Metabolic Engineering To Synthesize Methionine

Methionine(Met)is an essential amino acid for humans and animals,which has important physiological and biochemical functions.Most plants and microorganisms can synthesize Met,but the yield is low.In different organisms,the Met synthesis pathway has diverged to some extent.The previous studies on the synthesis of Met have made great progresses,but they are basically concentrated in the microorganisms.Furthermore,the output is still not enough to meet the needs of industrialization.Therefore,in this study,we tried to redesign and combine the Met synthesis pathways in different types of organisms(plants and microorganisms)to obtain a combined Methionine produced strain using metabolic engineering methods.The specific research results as follows:First,according to the differences of Met synthetic pathway in plants and microorganisms,the cystathionine-?-synthetase genes CGS and S-Methyl Methionine(SMM),which contains cysteine Methyltransferase(HMT)and Methionine Methyltransferase(MMT)were overexpressed in Escherichia coli W3110.CGS,which catalyzes O-phosphate homoserine to cystathionine,transfers the flux of threonine synthesis to Met.In this study,CGS was over-expressed and resulted in the strain ZM1-C.After 48 hours of shake flask fermentation,the yield of Met reached 146 mg/L,which is 2 times that of the wild type.Similarly,the HMT and MMT were overexpressed and the strain ZM1-HM was gotten,in which the yield of Met increased to 112 mg/L.And then in the strain ZM1-CHM,CGS,HMT and MMT are co-overexpressed,and the Met titer reached 287mg/L,which is 4 times higher than that of wild type.Therefore,CGS,HMT and MMT in plants are of great value for increasing Met production in microorganisms.Secondly,the internal Metabolic regulation of E.coli also seriously affects Met synthesis.In this study,CRISPR-Cas9 technology was used to modify its internal Metabolic pathways.For example,met J(encoding the repressor of Met synthesis),lys A and thr C in the competitive pathway of lysine and threonine synthesis pathway,and suc CD were knocked out.Meanwhile,the coding genes of the key regulatory enzyme aspartate kinase were also mutated to the feedback-insensitive lys C* and thr A*.After all of these genetic modification,the strain ZM15-C was produced and the Met titer reached 386 mg/L.Finally,the CGS-HMT-MMT combination in plants was overexpressed in the ZM15 strain(ZM15-CHM).The Met titer in strain ZM15-CHM reached 676 mg/L in a 48-hour shake flask fermentation.With the addition of lys C*,the Met shake flask fermentation titer of the strain ZM15-CHML can reach 635 mg/L.After the cell disruption,the maximal production of Met is 804 mg/L in ZM15-CHML.Therefore,the combination of metabolic pathways between different species can effectively improve the synthesis efficiency of Met.At the same time,the combination of metabolic engineering used in this study will also provide a reference for the synthesis of other compounds.