Engineering Precursor Supply For Enhancing S-adenosyl-L-methionine Synthesis In Saccharomyces Cerevisiae

S-Adenosyl-L-methionine（SAM）involves many biochemical metabolic reactions in organisms.In vivo,SAM is synthesized by L-methionine（L-Met）and adenosine triphosphate（ATP）catalyzed by S-adenosylmethionine synthetase（MAT,EC 2.5.1.6）.At present,the existing industrial production methods include enzyme catalysis method and microbial fermentation method.With the exploration of the significance of SAM in the treatment of liver diseases,neurological diseases and arthritis,the market demand for SAM is also increasing.However,the current production methods exist problems such as low MAT enzyme activity,high production cost,low substrate conversion efficiency,and low production intensity,which limits the expansion of industrial production scale.With the development of molecular biology and genetic engineering technology,exploring novel strategies to construct high-yielding SAM engineered strains through metabolic transformation has become the key to breaking the production bottleneck.Among them,it’s very important for improving SAM producton and reducing cost by exploring precursor supply strategies to enhance the synthesis of SAM.In this study,we started by screening chassis cells suitable for SAM production and metabolic transformation,and then explored strategies to improve the supply of L-Met and ATP precursors.The main research contents are as follows:（1）In order to find the optimal chassis cell for SAM production,the gene sam2 encoding MAT derived from Saccharomyces cerevisiae was overexpressed in Escherichia coli,Pichia pastoris and S.cerevisiae,respectively.The results showed that the engineered strain C2obtained by overexpressing sam2 with an integrated plasmid in S.cerevisiae has the highest production evaluation.At the 24th hour of fermentation,the SAM titer reached 623.98 mg·L^-1,which was 5.72-fold that of the orginal strain S.cerevisiae CEN.PK 2-1C.And the productivity of C2 was 25.99 mg·L^-1·h^-1.Thus,we selected S.cerevisiae as the chassis cell for further reaserch.（2）In order to explore the strategy of increasing the production of SAM by increasing the supply of precursor L-Met,the effect of met6 gene encoding methionine synthase were explored which includes the intracellular content of precursor L-Met and the consequent SAM production level.The results showed that met6 can effectively increase the intracellular L-Met precursor,thereby increasing the SAM production.The highest concentration of L-Met in C26was 528.50 mg·L^-1,which was 1.33-fold that of C2,the SAM titer of C26 was 837.20 mg·L^-1,increased by 34.17%compared with the expression of sam2 alone,and the productivity was34.80 mg·L^-1·h^-1,increased by 34.20%.On this basis,the effects of met25 encoding acetylhomoserine sulfhydrylase and str2 encoding cystathionine-γ-synthase on L-Met supply and SAM production were further compared.It was proven that overexpression of str2 was more significant in regard to the intracellular accumulation of L-Met and SAM.Compared with C26,the intracellular accumulation of L-Met in C262 was significantly increased by 55%.The SAM titer reached 1,070.82 mg·L^-1and the productivity reached 44.60 mg·L^-1·h^-1 increased27.90%and 27.89%respectively.（3）In order to explore the strategy of increasing the production of SAM by increasing the supply of precursor ATP,the effect of the adk1 gene encoding adenylate kinase on the intracellular content of ATP and its effect on the production of SAM were explored.Compared with the engineering strain C262,the intracellular ATP content of C2621 reached 50.04 mg·g^-1increased by 42.88%,and the SAM titer reached 1,185.82 mg·L^-1 increased by 10.73%.On this basis,the effect of the vgb gene encoding Vitreoscilla hemoglobin（VHb）on the accumulation of intracellular ATP and SAM was investigated.The results showed that the intracellular ATP content of C2621v reached 70.88 mg·g^-1 increased by 41.64%.However the cell growth was significantly reduced.and the maximum biomass further decreased by 10.03%,and the OD₆₀₀only reached 12.73.While the intracellular SAM content increased slightly,the titer of SAM did not decrease.（4）In order to improve the SAM yield of the engineered strain C2621v under shaking flask conditions,the types and concentrations of carbon sources and nitrogen sources,and the concentration of Mg SO₄ and sodium citrate in the fermentation medium were investigated in this sutdy.The results showed that in the fermentation medium that contains sucrose 90 g·L^-1,peptone 30 g·L^-1,yeast powder 15 g·L^-1,Mg SO₄ 1.5 g·L^-1,and no sodium citrate,the SAM titer reached a maximum of 1,980.20 mg·L^-1,increased by 71.70%compared with before optimization,and which was 18.18-fold that of the orginal strain S.cerevisiae CEN.PK 2-1C,and the biomass also reached a maximum OD₆₀₀=23.19.