Analysis Of Quorum Sensing Mechanism In Fermentation Process Of Acetobacter Pasteurianus

Acetobacter pasturianus is the main strain of vinegar industry fermentation and is often used in the liquid fermentation of vinegar.Quorum Sensing（QS）is a phenomenon that exists in some microorganisms and plays an important role in regulating the growth and metabolism of microorganisms.Our research group first detected quorum-sensing signal molecules in A.pasturianus,and identified them as N-（3-oxooctanoyl）-DL-homoserine lactone and butyryl-DL-homoserine lactone.In this study,the whole genome of A.pasturianus AC2005 was analyzed,and the functions of related genes were verified by combining exogenous recombination expression and endogenous overexpression methods,so as to further analyze the quorum sensing regulation mechanism of A.pasturianus,deepen and expand the understanding of acetic acid bacteria fermentation regulation.It has important scientific and applied value.The whole genome of A.pasturianus AC2005 was sequenced.The sequencing results showed that the whole genome was about 3.3Mb in length and contained four plasmids encoding 3,296 genes.Bioinformatics analysis found that the encoded protein had low homology with the reported Lux I/Lux R type quorum-sensing-related proteins,and the putative AHLs synthase gene 0444 was recombinantly expressed in E.coli,but no signal molecule was detected.A transposon mutation method of A.pasturianus was established,which laid the foundation for further screening of quorum sensing-related proteins.0046protein in A.pasturianus AC2005 with a potential quorum quenching function was preliminarily identified,and its amino acid homology with the reported quencher in Komagataeibacter europaeus was 62.5%.The recombinant strains AC2005(p BBR1-P₄₅₂-Aii A)and AC2005(p BBR1-P₄₅₂-0046)of A.pasturianus were constructed,and the chromogenic analysis showed that the protein 0046 had the function of quenching the quorum sensing signal molecule.The effect of overexpression of quenching enzyme on the acetic fermentation of A.pasturianus was further analyzed.Compared with the blank plasmid AC2005(p BBR1-P₄₅₂),the biomass of the recombinant 0046 gene strain increased by 39.6%and 41.8%at 36h and 48h.At the same time,the fermentation time was greatly shortened,and the average acid production rate increased by 50.5%.Compared with the blank group,the bacterial biomass of the recombinant Aii A gene strain increased by 17.8%at 96 h.And,the average acid production rate of the recombinant Aii A strain increased by19.3%.It indicated that the recombinant expression of quenching enzyme mainly increased the biomass of recombinant bacteria by degrading the concentration of signal molecules in the fermentation broth,thereby promoting the effect of acetic acid fermentation.The effect of exogenous addition of homoserine lactone signaling molecule on gene expression of A.pasturianus was analyzed by transcriptomic method.The analysis showed that exogenous addition of signal molecules could down-regulate the expression of ribosome-related proteins of A.pasturianus,thereby affecting the synthesis of bacterial proteins and the production of metabolites,resulting in a decrease in bacterial biomass.At the same time,it can induce the up-regulation of arylesterase expression,thereby enhancing the response of phosphorylation signaling pathway and promoting cell signaling.At the same time,the addition of exogenous signal molecules led to the down-regulation of the expressions of alcohol dehydrogenase and aldehyde dehydrogenase,which weakened the production capacity of the ethanol respiratory chain in the acetic acid fermentation,while the genes such as acetyl-Co A synthase and citrate synthase in the TCA cycle were up-regulated.Strengthens the TCA cycle to maintain the energy needs of cells.