Cofactor-Based Regulation For S-Adenosylmethionine Production

S-adenosylmethionine (SAM) is an essential regulatory compound in many biological reactions and an active small molecule widespread in organisms. Clinically, SAM is used in the treatment of arthritis, liver disease, depression and other diseases, and the effect is remarkable. At present, SAM is mainly prepared by microbial fermentation, but there are serious problems, such as low yield, insufficient supply of substrate L-methionine and ATP.Researchers tried to improve the capacity of strains for SAM production through metabolic engineering. However, SAM is not the terminal metabolites and the intracellular metabolic pathway of SAM is lengthy and complex. It’s hard to improve the SAM yield with a significant increase by manipulating the genes involved in main metabolic pathways.The cofactors of ATP/ADP, NADH/NAD+and NADPH/NADP+are key components of intracellular micro environment, and they involve in the amounts of biological reaction processes which linked metabolic pathways to form a complex network system. Eventually, carbon flow distribution is subjected to the form and the concentration of cofactor. Therefore, it is of great potential to regulate the synthesis of intracellular metabolites by cofactor engineering.We aimed to explain the mechanism of cofactor regulation for enhancing SAM production in this work. The form and concentration of cofactor were disturbed through gene recombination technique in Escherichia coli and Saccharomyces cerevisiae. Principles of cofactor on the product synthesis, energy metabolism and carbon metabolism were analyzed and summarized to provide a theoretical basis for the metabolic engineering of SAM producing strains. Main research work are as follows:1. The synthetic sRNA-based regulation system for cofactor regulation was developed. The cofactor level was regulated by interference gene transcription level. The red fluorescent protein and relative target binding sequence was introduced to testify the efficiency of the repression system. The fluorescence intensity detected by Flow Cytometer and the qPCR data confirmed the viability of sRNA-based repression system, and the repression efficiency could reach to 85%. The synthetic sRNA-based strategy was applied for the regulation of the intracellular ATP and NADPH concentration in order to enhance SAM production. The synthetic sRNAs of anti-proB, anti-glnA, anti-argB, anti-aroE, anti-argC, anti-proA, anti-ilvC and anti-proC were constructed. The qPCR data confirmed the decrease of transcription levels of target genes. The flask results showed that intracellular levels of ATP and NADPH were increased in recombinant strains, and SAM titer doubled comparing with the control strain.2. We constructed two kinds of NADPH regenerators of heterologous NADH kinase (Pos5p) and NADH kinase-like enzymes (combination of transhydrogenase PntAB and NAD kinase YfjB) to increase the availability of NADPH in SAM biosynthesis in E. coli. It was also proposed that the production of SAM is not only dependent on the absolute value of NADPH but also dependent on the NADPH/NADP+ratio. Modulating of Pos5p resulted in a superior SAM production and led to approximately 5.30 mg/L SAM (increased by 13 fold) in E. coli without L-methionine addition.3. NADPH metabolism in S. cerevisiae is relatively independent in mitochondrion and cytoplasm. Therefore, the effect of intracellular NADPH on the SAM production was studied in compartmentalized region in S. cerevisiae BY4741 based on the study of E. coli. The laser scanning confocal microscope confirmed the expression of NADH kinase in mitochondrion and cytoplasm separately. The results showed that the SAM titer with NADH/NAD ratio in cytoplasmic NADPH regeneration strain NBYSM-1 were significantly higher than that of mitochondrial NADPH regeneration strain NBYSM-2. However, NADPH/NADP ratio in NBYSM-1 was lower than NBYSM-2. These indicated that NADPH regulation for SAM production has stimulative effect, but the ATP supply is more important in SAM production.4. To study the effect of intracellular ATP on SAM synthesis in S. cerevisiae, a variety of different forms of ATP control systems were constructed on the basis of over expression of saml. The recombinant plasmids pRS425-PHXT7-noxEopt-THXT7, pRS425-PHXT7-vhb-THXT7, PRS425-PHXT7-ptxD-THXT7, and pRS425-PHXT7-fdh-THXT7 were constructed. The results showed that the activity of methionine adenosyltransferase is extremely important for the synthesis of SAM, and the intracellular SAM titer of sam2 expression strains doubles. Gene of vhb and ptxD have more significant role in promoting, SAM production. The intracellular concentration of SAM in the strain ABYSM-2 was the highest at 28 h, and it was improved by 67%, which was 54.92 mg/L, compared with the control strain. Metabolites were determined by LC-MS/MS combined with the analysis of the metabolomics. The difference of metabolites and amino acids concentration were significant, which contribute to different capacity for SAM production. In addition, it was found that higher levels of NADH and ATP significantly inhibited key enzymes in glycolysis and TCA cycle at the transcriptional level. The genes of tdh1,pyk2 and idh1were affected by NADH level significantly. The transcriptional level of gene zwfl in the pentose phosphate pathway was not affected in the strain that ATP level was improved, but it was reduced by NADH effect, which also affected the SAM production.