Metabolic Engineering Corynebacterium Glutamicum To Produce S-adenosyl-L-methionine

S-Adenosyl-L-methionine(SAM), a methyl group donor, is important in all living organisms. SAM involves in synthesis and metabolism of nucleic acids, proteins and phospholipids, and improves cell metabolism. It is the important substance to maintain cell physiological activities. SAM is mainly produced by yeast fermentation for more than 100 h, and the expensive L-methionine needs to be added. Moreover, the structure of yeast cell wall is complex, leading the difficulty for SAM extraction and purification. In order to overcome the disadvantages, in the current study Corynebacterium glutamicum was employed to biosynthesize SAM. C. glutamicum ATCC 13032 can accumulate a high level of L-methionine, precursor of SAM, and the genome sequence of C. glutamicum has been determined, therefore, it has theoretical value and application prospect to produce SAM by metabolic engineering C. glutamicum. In this study, a series of metabolic engineering strategies have been employed to increase SAM production level in C. glutamicum ATCC 13032 and L-isoleucine production strain IWJ001. The main work was described as follows:(1) SAM synthetases from Saccharomyces cerevisiae, C. glutamicum, Escherichia coli and Bacillus subtilis were overexpressed in E. coli JM109. The highest SAM synthetase activitie was observed in E. coli overexpressing SAM2 from S. cerevisiae and metK from C. glutamicum. Therefore, SAM2 and metK were cloned into the shuttle vector pDXW-8, and transformed into ATCC 13032, resulting ATCC 13032/pDXW-8-SAM2 and ATCC 13032/pDXW-8-metK, respectively. In ATCC 13032/pDXW-8-metK, MetK activity was 7.9 U·g-1, SAM accumulation reached 97 mg·L-1, which is 5.27 times higher than that in the control ATCC 13032/pDXW-8. In ATCC 13032/pDXW-8-SAM2, however, SAM synthetase activity and SAM production are the same levels to the control strain ATCC 13032/pDXW-8.(2) Firestly, RT-PCR and SDS-PAGE analysis showed that SAM2 gene could be transcribed in ATCC 13032/pDXW-8-SAM2, but not translated into proteins. Thus codons in SAM2 gene were optimized to be suitable for C. glutamicum, resulting SAM2 C. SAM2 C was overexpressed into ATCC 13032, resulting ATCC 13032/pDXW-8-SAM2 C. SAM2 C was successfully overexpressed in ATCC 13032/pDXW-8-SAM2 C, enzyme activity was 2.2 U·g-1 and SAM production reached 105 mg·L-1. Later the fed batch fermentation was employed. The results showed that ATCC 13032/pDXW-8-metK produced 316 mg·L-1 SAM after 36 h, while ATCC 13032/pDXW-8-SAM2 C produced 239 mg·L-1 SAM after 60 h. Then MetK and SAM2 C were purified, respectively. Optimum activity for both enzymes was observed at pH 8.5 and 35°C. Km values of MetK and SAM2 C for L-methionine were 0.51 and 1.04 mM, respectively; Km values of MetK and SAM2 C for ATP were 2.07 and 1.99 mM, respectively. The affinities of MetK and SAM2 C for ATP were similar, while the affinity for L-methionine MetK was higher than SAM2 C.(3) A series of C. glutamicum strains were constructed to synthesize SAM, using metabolic engineering. Firstly, WHQ103 was constructed from ATCC 13032 mutant WTQ102 by gene deletion. Genes mcbR encoding regulatory protein McbR, thrB encoding homoserine kinase and metB encoding cystathionine γ-synthase were deleted in WHQ103. WHQ103 produced 74.3 mg·L-1 SAM. Secondly, WHQ104 was constructed from WHQ103 by deleting gene Ncgl2640, and SAM production reached 95.4 mg·L-1. WHQ104/pJYW-4-metK-vgb was constructed by overexpression of metK and vgb encoding Vitreoscilla hemoglobin in WHQ104. WHQ104/pJYW-4-metK-vgb-metYX-homm-lysCm was constructed by further overexpression of methionine biosynthesis key genes metX, metY, feedback resistant genes homm and lysCm. WHQ104/pJYW-4-metK-vgb produced 181.8 mg·L-1 SAM after 36 h cultivation, while WHQ104/pJYW-4-metK-vgb-metYX-homm-lysCm produced 180.6 mg·L-1 SAM after 60 h cultivation. Addition of methionine did not improve SAM production, suggesting that not the methionine supply but the intracellular ATP level became the limited factor for SAM biosynthesis.(4) C. glutamicum strains that can produce both SAM and L-isoleucine were developed from L-isoleucine-producing strain IWJ001 by overexpression of metK and vgb. Compared to the control strain IWJ001/pDXW-8, SAM production increased 11.65 times in IWJ001/pDXW-8-metK, and increased 14.95 times in IWJ001/pDXW-8-metK-vgb. Strains IWJ001/pDXW-8-metK-vgb-pyk, IWJ001/pDXW-8-metK-vgb-mqo and IWJ001/pDXW-8-metK-vgb-mdh were also constructed by co-overexpressing pyk, mqo and mdh; but they did not produce more SAM than IWJ001/pDXW-8-metK-vgb. In 72-h flask cultivation, IWJ001/pDXW-8-metK-vgb produced 0.55 g·L-1 SAM and 4.24 g·L-1 L-isoleucine; in 72-fed-batch cultivation, IWJ001/pDXW-8-metK-vgb produced 0.67 g·L-1 SAM and 13.8 g·L-1 L-isoleucine.