Dynamic Pathway Regulation Using a Quorum-Sensing Circuit to Improve the Production of L-Homoserine in Escherichia coli.

Abstract

L-Homoserine has diverse applications in the fields of agrochemicals, pharmaceuticals, and animal feed; therefore, microbial fermentation using engineered cell factories has attracted widespread and intense attention. In this study, the nonauxotrophic strain for higher L-homoserine production was developed based on the previously constructed strain Escherichia coli HS. First, key genes involved in the biosynthesis pathways of essential amino acids were replenished with different strategies to address the growth deficiencies. Subsequently, carbon flux through the l-aspartate to L-homoserine was amplified by thrA overexpression. Furthermore, the supply of NADPH and ATP was optimized to synergistically enhance L-homoserine biosynthesis. Finally, the quorum sensing (QS) system esaI/esaR from Pantoea stewartii was introduced to dynamically regulate the carbon flux of l-threonine biosynthesis. And the results indicated that optimizing the regulatory efficiency of the QS system triggered autonomous downregulation of thrB during the high-cell-density phase, achieving a balanced metabolic flux competition between the l-threonine and L-homoserine biosynthesis pathways. The QS-regulated strain E. coli HS27/PA-P7QS produced 101.81 g/L L-homoserine with a yield of 0.41 g/g glucose after 96 h of fermentation in a 5-L bioreactor. This study demonstrates the feasibility of applying the QS system in E. coli for metabolic flux control, thereby providing novel insights into the rational design of amino acid biosynthesis pathways.