Screening And Functional Research On Related Genes Of Soy Sauce Flavor Produced By Bacillus Subtilis

B.subtilis is widely distributed in the environment and is mainly used in food,agriculture and environmental protection.As a functional strain of maotai-flavor,its products,such as soy sauce,soy,fermented black beans,liquor and other maotai-flavor foods,are deeply loved by consumers.So far,the mechanism of B.subtilis producing maotai-flavor is still unclear.By sequencing the transcriptome of the industrial strain B.semtilis BJ3-2 at medium and high temperature(45?)and medium temperature(37?),enrichment analysis was carried out for their differences,and multiple genes with significant differences were screened out.Bioinformatics analysis and screening showed that three genes might be related to soy sauce aroma,and the function of soy sauce aroma was studied after its knockout.The results obtained are as follows:1.Perform analysis of expression level,GO analysis of different genes,KEGG annotation and enrichment analysis on the early stage of the transcriptome data,and control the screening conditions of Fold-change?2 and FDR?0.05 and genes highly expressed at45?.Then 15 genes were initially screened out with significant differences.Analyzed these genes comprehensively and 3 candidate genes for sauce production were obtained,cdoA(cysteine dioxygenase),panE(Keto ubiquitinate reductase),lspA(lipoprotein signal peptidase II).2.Fluorescence quantitative PCR was used to detect the expression levels of 3candidate genes,cdoA,panE and lspA of B.subtilis BJ3-2 cultured at 37? and 45?,with16 S rRNA as the reference gene.The results showed that the expressions of cdoA,panE and lspA at 45 ? were 1.81,1.38,and 1.51 times higher than those of 37 ?,which were consistent with transcriptase results.Three genes,cdoA,panE and lspA,were selected to study the function of producing soy sauce flavor.3.cdoA,panE and lspA were constructed as homologous recombination double-exchange knockout vectors,pUC18+HLarm+cat+HRarm.Using pUC18 as the initial vector,respectively designed and synthesized with enzyme loci of specific primers,PCR amplification BJ3-2 genome to obtain corresponding genes homologous left arm(HLarm)and homologous(HRarm)in her right arm and at the same time with pHT01cas9-p43 carrier for template amplification of chloramphenicol gene(cat),which were connected and transformed into the E.coli DH5?.By colony PCR,plasmid PCR,double enzyme detection and sequencing validation,finally respectively obtained 3 genes' homologous recombination double knockout exchange vector pUC18+HLarm+cat+HRarm.4.The three double-exchange knockout vectors were transformed into B.Simtilis BJ3-2by chemical method,and the gene knockout strains of BJ3-2?cdoA,BJ3-2?panE and BJ3-2?lspA were obtained by gram-staining,PCR validation and plasmid sequencing validation.5.The three strains were inoculated with soybeans and fermented at 45? for 72 h to obtain tempeh.Compared with the original strain,BJ3-2?cdoA fermented tempeh became paler in color and less fragrant in soy sauce.BJ3-2?panE fermented black beans showed no significant color change,but the viscosity increased and the aroma of soy sauce decreased.BJ3-2?lspA fermented fermented black beans had the lightest color,significantly enhanced viscosity,more viscose and bright,and significantly weakened sauce flavor.The results of GC-MS detection of volatile substances in fermented black beans showed that the content of2.3 butanediol in BJ3-2?cdoA,bJ3-2 ?panE,and BJ3-2?lspA were decreased by 0.437%,0.108%,and 0.474%,respectively;the content of acetoin was decreased by 5.415%,6.259%,and 1.101%,respectively;and the content of tetramethylpyrazine was decreased by 0.768%,0.025%,and 4.063%,respectively,compared with the control group.In summary,B.subtilis BJ3-2 can produce significant soy sauce flavor at medium and high temperature,and enzymes encoded by the three genes can regulate the formation of soy sauce flavor.Among them,cdoA regulates the formation of L-cysteine sulfonate in themetabolism of cysteine and methionine,affects the metabolism of pyruvate and thus affects the formation of the flavor of soy paste.panE controls the biosynthesis of pantothenic acid and CoA and influences pyruvate metabolism in the tricarboxylic acid cycle to regulate sauce flavor;lspA affects pyruvate metabolism and regulates sauce flavor by controlling the transport of carbohydrates,proteins and other substances.It is generally believed that soy sauce aroma is positively correlated with the Browning degree of food.After the cdoA and lspA genes are knocked out,the flavor of soy sauce decreases and browning degree are weaken,but after the panE gene is knocked out,the Browning degree of soy sauce flavor remains unchanged,indicating that the soy sauce flavor and the Browning degree of soy sauce are not necessarily linearly correlated.