High-yield Strain Screening And Metabolic Regulation Component Mining Of Vitamin B₁₂

Vitamin B₁₂ is an essential nutrient for human growth and development and is widely used in medical,feed,food and other fields.The domestic and international market is increasing.The synthetic route of vitamin B₁₂ is very complicated and requires more than 30 steps of enzyme catalytic reaction.At present,industrial production can only be obtained by microbial fermentation.Through mutation breeding and high throughput screening,we obtained three strains with different production capacity of vitamin B₁₂.By detecting their intermediate metabolites,We speculate that the accumulation of ALA and the conversion of HBA to HBAD are positively correlated with the production of vitamin B₁₂.Therefore,we overexpressed the hem A gene and cob B gene and detected the production of vitamin B₁₂ by shake flask fermentation.Sinorhizobium meliloti 320(S.meliloti 320)was isolated and identified in our previously study.It contains all the genes of vitamin B₁₂ synthesis pathway.However,it lacks regulatory elements that effectively promote gene expression in S.meliloti 320.In this paper,we constructed plasmids harboring P_Aor P_B from S.meliloti 320 based on a promoter-probe vector with GFP as a reporter protein and measured expression levels.Then,compared them with P_{ara A}promoter from S.meliloti 320,they both had higher levels of induced expression and lower basal expression than P_{ara A}.The effect of glucose or sucrose on the expression of these three promoters also was determined.Glucose obviously repressed expression of these three promoters and the expression of P_{ara A} was the lowest.The expression level of P_Ais the highest and is 1.13-fold than that of P_{ara A},although sucrose obviously repressed expression of P_A by34.8%while improved the expression of P_{ara A} by 16%.Then,overexpressed hem A gene and cob B gene using P_A promoter in S.meliloti 320,and found the VB₁₂ production of the engineered strain increased by 10.9%and 19.36%,respectively,compared with S.meliloti 320,It also enriches the tool library that can be used to analyze S.meliloti 320.Sinorhizobium meliloti 320 has good industrial production potential of vitamin B₁₂,and currently lacks effective tools for gene editing.The development of CRISPR/Cas9 technology provides technical support for the genetic transformation of Sinorhizobium meliloti 320.In this paper,we construct CRISPR/Cas9 system and NHEJ connection system,and by transferring the plasmid containing the Cas9 system into the S.meliloti 320 by a three-parent transformation method,verified the cutting efficiency of CRISPR/Cas9 system.We found that Cas9 system has cutting activity in Sinorhizobium meliloti 320,but in Sinorhizobium meliloti 320,there may also be unknown sequences similar to the Cas9 targeting sequence.However,we combine CRISPR/Cas9 system with NHEJ connection system,No mutations were found in the target position of Cas9 by gene sequencing.We suspected that the colony after Cas9 cutting was not successfully connected by the NHEJ system,and the grown colonies were not cut by Cas9.