代谢工程改造大肠杆菌从头合成1,4-丁二醇

Metabolic engineering of Escherichia coli for de novo synthesis of 1,4-butanediol

摘要:

1,4-丁二醇是一种重要的中间体，广泛应用于化工、农业、医药等领域。本研究将酶工程和代谢工程相结合，构建了一条以葡萄糖为底物生产1,4-丁二醇的新途径。首先，通过数据库挖掘设计了一条包含α-酮酸脱羧酶(α-ketoglutarate decarboxylase, SucA)、羧酸还原酶(carboxylate reductase, Car)、乙醇脱氢酶(alcohol dehydrogenase, YqhD)的新型催化途径，引入底盘细胞W3110 (K-12)后，实现了1,4-丁二醇的从头合成。为进一步提高该路径的合成效率，敲除了乳酸脱氢酶(lactate dehydrogenase A, LdhA)、丙酮酸甲酸裂解酶(pyruvate formate lyase B, PflB)基因，阻断旁路代谢途径；强化表达柠檬酸合酶(citrate synthase, GltA^R163L)，增加α-酮戊二酸代谢通量；强化底盘细胞中关键辅酶NADPH合成量并替换强启动子强化sucA、car、yqhD基因表达量，改善了1,4-丁二醇合成前体的供给效率。最终，重组菌株摇瓶发酵48 h最高合成770 mg/L的1,4-丁二醇，在5 L发酵罐上发酵60 h ，1,4-丁二醇产量达4.22 g/L，得率为12.46 mg/g葡萄糖。本研究设计了一条新的1,4-丁二醇从头合成路径，与已报道的路径相比，该路径无需乙酰辅酶A参与，避免了副产物乙酸的积累，同时避免了氨的添加，为代谢工程改造生产1,4-丁二醇及其高附加值衍生产品提供了一种新的思路。

Abstract:

1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltA^R163L) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA, car, and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.