Metabolic Engineering Transforms E.coli To Produce ?-Aminoadipate

?-Aminoadipic acid(AAA)is a non-protein amino acid.AAA is a key biosynthetic precursor of ?-lactam antibiotics such as penicillin and cephalosporin.It's also used for chemical synthesis of antirheumatic and carcinostatic methotrexate derivatives and used as terminal modifiers of physiologically active peptides(such as peptide antibiotics and peptide hormones).Currently,AAA is manufactured by chemical synthesis.However,the processes encounter problems such as low yield and high cost.In this study,we first realized the de novo synthesis of AAA in E.coli,and the gradual increase in production was achieved through systematic metabolic engineering.1.Construction of the downstream pathway from lysine to AAA:Introduced lysine ketoglutarate reductase(Lys1)and saccharopine dehydrogenase(Lys9)from S.cerevisiae and ?-aminoadipate semialdehyde dehydrogenase(reAASADH)derived from R.erythropolis into E.coli,and production of AAA was achieved by feeding precursor lysine.The expression level of the enzymes was optimized through genes modules optimization,the optimized plasmids combination was transformed into BW25113 for fermentation in M10 medium,and 249.93 mg·mL-1 of AAA was synthesized de novo.2.Boosting the supply of lysine:By knocking out the genes(cadA and ldcC)encoding lysine decarboxylase,the degradation of lysine was reduced.By enhancing the expression of the rate-limiting enzymes aspartate kinase(LysC)and 4-hydroxy-tetrahydrodipicolinate synthase(DapA)in the diaminopimelate(DAP)pathway,AAA titer was improved to 453.73 mg·mL-1.By replacing the native promoters of lysC and dapA on the chromosome with the strong T7 promoters,an integrated strain was obtained,and AAA titer was improved to 1.88 g·L-1.By introducing the multifunctional enzyme Ddh from C.glutamicum to replace the four enzymatic steps pathway in E.coli,AAA titer was further improved to 2.35 g·L-1.3.Facilitate the product efflux:By introducing the glutamate efflux protein MscCG derived from C.glutamicum,the product output was accelerated,and the final titer was improved to 2.96 g·L-1.