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

作　　者：姜君逸 郭艺鸣 杨套伟[1,2] 饶志明[1,2] JIANG Junyi;GUO Yiming;YANG Taowei;RAO Zhiming(Key Laboratory of Industrial Biotechnology of Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2024年第9期3142-3157,共16页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(32370040,32100055);江苏省自然科学基金(BK20221537);江苏高校优势学科建设工程项目和江苏高校品牌专业建设工程项目。

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。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 byα-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 theα-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.

关 键 词：大肠杆菌 代谢改造 1 4-丁二醇 路径优化 

分 类 号：TQ923[轻工技术与工程—发酵工程]