产不兼容。在这里，构建了一种人工微需氧代谢，用于甘油到 MCFAs 的能量和碳高效转化，以解决这些障碍。在微需氧条件下评估了许多细菌途径后，构思并成功构建了一个同时合成乙酰辅酶 A 和 NADH 的合成代谢步骤，无需任何能量成本和额外的碳需求，并减少了碳向二氧化碳排放反应的损失。鉴定并生化表征了来自粪肠球菌的丙酮酸脱氢酶，证明了其最适合的特性。此外，通过成簇规律间隔短回文重复干扰系统重新布线大肠杆菌的碳和能量代谢，抑制了天然发酵途径对该合成步骤的竞争。与初始菌株相比，该工程菌株的 MCFA 产量增加了 15.7 倍，并且在没有外源乙酸盐或甲酸盐的情况下，从生物柴油副产物甘油中生产了 15.67 g/L 的 MCFAs，这是迄今为止报道的最高 MCFA 产量。这项工作表明，这种人工微需氧代谢具有实现脂肪酸和其他增值还原化学品的经济高效、大规模生产的潜力。
