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

Abstract Aims 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. Methods and results 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. Conclusions 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.