title: Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.
abstract: S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Wang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
authors.forename: Zhilai
authors.initials: Z
authors.lastname: Wang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Dou
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China. cl_zhou@cpu.edu.cn.
authors.forename: Changlin
authors.initials: C
authors.lastname: Zhou
mesh_terms: D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019149:Bioreactors; D002951:Citrates / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D005240:Feasibility Studies; D005285:Fermentation; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D008716:Methionine Adenosyltransferase / Q000235:genetics* / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D000077559:Sodium Citrate
chemical_list: D002951:Citrates; D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins; D000077559:Sodium Citrate; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D008715:Methionine; D007521:Isocitrate Dehydrogenase; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D008716:Methionine Adenosyltransferase
keywords: Co-expression; Intracellular ATP levels; Intracellular methionine availability; Methionine adenosyltransferase; Methionine synthase; S-adenosyl-L-methionine; Sodium citrate feeding