Metabolic Engineering of Escherichia Coli for Dihomomethionine Production

This study aimed to metabolically engineer Escherichia coli to produce dihomomethionine (DHM). Towards this aim, genes involved in DHM biosynthesis pathways in plant cells were introduced to E. coli. Utilization of E. coli as an engineering host has two potential advantages in this study. First, E. coli has a small number of methionine-consuming metabolic pathways, of which fluxes could be more easily directed to methionine chain-elongation pathways. Second, E. coli does not have cellular compartments, which might increase the synthesis efficiency of α-keto acids. In this work, production of DHM was detected and quantitated in E. coli by mass spectrometry. To optimize DHM production level in E. coli, three strategies were applied. First, optimal strength of promoters was determined by steady-state modeling, and various combinations of promoters were experimentally tested. Second, methionine-metabolizing pathways were sequentially disrupted by gene-knockout mutagenesis. Third, metabolic fluxes in the knockout mutants were modified by in silico flux balance analysis. Furthermore, culture conditions including temperature and medium compositions were optimized. In particular, effects of methionine supplementation on the production yield were investigated. These results represent the first report of DHM production in microbes and provide new insights into the feasibility of DHM production in microbial hosts.