Metabolic Analysis And Intensification Of Vitamin B₁₂ Produced By Propionibacterium

Vitamin B₁₂ is one of the essential cofactors for many life activities in mammals.However,mammals are unable to synthesize vitamin B₁₂ by themselves,which is synthesized exclusively by a few microorganisms.Propionibacterium freudenreichii meets the requirements of GRAS of FDA and is used to produce vitamin B₁₂ by large-scale anaerobic fermentation.The vitamin B₁₂ produced can be directly used in the pharmaceutical and food industries.With the increasing demand of vitamin B₁₂,it is particularly urgent to produce vitamin B₁₂ by P.freudenreichii.Studies have shown that P.freudenreichii mediates the biosynthesis of vitamin B₁₂ by special metabolic pathways.Therefore,it is necessary to analyze its metabolic regulation from the perspective of whole cell level.However,changes in metabolites during vitamin B₁₂fermentation are still unknown.In this paper,the fermentation medium for producing vitamin B₁₂ was optimized by statistical method,taking P.freudenreichii as the research strain and vitamin B₁₂ production as the standard of measurement.The effect of medium composition on vitamin B₁₂ production was analyzed by metabolomics,and the yield of vitamin B₁₂ was increased by genetic engineering.The main research contents and results are as follows:A series of optimization strategy,based on statistical design methods,was used to optimize the fermentation medium for vitamin B₁₂ production.Four components,including glucose,yeast extract,KH₂PO₄ and glycine were determined to have significant effects on vitamin B₁₂production according Plackett-Burman design.Box-Behnken design was used to optimize the above four mediums and constructed a mathematical model to obtain an optimized medium for vitamin B₁₂ production.P.freudenreichii using optimized medium yielded vitamin B₁₂ of 8.32mg/L,which was 2.2 times higher than that of the original medium?3.81 mg/L?.Using the metabolomics profling method based on liquid chromatography-mass spectrometry,the difference of intracellular metabolites in the fermentation of P.freudenreichii in the original and optimized medium was studied.72 intracellular metabolites were identified by this method.Metabonomics analysis was valid to distinguish various culture phases(logarithmic phase,stable phase,and vitamin B₁₂ production phase)by principal components analysis of negative ions ESI-MS data.In addition,significant metabolites of three phase?46,26,and 23?were determined by orthogonal partial least squares discriminant analysis?OPLS-DA?.Pathway enrichment analysis,hierarchical Clustering Analysis and metabolic pathway comprehensive analysis were used to determine that TCA cycle,pentose phosphate pathway and amino acid metabolism were the metabolic pathways with significant influence on vitamin B₁₂ production.Finally,the endogenous genes?cobA,cbiK and cobR?,exogenous genes?RccobA and SmfldA?and endogenous gene cluster?cobalt transporter gene cluster?in the vitamin B₁₂synthesis pathway were respectively expressed by genetic engineering methods.Among them,the vitamin B₁₂ production of strain expressing cobalt ion transporter gene cluster was 10.23mg/L,which was 24.8%higher than that of the control strain?8.2 mg/L?.In this study,the culture medium optimization strategy and genetic engineering methods were used to improve the ability of vitamin B₁₂ production.Based on the metabolomics analysis of the fermentation process of the original and optimized medium,the regulation mechanism of vitamin B₁₂ production by P.freudenreichii was analyzed.It provides a valuable theoretical basis for the comprehensive analysis of the regulation mechanism of vitamin B₁₂ production by P.freudenreichii.