期刊
BIOCHEMICAL ENGINEERING JOURNAL
卷 107, 期 -, 页码 26-34出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bej.2015.11.016
关键词
S-Adenosyl-L-methionine; Biosynthesis; Intracellular acetyl-CoA levels; Recombinant DNA; Yeast; Metabolite over production
资金
- Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)
- State Key Laboratory of Bioreactor Engineering
S-Adenosyl-L-methionine (SAM), with diverse pharmaceutical applications, is biosynthesized from L-methionine and ATP. To enhance SAM accumulation in Saccharomyces cerevisiae CGMCC 2842 (2842), a new strategy based on yeast acetyl-CoA metabolism combined with introducing a methionine adenosyltransferase (metK1) from Leishmania infantum, was presented here. It was found that over-expressing acs2 (encoding acetyl-CoA synthase) and deleting mls1 (encoding malate synthase) increased SAM by 0.86- and 1.30-fold, respectively. To eliminate feedback inhibition of SAM synthase, a codon-optimized metK1 was introduced into 2842, and an increase of 1.45-fold of SAM was observed. Subsequently, metK1 and acs2 were co-expressed in the mls1 deleted strain, obtained the highly SAM-productive strain Ymls1 Delta GAPmK, and 2.22 g/l of SAM accumulated, which was 3.36-fold that in 2842. Moreover, the Ymls1 Delta GAPmK strain yielded 6.06 g/L SAM, which was 9.18-fold that in 2842, by fed-batch fermentation in a 10-L fermenter. Finally, the isolation and purification of SAM from yeast cell and preparation of SAM sulfate were preliminarily investigated. This study demonstrated that up-regulating acs2 and deleting mls1, which elevated intracellular acetyl-CoA levels, effectively enhanced the intracellular methionine biosynthesis. The elevated intracellular acetyl-CoA levels ultimately enhanced SAM accumulation, whereas the introduction of metK1 enhanced the redirection of acetyl-CoA to SAM biosynthesis in Ymls1 Delta GAPmK strain. (C) 2015 Elsevier B.V. All rights reserved.
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