Studies On Improving The Biosynthesis Of S-adenosyl-L-methionine By Metabolic Engineering In An Industrial Saccharomyces Cerevisiae Strain

S-adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulphuration and polyamine synthesis in all living cells, and it’s also an effective cure for liver disease, depressive syndromes and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, metabolic engineering technology was used to optimize the pathways involving SAM synthesis in a industrial Saccharomyces cerevisiae strain ZJU001.SAM2 was overexpressed by intoducing the mutiple-copy integretive plasmid pYMIKP-SAM2 into ZJU001 and the enzyme activty of SAM synthetase was increased, which was responsible for the improved SAM production. In lOL-tank fermentation, the recombined strains could produce 8.81 g/L SAM, which was 27.1% more than its parent strain ZJU001.In order to have an insight into metabolic networks involving SAM synthesis in Saccharomyces cerevisiae, haploid yeast strain BY4741 was taken as the object of this study firstly. Considering the substrates in SAM synthesis, location where SAM accumulates and the pathways of SAM degradation, four mutants were constructed, in which genes of PMRI(P-type ATPase), GLC3(glycogen branch enzyme), PHO85(cyclin-dependent kinase) and SPE2(SAM decarboxylase) were disrupted respectively. It was found that deletion of either GLC3or SPE2 could benefit SAM accumulation in BY4741.Inorder to improve the stability of gene disruption in ZJU001, an effective gene disruption method for industrial diploid yeast strains was invented. Using this method, GLC3, SPE2, and two key genes in ergosterol synthesis, ERG4 and ERG6, were disrupted in ZJU001 respectivly. Besides, a transcription factor GAL11 was overexpressed in ZJU001 too. SAM Productivities of five engineerd strains were measured. Data showed that SAM production of GLC3 mutant and SPE2 mutant increased 20.1% and 12.4% respectively, comparing to their parent strain ZJU001.Mark-rescure gene disruption technology was used for disrupting both GLC3 and SPE2 in ZJU001.Besides, SAM2 was overexpressed in the final engineered strain, ZJU001-GS-SAM2. In flask medium, ZJU001-GS-SAM2 could produce 1.14 g/L SAM, which was 0.7-fold more than its parent strain ZJU001. It idicated that modification of the three pathways showed synergitc effect on promotion of SAM production.SAM productivitis in 1 OL-tank were further measured among constructed strains using fed-batch fermentation strategy with feedback control. Experiment data showed that ZJU001-GS-SAM2 produced the most SAM, which was as much as 10.32 g/L. Base on the former fermentation data, a Pseudo-exponential feeding strategy was constructed. When the specific growth rate was set as μ=0.125h-1, the biomass of ZJU001-GS-SAM2 could grow up to 121.0 g DCW/L and the SAM podution reached 12.47 g/L.In this study, metabolic engineering technology was used to optimize metabolism network involving SAM synthesis in the yeast industrial strain ZJU001. And by this means, productivity of SAM was greatly enhanced in this industrial strain, which laid a solid foundation for SAM production on industrial scale.