The Effect Of Amino Acid Transporter System Modification On Extracellular L-methionine Accumulation In Escherichia Coli

The transformations of microbial amino acid transport systems were widely applied in the research of amino acid manufacture strain breeding,for the possible result of improving amino acids titers and extracellular accumulation.First of all,we compare the L-Met absorption rate of different E.coli strains deleting gene metN,metI or metQ of MetD transport system respectively.Then on this basis,YjeH and YeaS secretory systems of L-Met were strengthened in E.coli for more L-Met accumulation.At last,E.coli local CysB activator protein was replaced by that from Salmonella typhimurium and the resulting expression levels of relative genes in sulfur metabolism along with L-Met titers were studied.The main results in this research were as follows:The damage of MetD transport system by individual gene deleted could reduce the absorption of L-methionine.By the RED homologous recombination method,we knocked out gene metI,metN and metQ respectively,obtaining single-gene deletion mutants for L-methionine uptake capacity comparation.While the M12 mutant with metI gene damaged turned out to be a best performance with 16.7%decrease among all the effective mutants.Genomic integration expression of yjeH could promote the secretion of L-methionine.The mutant strain M12?pKK-yjeH/pET-ABY?was constructed leading to a L-Met titer of 0.21 g·L^-1.While the mutant M14 with gene yjeH integration harbouring recombinant plasmid pET-ABY produced L-methionine for 0.48 g·L^-1,which increased the titer and productivity for 128%and 85.7%compared with M12?pKK-yjeH/pET-ABY?.On the other side,the mutant M15 with gene yeaS integration harbouring recombinant plasmid pET-ABY produced L-methionine for 0.25 g·L^-1,which increased the titer for 38.9%compared with M12?pKK-yeaS/pET-ABY?,0.18 g·L^-1.Based on these results,an available strategy of L-methionine producing strain constructing has been built by weakening uptake pathway and enhancing secretion pathway.And with the uptake system knockout in E.coli,integration expression of YjeH system seemed to be more positive in L-methionine transportation instead of YeaS system.Overexpression related genes of cys operon could improve the supplement of sulfur and synthesis of L-methionine.As cys operon in sulfur metabolism was regulated by CysB activator protein,overexpression of cysC,cysK,cysM,cysP,cysU and cysE caused increased L-methionine titers up to 0.57,0.63,0.60,0.63,0.64 and 0.72 g·L^-1 respectively.This shows that in E.coli L-methionine metabolism,the total sulfur supplement would be improved by alleviation of cys operon regulation which also lead to a reinforce synthesis of L-methionine.Furthermore,CysB was replaced by exogenous gene cysB^T149P resulting to M16 mutant,improving the gene cysC,cysK,cysM,cysP,cysU expression level to 7.12,3.72,4.67,4.43and 4.24-folds compared with that of M14 mutant.While the L-methionine titer of final recombinant strain M16?pET-ABY?got to be 0.53 g·L^-1,increasing 15.2%compared with that of M14?pET-ABY?.