The Screening And Preliminary Study On Genes Related To Mao Tai-flavor In Bacillus Subtilis

In order to isolate genes related to the forming of MaoTai-flavor in Bacillus subtilis, Bacillus subtilis E20, responsible for producing strong Moutai-flavor served as a tester, Bacillus subtilis 10075, which was identified as producing non-Moutai-flavor served as a driver, a PCR-select genomic DNA subtraction method(called suppressive subtractive hybridization, or SSH) was performed. A DNA subtractive hybridization librariy was successfully obtained, 77 unique gene segments were identified from Bacillus subtilis E20 which was identified as producing strong Moutai-flavor, and the differential fragments obtained was used for sequencing and bioinformatics analysis.Blast analysis showed that there were 65 differential gene fragments obtained an accurate annotation, and the average similarity was 98%. There were 26 enzymes among the 65 sequences, and 245 metabolites of 19 of these enzymes were found in the KEGG database. First, extracted 45 metabolites related to MaoTai-flavor from the published papers; second, used HMDB and KEGG database to converse after geting its English name though name matching; last, obtained correlation analysis using the signal pathway of KEGG. Find out the corresponding metabolic pathway by the enrichment analysis of these metabolites. Used the information of library’s sequencing genes, the enzymes found in genes, the enzyme’s metabolites, the enzyme’s metabolic pathways, the metabolic pathway that enzyme’s metabolites involved in; the metabolites related to MaoTai-flavor in published papers,the relevant metabolites’ corresponding metabolic pathway, the corresponding metabolites’ corresponding enzymes and some known genes that related to MaoTai-flavor to construct multi-level commol/Lon regulatory networks. The results were as follows:1. The metabolic pathways that library’s sequencing genes involved in were phenylalanine metabolism, amino sugars and sugar nucleotides metabolism, nitrogen metabolism, histidine metabolism, starchand sucrose metabolism, ect.2. The metabolites of library’s sequencing genes mainly included a large number of amino acids, carbohydrates, acetic acid, butyric acid, propionic acid, othersmall molecular compounds like catechol as well as HTH type transcriptionfactor yyb E(yybE), etc.3. The library’s sequencing genes had a certain correlation relatioship with acetoin’s metabolites and alsS’s metabolism.4. The library’s sequencing genes provided precursors and reaction conditions for the Maillard reaction.The experiment successfully isolated gene sequences that had highly correlationwith MaoTai-flavor by suppression subtractive hybridization, some gene fragments directly display its potentiality as precursors in the production of MaoTai-flavor, such as phenylalanyl tRNA synthetase beta chain(pheT), DNA gyrase A subunit(gyrA) and HTH type transcription factor yyb E(yybE), and the related geneswere an important reason whether strains producing MaoTai-flavor or not, although that information cannot be used as a direct evidence for the phenotypic difference of strains producing MaoTai-flavor, but the information of differential genes library related to producing MaoTai-flavor, can provide clues for a further study of these genes’ role in MaoTai-flavor producing in Bacillus subtilis, and also laid the foundation for the future in-depth study on the mechanism of MaoTai-flavor generation.