代谢工程改造大肠杆菌高效合成l-瓜氨酸

Metabolic engineering of Escherichia coli for efficient biosynthesis of l-citrulline

摘要:

l-瓜氨酸是一种非蛋白质氨基酸，在人体健康方面发挥着重要作用，具有很大的市场需求。尽管微生物细胞工厂已被广泛用于生物合成，但在l-瓜氨酸生物合成方面仍存在着遗传不稳定和效率低等挑战。本研究以大肠杆菌BL21(DE3)为出发菌株构建了一株高效、无质粒、无需诱导的l-瓜氨酸生产菌株。首先阻断l-瓜氨酸降解并解除反馈抑制，构建出l-瓜氨酸合成底盘菌株，其产量达到0.43 g/L。随后采用推拉抑制策略增强了l-瓜氨酸生物合成，使产量提高到6.0 g/L。接着，强化了NADPH合成和l-瓜氨酸转运系统以提高合成效率，最终l-瓜氨酸产量达到11.6 g/L。最后，在3 L发酵罐中进行分批补料发酵，l-瓜氨酸产量达到44.9 g/L。本研究为l-瓜氨酸的工业化生产奠定了基础，为其他氨基酸代谢网络的改造提供了思路。

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.