The Biosynthesis Of L-2,3-diaminopropionic Acid In E.coli

In recent years, E.coli has been widely used as a model organism in the production of various functional compounds, and amino acids have attracted much attention because of its wide medicinal value. In this paper,we used synthetic biology method to introduce L-2,3-diaminopropionic acid exogenous pathways into E.coli for the first time to realize its biosynthesis. The specific work is as follows:Three exogenous synthetic pathways were designed. The foreign gene was ligated into pETDuet-1 vector, and BL21 (DE3) was used as host to express exogenous enzyme. Three groups of exogenous enzymes were obtained by purification of nickel column, and the enzyme activity was tested in vitro. Among the three group exogenous enzymes, SbnA and SbnB derived from Staphylococcus aureus could use glutamic acid and phosphoserine as the substrate, and transformed them into the target product L-2,3-Diaminopropionic Acid, but no activity detected in the other enzymes.Transform the exogenous genes sbnA and sbnB into wild-type E. coli BW25113 successfully achieved L-DAP biosynthesis, and then via overexpression of the endogenous gene and knockout of the branch gene to further increase the yield of the target product. The titer of L-DAP was increased from the initial 178.4 mg·L-1 to 378.6 mg-L-1. The fermentation ability of BL21 (DE3) strain was tested and we optimized the strain.Finally, BW25113 was used as host bacteria.The metabolic pathways were divided into two modules, The foreign gene and the endogenous gene module. By module optimization to regulate the expression of endogenous gene and exogenous gene. Finally,The optimal combination is that use the high copy number of plasmid pZE12-luc to express exogenous gene and the pCS27 plasmid to express endogenous gene.The inhibitory effects of L-DAP on cell growth were investigated under different yeast concentrations. It was found that the addition of sufficient amount of yeast could restore normal growth of E.coli,indicating that L-DAP inhibited the synthesis of certain amino acids, and the addition of yeast powder made up for the deficiency of amino acid synthesis. The knockout of the ygeX gene did not relieve the decrease of L-DAP concentration in the culture medium, which indicated that ygeX gene expression in E.coli is weak and had no obvious degradation to L-DAP.We tried to use antisense RNA to regulate TCA cycle, Three different targeting sequences were designed to interfere with the eno gene,and the pCS-SerA-SerC-EnoasRNA vector was successfully constructed,but the production did not meet the expected results. At last a specific color reaction was established to determine the L-DAP concentration, and the reaction time is 30 min.