Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

谷氨酸棒杆菌 生物化学 代谢工程 生物合成 大肠杆菌 发酵 精氨酸 瓜氨酸 代谢途径 生物 氨基酸 基因
作者
Shuai Jiang,Wang De-hu,Ruirui Wang,Chunguang Zhao,Qian Ma,Heyun Wu,Xixian Xie
出处
期刊:Metabolic Engineering [Elsevier]
卷期号:68: 220-231 被引量:21
标识
DOI:10.1016/j.ymben.2021.10.009
摘要

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.
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