Metabolic engineering of <i>Escherichia coli</i> for efficient biosynthesis of L-citrulline.

Linfeng XU; Wenwen YU; Xuewen ZHU; Quanwei ZHANG; Yaokang WU; Jianghua LI; Guocheng DU; Xueqin LV; Jian CHEN; Long LIU

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Metabolic engineering of Escherichia coli for efficient biosynthesis of L-citrulline.

Author: Linfeng XU ¹ ; Wenwen YU ¹ ; Xuewen ZHU ¹ ; Quanwei ZHANG ¹ ; Yaokang WU ¹ ; Jianghua LI ¹ ; Guocheng DU ¹ ; Xueqin LV ¹ ; Jian CHEN ¹ ; Long LIU ¹
Author Information

1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: Escherichia coli; L-citrulline; L-proline; feedback inhibition; metabolic engineering
MeSH: Citrulline/biosynthesis*; Escherichia coli/genetics*; Metabolic Engineering/methods*; Fermentation; NADP/biosynthesis*
From: Chinese Journal of Biotechnology 2025;41(1):242-255
CountryChina
Language:Chinese
Abstract: L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.