High-throughput Screening And Directed Evolution Of S-adenosylmethionine Synthase

S-adenosyl-L-methionine(SAM)is the active form of methionine,which participates in various metabolic reactions and play an important role.It is mainly used as a precursor by three key metabolic pathways: methylation,transsulfuration and transamidopropylation.Studies have shown that SAM has a good therapeutic effect on depression,osteoarthritis,liver disease,etc.At present,the output of SAM is very low and the price of SAM is expensiv.SAM is mainly obtained by fermentation of Saccharomyces cerevisiae,so a more efficient synthetic route is needed.In vitro enzymatic synthesis of SAM has the advantages of short reaction time,high conversion rate,high product concentration,easy separation and purification,and less environmental pollution.However,when the accumulation of SAM exceeds 1m M,most SAM synthases have product inhibition.Directed evolution is a green technology that can quickly obtain a large number of beneficial mutations,the key is a high-throughput screening method,while there is no suitable method for high-throughput detection of SAM.In order to obtain mutants with higher accumulation ability of SAM,we innovatively established a high-throughput screening method,which use glycine oxidase and glycine/sarcosine methyltransferase to detect SAM content,and optimized this method.Then,we used this method to screen the random mutation library with I303 V as the template,and obtained a mutant I303V/Q22R/M255 K with stronger SAM accumulation ability.Compared with the wild type,the SAM accumulation capacity of the mutant I303V/Q22R/M255 K was increased by about 86.3%,and the I303 V mutation was increased by about 56.8%compared with the wild type.Then,iterative point saturation mutation and combinatorial mutation were performed on positions 22 and 255 to obtain four dominant mutants,I303V/Q22 R,I303V/Q22 S,I303V/Q22 V and I303V/Q22 F.Finally,the enzymatic properties of wild type,I303 V,I303V/Q22 R and I303V/Q22 F were measured and their mutation mechanisms were analyzed.