| Bacillus subtilis(B.subtilis)is a common strain in the fermentation industry.It has strong ability to hydrolyze starch and protein,which is closely related to the formation of soy sauce flavor.At present,the mechanism of production of soy sauce flavor by Bacillus subtilis is still unclear.Soybeans fermented with B.subtilis BJ3-2strain exhibited strong ammonia taste below 37? and prominent soy sauce flavor above 45?.B.subtilis BJ3-2 strains that can produce soy sauce flavor and ammonia taste were used.Combining the transcriptome and metabolome analysis of the production of B.subtilis BJ3-2 at 37? and 45?.24 differentially expressed genes(DEGs)were screened,uxa C(glucuronate isomerase)and uxa B(tagaturonate reductase)were selected as flavor candidate genes.The two genes were verified by reverse transcription quantitative PCR(RT-q PCR)and their effects on the soy sauce flavor produced by Bacillus subtilis were studied.The main results are as follows:1.Transcriptome sequencing and metabolome detection of B.subtilis BJ3-2(cultured at 37°C and 45°C)were used.A total of 371 DEGs were screened out of3646 genes from the transcriptome sequencing data(P<0.05),including 255up-regulated and 116 down-regulated genes.25 up-regulated metabolites and 125down-regulated metabolites were identified by analyzing the metabolome detection data(P<0.05,VIP>1)with the ineffective metabolites removed.Meanwhile,a total of58 metabolic pathways in which all differential metabolites participated were found by KEGG analysis,and further 38 key metabolic pathways highly correlated with the differential metabolites were screened.2.According to the fold change,FPKM value and enriched metabolic pathway,100 DEGs and 95 differential metabolites were selected for the correlation analysis of the transcriptome and metabolome,and 25 metabolic pathways shared by the transcriptome and metabolome were obtained,including phenylalanine,tyrosine and tryptophan biosynthesis,citrate cycle,pentose and glucuronate interconversions,butanoate metabolism,fatty acid metabolism and so on.According to the pathways existed in the overlap of differential genes and differential metabolites,24 DEGs were obtained,of which 8 up-regulated genes,16 down-regulated genes.3.According to the gene fold change,gene expression and the metabolic pathways with the literature,uxa C(glucuronate isomerase)and uxa B(tagaturonate reductase)were finally selected as the candidate genes.4.uxa C and uxa B were verified by RT-q PCR,and 16 S r RNA was selected as the internal reference gene.The results showed that the expression levels of uxa C and uxa B at 45°C were 2.65 times and 2.82 times higher than those at 37°C respectively,which was consistent with the trend of transcriptome sequencing results.5.The homologous recombination knockout vectors of uxa C and uxa B were constructed and then transformed into B.subtilis BJ3-2.The BJ3-2?uxa C and BJ3-2?uxa B knockout strains were finally obtained through gram staining,PCR verification and sequencing verification.6.The sensory evaluation and GC-MS results showed that the soybeans fermented by BJ3-2?uxa C and BJ3-2?uxa B had stronger soy sauce flavor,more sticky silk and darker color than tha by BJ3-2.The content of acetoin decreased by6.826% and 6.222%,and the content of tetramethylpyrazine increased by 4.211% and5.104% respectively.The study successfully knocked out uxa C and uxa B,which could effect the production of soy sauce flavor at 45? by the pentose and glucuronate interconversion pathway.The changes of soy sauce flavor from multiple levels were analyzed by combining genomics,transcriptomics and metabolomics analysis,which reveals the formation mechanism of soy sauce flavor is important theoretical value and production guidance for flavor control and quality control of soy sauce flavor foods significance. |
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