Improved Batch Co-production Of SAM And GSH Based On Energy Metabolism Regulation

Both S-adenosylmethionine(SAM)and glutathione(GSH)are important active sulfur-containing compounds in vivo.SAM is a principal methylating agent in various intracellular transmethylation reactions and is biosynthesized from L-methionine and ATP by the catalytic enzyme of S-adenosylmethionine synthetase.GSH is an active tripeptide compound composed by glutamic acid,cysteine and glycine,and serves in many cellular processes including maintaining the appropriate redox environment of the organism,protecting the hydrogen sulfide of the protein,removing the cell's oxygen radical and so on.Because SAM and GSH have such important physiological functions in the cell,they have a wide range of applications in many fields such as clinical medicine,sports health care,food processing and cosmetics.In contrast to the former approach of mutation breeding or using genetic engineering technology to transform the material metabolism process,the energy metabolism process of the yeast strain was modified to improve the yield of energy-consuming synthetic compounds in this study.Candida utilis CCTCC M 209298 was used as the original strain whose energy metabolism genes were modified.The strains with high yield of SAM and GSH were selected and analyzed from the aspects of fermentation process,key enzyme activities,energy metabolism and gene expression levels.Finally,the mechanism of energy metabolism in the fermentation process was further explored by constant dissolved oxygen fermentation.The main results of this study are summarized as follows:In this study,energy metabolism associated genes of C.utilis CCTCC M 209298 were modified to select a lot of mutants of the parental strain.First,an expression vector which has expressing activity in C.utilis was constructed,and then the ATP6 gene in Arabidopsis thaliana and the gap gene encoded 3-phosphoglyceraldehyde dehydrogenase in Saccharomyces cerevisiae were ligated to the expression vector,and then converted into C.utilis CCTCC M 209298 to obtain the ATP6 gene overexpressing strain C.utilis ATP6 and the gap gene overexpressing strain C.utilis gap.The por1 gene knockout component was constructed by cloning the upstream and downstream fragments of mitochondrial membrane porin protein por1 gene in C.utilis and linking them to the marker gene.Next,the por1 knockout component was used to knock out the por1 gene of C.utilis CCTCC M 209298,C.utilis ATP6 and C.utilis gap and the mutant strains C.utilis ?por1,C.utilis(ATP6 ?por1)and C.utilis(gap ?por1)were successfully constructed.Finally,the performance of SAM and GSH synthesized by five mutants was investigated in flasks.The results showed that the intracellular contents of SAM and GSH increased only in por1 gene knockout strain C.utilis ?por1,and SAM and GSH production reached 358.1 mg/L,which was 14.2% higher than that of the control of parental strain.Batch co-production of SAM and GSH with the parental strain and mutants were carried out in a stirred fermentor.We found that co-production of SAM and GSH also increased significantly only in C.utilis ?por1,and SAM and GSH reached the highest titer of 243.1 and 294.1 mg/L,which were 28.4% and 39.1% higher than those achieved by the parental strain,respectively.With regard to efficient co-production of SAM and GSH,the maximum co-production of SAM and GSH reached 523.1 mg/L,which was 34.9% higher than that achieved by the parental strain.Then the batch fermentation processes were further analyzed to explain the mechanism of high yield.We found that por1 knock-out could increase the basal metabolic rate of energy and the ATP concentration in mitochondria,which increased the intracellular NADH and ATP levels.Meanwhile,it could significantly improve the activity of the key enzyme MAT for the biosynthesis of SAM and the key enzyme ?-GCS for GSH biosynthesis.The result of RNA-seq indicated that the por1 gene knockout increased the expression of many genes involved in ligand binding,catalysis and protease regulators in subcellular organosomes.In addition,it could also speed up the biosynthesis of fatty acid and slow down the metabolism of secondary metabolism,antibiotics and a variety of amino acid.These results provide evidence for the high yield of SAM and GSH at the molecular level.The batch fermentation process of SAM and GSH were further investigated under different constant dissolved oxygen(DO)conditions.The results showed that a controlled DO level of 30% with an initial agitation rate of 350 r/min was found to be the optimum for improved co-production of SAM and GSH.The maximum co-production of SAM and GSH reached 456.8 mg/L in the parental strain,which was 11.9% higher than that obtained at 350 r/min.In the mutant of C.utilis ?por1,the maximum co-production of SAM and GSH reached 514.2 mg/L,which was 11.8% higher than that obtained at 350 r/min.The mechanism of high yield of SAM and GSH under 30% was investigated.The results revealed that co-production of SAM and GSH was increased mainly by increasing intracellular ATP level and the activity of ?-GCS in the parental strain,which resulted in a significant increase in intracellular GSH content.However,according to the increased co-production of SAM and GSH in C.utilis ?por1,intracellular NADH level was enhanced in the late phase of batch fermentation,besides,intracellular ATP level and the activity of MAT were also improved.Base on the above analysis,the DO of 30% favored the formation of sufficient energy substrates,which resulted in increased co-production of SAM and GSH.