Identification and engineering efflux transporters for improved L-homoserine production in Escherichia coli

This study aimed to functionally identify the potential L-homoserine transporters in Escherichia coli, and to generate the promising beneficial mutants by targeted directed evolution for improving the robustness and efficiency of microbial cell factories.By constructing a series of gene deletion and overexpression strains, L-homoserine tolerance assays revealed that RhtA was an efficient and major L-homoserine exporter in E. coli, whereas RhtB and RhtC exhibited relatively weak transport activities for L-homoserine. Real-time RT-PCR analysis suggested that the expression levels of these three target mRNAs were generally variably enhanced when cells were subjected to L-homoserine stress. Based on in vivo continuous directed evolution and growth-couple selections, three beneficial mutations of RhtA exporter (A22V, P119L, and T235I) with clearly increased tolerance against L-homoserine stress were quickly obtained after two rounds of mutagenesis-selection cycles. L-homoserine export assay revealed that the RhtA mutants exhibited different degrees of improvement in L-homoserine export capacity. Further studies suggested that a combination of these beneficial sites led to synergistic effects on conferring L-homoserine-resistance phenotypes. Moreover, the introduction of RhtA beneficial mutants into the L-homoserine-producing strains could facilitate increased amounts of L-homoserine in the shake-flask fermentation.In this study, we provided further evidence that RhtA serves as a major L-homoserine exporter in E. coli, and obtained several RhtA beneficial mutants, including A22V, P119L, and T235I that contributed to improving the L-homoserine resistance phenotypes and the production efficiency in microbial chassis.