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

• Published in: Metabolic engineering 2021-11, Vol.68, p.220-231
• Main Authors: Jiang, Shuai, Wang, Dehu, Wang, Ruirui, Zhao, Chunguang, Ma, Qian, Wu, Heyun, Xie, Xixian
• Format: Article
• Language: English
• Subjects: Dynamic regulation; Escherichia coli; L-citrulline; Quorum sensing; Recycling biosynthetic pathway
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2021.10.009

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. •A nonauxotrophic L-citrulline production strain was engineered in E. coli.•Reconstruction of a superior recycling biosynthetic pathway involved multi-module optimization.•A dynamic toggle switch was developed to achieve L-citrulline fermentation without extra L-arginine supply.•82.1 g/L L-citrulline with a yield of 0.34 g/g glucose and productivity of 1.71 g/(L h) was produced in a 5-L bioreactor.