Study Of Betaine And Sodium Citrate On The Metabolic Mechanism Of Vitamin B₁₂Producing By Pseudomonas Denitrificans

Betaine?N,N,N-Trimethylglycine?is the most common methyl group donor in the biosynthesis of vitamin B₁₂,which contains three methyl group.As intermediate metabolites,citric acid regulates important point in citric acid cycle,and people often regulate the metabolic flux distribution of the microbial fermentation process by means of adding exogenous sodium citrate.This paper reveals the informations of iconic difference metabolites and protein expression differences and others by metabolomics technology and proteomics technology.It mainly explains the mechanism of betaine in promoting the efficient synthesis of vitamin B₁₂2 by Pseudomonas denitrificans,and it explores the metabolic regulation of sodium citrate by enzyme analysis technique.?1?The mechanism of betaine inhibiting bacteria cell growth and promoting the synthesis of vitamin B₁₂2 is explained.Firstly,the effects of betaine on the cell growth and product synthesis had explored by shake flask fermentation experiment.The results show:betaine is essential component in the synthesis of vitamin B₁₂.However,if betaine is used by cell,it will cause the accumulation of high concentrations of glycine in the fermentation broth and seriously inhibites cell growth because of nitrogen repression effect.Further studying had explored the changes of intracellular metabolites of Pseudomonas denitrificans under the addition of 0 g/L and 10 g/L betaine by using metabolomics analysis technology based on GC-MS and combining with intracellular free amino acid analysis.The results show:betaine can significantly promote the generation of precursor amino acids related to the synthesis of vitamin B₁₂,such as glutamic acid and glycine which participate the synthesis of aminolevulinic acid,and methionine which participates in the methylation reaction.At last,using proteomics technology based on iTRAQ to explore the difference of protein expression of Pseudomonas denitrificans under the addition of 0 g/L and 10 g/L betaine.The results show:betaine addition can contribute to enhance oxidative phosphorylation of the cell,and significantly reduce the expression of the ribosomal protein;though analyzing the porphyrin metabolic pathways,it finds that the expression of protein related to the synthesis of vitamin B₁₂2 was up-regulate,especially uroporphyrinogen III methyltransferase,precorrin-4methyltransferase,precorrin-2 methyltransferase and so on.According to the above research results,it can be concluded that,in addition to being methyl group donor required for the synthesis of vitamin B₁₂,betaine can also promote the generation of precursor amino acid and the expression of enzyme protein which are closely related to the synthesis of vitamin B₁₂,thereby contributing to the biosynthesis of vitamin B₁₂.?2?The metabolic regulation of Pseudomonas denitrificans by sodium citrate is revealed.The effect of different concentrations of sodium citrate on the fermentation of Pseudomonas denitrificans is studied by shake flask experiment.The results show:0.3%-0.4%sodium citrate can effectively stabilize the pH at 6.5-7.0 in fermentation process,and it is helpful for cell growth and the synthesis of vitamin B₁₂.The determination results of organic acids show that,the addition of sodium citrate significantly reduces the accumulation of citric acid and succinate acid in the cell and accelerates the consumption of malic acid.It not only can avoid the pH of the fermentation liquid decreased rapidly,but also provide enough NADH to the synthesis of vitamin B₁₂.The enzymology test results of key enzymes in the pathway of glucose metabolism show that,exogenously added sodium citrate can significantly increase the activity of glucose-6-phosphate dehydrogenase in the HMP pathway,and simultaneously reduce the activity of pyruvate kinase and fructose kinase in the EMP pathway and the activity of citrate synthase in TCA cycle.So,sodium citrate can contribute to increase the metabolic flux of HMP pathway in Pseudomonas denitrificans,which can provide sufficient of NADPH for the synthesis of vitamin B₁₂,thereby contributing to improve the yield of vitamin B₁₂2 fermentation.