Screening And Functional Study Of The Genes Related To The Production Of Soy Fragrance From Bacillus Subtilis

Bacillus subtilis is widely used in food,agriculture,feed,environmental protection and other fields.The unique flavor of the bacterial tempeh in Guizhou is deeply loved by people.However,there are few reports on the aroma-producing mechanism of Bacillus subtilis.The research uses the patented strain B.subtilis BJ3-2to perform mid-temperature(45 ?)and mid-low temperature(37 ?)transcriptome sequencing results,bioinformatics analysis and screening to obtain 16 significantly different genes,combined with literature reports and gene metabolic pathway analysis,Finally,3 genes were obtained as candidate genes for soybean flavour to study,and the results obtained are as follows:1.Through the previous analysis of the second-generation transcriptome sequencing results of B.subtilis BJ3-2 at 45? and 37?,including gene expression,differential gene KEGG,GO annotation and enrichment analysis,control the screening conditions: whether the gene is meaningful,Gene function description(excluding conservative hypothetical proteins),Log2FC(BJ 3-2 45°C/BJ 3-2 37°C)?2.5,16 significantly different genes were screened,combined with literature and metabolic pathway analysis,3 significant differences were selected The genes cysC(adenosyl sulfate kinase),uxuA(D-mannitol dehydratase),and rapF(reaction modifier aspartate phosphatase f)are candidate genes for soy.2.Validate the expression of cysC,uxuA,and rapF by fluorescent quantitative PCR.Using 16 S rRNA as an internal reference gene,the results showed that cysC gene expression was down-regulated by 0.22 times,and the gene expression of uxuA and rapF were up-regulated by 41.35 times and 2.21 times,respectively.The results are consistent with the trend of transcriptome sequencing results.3.Successfully constructed homologous recombination double crossover knockout vectors of cysC,uxuA and rapF.Using pUC18 as the original vector,designthe target gene 5'and 3'restriction sites(Sac I,BamH I,Pst I,EcoR I,Xba I),respectively,and each candidate obtained by BJ3-2 genome amplification The gene homology left arm(HLarm)and right arm(HRarm)and the chloramphenicol acetyltransferase gene(cat)on the pHT01 cas9-p43 vector were digested and connected.After colony PCR,plasmid PCR,double enzyme digestion and sequencing verification,the recombinant knockout vector pUC18 + HLarm + cat + HRarm of cysC,uxuA and rapF was finally constructed.They were transformed into B.subtilis BJ3-2 by chemical method.Through colony PCR screening,plasmid PCR and sequencing verification,Gram staining finally successfully screened the knockout strains of BJ3-2?cysC gene.4.The sensory evaluation showed that the fermented tempeh with BJ3-2?cysC was more fragrant than the fermented tempeh with BJ3-2,the color was darker,the skin was slightly wrinkled,the hyphae was less,and the silk was not drawn.GC-MS shows that volatile substances include: pyrazines,acids,quinones,esters,ketones,alcohols,etc.Analysis Since cysC exists in sulfur metabolism and purine metabolism,knocking out this gene will cause sulfate to fail to produce cysteine,which will affect the metabolism of pyruvate and affect the flavor of soy.Further analysis showed that trimethyl pyrazine,2,5-dimethyl-pyrazine,and tetramethyl pyrazine increased by12.774% and 8.901%,respectively,4.362%.Trimethylpyrazine and2,5-dimethylpyrazine contribute hazelnut aroma,cocoa aroma or chocolate aroma,and roasted peanut aroma to soy.2,3-butanediol,1,3-butanediol,and acetoin are reduced by 11.489%,5.760%,and 3.758%.Acid substances include 2-ethylbutyric acid,acetic acid,valeric acid and isobutyric acid,respectively Decrease 3.4%,2.715%,1.255%,0.817%.The synthesis of 2,5-dimethylpyrazine and trimethylpyrazine in vivo is related to L-threonine.The changes of pyruvate are directly related to the changes of 2,3-butanediol,acetoin and acetic acid.The analysis of these substances ultimately affects the TCA cycle through multiple metabolic pathways such as pyruvate metabolism and amino acid metabolism,and ultimately affects the flavor of soy.5.Benzaldehyde and 3-octanol were not detected in fermented tempeh fromBacillus subtilis BJ3-2,but were detected in fermented tempeh from Bacillus subtilis BJ3-2 ? cysC.It is inferred that the bitter almond flavor and strong cherry aroma possessed by benzaldehyde and 3-octanol have the special odor of oil,nut and herbal flavor,and have an important contribution to the flavor of black soybean.In summary,cysC is a functional gene of soybean flavour,and the enzyme it encodes negatively regulates the flavour of soybean fragrant.Pyrazines and acids are important flavour substances of soybean fragrant.Sulfur metabolism,amino acid metabolism,and pyruvate metabolism can affect the formation of black bean flavor.