Efficient Selection And Rational Transformation Of The Strain Producing Vitamin B₁₂

Cobalamin,also known as vitamin B₁₂,is a kind of vitaimin.It has been widely used in pharmaceutical,food and other industries due to its important physiological functions and.At present,VB₁₂ is mainly produced by fermentation of bacteria or archaea,such as Ensifer adhaerens,Pseudomonas denitrificans and Propionibacterium shermanii.In this study,an industrial VB₁₂producer E.adhaerens HY-1 was used as the starting strain to further improve its ability to produce vitamin B₁₂.Firstly,a high-throughput screening method for VB₁₂ was constructed.Then the E.adhaerens was mutagenized with atmospheric and room temperature plasma（ARTP）,and a mutant strain 46H-1 with high vitamin B₁₂ production was obtained.To further enhance the production of VB₁₂,the molecular biology was used to optimize the metabolic pathway of VB₁₂ in E.adhaerens.The genome sequencing and RNA-Seq analysis of E.adhaerens HY-1 were conducted.Through the results of genome sequencing,we obtained the metabolism-related genes of VB₁₂,and overexpressed these genes in HY-1.Through the result of RNA-Seq,we screened and obtained 65 promoters with different expression intensities.Finally,the key genes were expressed in combination with different strength promoters,which further improved the production of VB₁₂.The main research results of the thesis were as follows:1.A high-throuput screening method for VB₁₂ was established,and high-yielding strains were successfully.Based on the absorption peak of VB₁₂ at 361 nm,a high-throughput detection system for VB₁₂ was established.Then the lethality curve of HY-1 was determined with ARTP treatment,and the treatment time of 80s was chosed.The obtained mutant library was compared through 48 deep-well plates,and 29 high-yielding strains were obtained by the preliminary screening.Then through the shake flask fermentation,the high-yielding VB₁₂ and genetically stable strain 46H-1 was selected,of which VB₁₂ production reached 110.25 mg·L^-1.2.The key steps in the synthesis of VB₁₂ in HY-1 were determined by overexpression.By analyzing the results of genome sequencing,the synthesis method of VB₁₂ in HY-1 and related gene sequences were obtained.In this study,p BBR-MCS-5 was used as an expression vector,and the medium-strong promoter P_dnak was used to overexpress genes related to VB₁₂ synthesis.Finally,three key VB₁₂ synthesis genes were obtained,namely cob SV,cob Q and cob W.3.The reporter gene mCherry was used to characterize the promoter strength of endogenous genes in strain HY-1.Through the analysis of the RNA-Seq,the expression levels of all genes in HY-1 at different times of strain growth were obtained.We selected 65 promoters corresponding to genes with relatively stable expression and used the reporter gene mCherry to verify the expression intensity of these promoters at different times.4.The key steps in the synthesis of VB₁₂ in HY-1 were expressed in combination with promoters of different strengths to obtain high-yielding strains.Based on the result 3,three strong promoters,medium strong promoters and weak promoters were selected.These promoters were combined with the 3 key genes in result to form 27 combinations,which were all expressed in HY-1 using the vector p BBR-MCS-5.Finally,an engineered strain hmm(genotype is p BBR-P_{met H}-Cob SV-P_{ibp A}-Cob Q-P_mdh-Cob W)was obtained.It could produce 143.8 mg·L^-1 of VB₁₂,which increased 41.0%compared to the original strain.Finally,the production was increased to 171.18 mg·L^-1 through fed-batch fermentation.