Screening Of B.subtilis Soybean Flavoring Enzyme Gene And Construction Of Knockout Vector

Bacillus subtilis,as a food safety microorganism(GRAS),is applied to biological control,food fermentation,and industrial protease production.Flavor is a compound aroma.B.subtilis is the main microorganism formed by Maotai flavor.So far,the molecular mechanism of Maotai flavor formation is still unclear.In order to explore the mechanism of the production of Maotai flavor,9 Maotai flavor gene and the target gene rocG(glutamate dehydrogenase)was established by using the third generation genome sequencing and transcriptome sequencing of B.subtilis BJ3-2 and B.subtilis 10075.Combining the pre-laboratory study and the acetolactate synthase alsS,four CRISPR/Cas9 vectors by molecular cloning were successfully constructed for the target genes rocG and alsS.The main contents and results were as follows:1.By comparing the common protein of B.subtilis BJ3-2 and B.subtilis 10075,the preliminary analysis of the flavor of soy sauce may be related to the gene rearrangement.2.Analysis of the genome sequencing of the third generation and the transcriptional sequence,by comparing the genomic differences,the difference of gene expression and the multiplier of transcriptional differences of B.subtilis BJ3-2 and B.subtilis 10075,and combining with the complete genome sequence of B.subtilis168 and the database GO?KEGG.Fixed sauce flavor glutamate dehydrogenase rocG gene.we determined the Maotai flavor gene of speD?rocF?hutG?PRODH?celF?hutI?celB?OxdD?rocG.3.Comply with the design principle of sgRNA and donor,meanwhile consider sequences of the wild strain BJ3-2 and target genes rocG and als S,we finally selected four sgRNA of als S 110 f,alsS 527 f,rocG 250 r,rocG456f and the left and right arms of donor.The donor L+R of rocG and alsS was amplified by overlapping PCR,and the complete homologous replacement arms were obtained.4.After two enzyme digestion,PHT01cas9-p43 was connected to sgRNA and donor inturn,and four recombinant plasminds p43/sgRNA110/donor,p43/sgRNA527/donor,p43/sgRNA250/donor,p43/sgRNA456/donor were obtained.Screening of transfected bacteria by bacteria colonies PCR,plasmind PCR and sequencing,and results showed that the CRISPR/Cas9 plasmids was successfully completed.The research laid a foundation for the functional study of the rocG and alsS sauce flavor genes,and provided an effective theoretical basis for exploring the mechanisms of the production of Maotai and the genetic improvement of GRAS strains.