Screening And Functional Verification Of Acid-resistant Candidate Genes In Mutagenized Oenococcus Oeni

Oenococcus oeni(O.oeni)is the main strain of malolactic fermentation(MLF)in wine.It can adapt to the harsh environment of wine and play a role in reducing acidity and soft taste in wine.The low p H in wine is an important factor limiting the growth of O.oeni.Therefore,screening and identifying the acid-resistant genes of O.oeni can lay a good foundation for screening the excellent fermentation strains.Three pairs of acid-resistant and acid-sensitive O.oeni strains were re-sequenced in previous research,and 178 acid-resistant candidate genes were localized by SNP differences between the acid-resistant and acid-sensitive strains.In this study,the 178 acid-resistant candidate genes were screened by removing sequences with only synonymous mutations and reviewing the literature.Then,real-time fluorescence quantification(RT-q PCR)was used to determine the expression level of the acid-resistant candidate genes in the two strains under the conditions of p H 4.8 and 3.0,and the range of acid-resistant candidate genes was further narrowed by the difference in expression level.The target genes for subsequent analysis were selected.Based on the amino acid sequences of the target genes,the sequences derived from the acid-resistant strain and the acid-sensitive strain are aligned and bioinformatics analysis of the functional domain,the transmembrane domain and the phylogenetic tree is performed.Finally,the overexpression vector of the target sequences was constructed and electroporated into Lactobacillus plantarum(L.plantrum).The growth ability of recombinant L.plantrum was determined under stress conditions(MRS medium at p H 3.2).The main results were as follows:1)Using the Perl script to extract the DNA sequences of 178 acid-resistant candidate genes from the library and converted them into amino acid sequences.After comparison,20acid-resistant candidate genes were synonymous mutations only and 158 acid-resistant candidate genes were non-synonymous mutations.According to published literature reports,a total of 38 genes were selected from 158 acid-resistant candidate genes for RT-q PCR.The resulst showed that the expression trends were represented by three types: I)The expression of the acid-resistant strain and the acid-sensitive strain showed the same trend,which were both significantly up-regulated or down-regulated;II)The expression trends of acid-resistant and acid-sensitive strains were opposite,the expression of the acid-resistant strain was significantly up-regulated while that of the acid-sensitive strain was significantly down-regulated,or the expression of the acid-resistant strain was significantly down-regulated while that of the acid-sensitive strain was up-regulated;III)Only the expression level of the acid-resistant(acid-sensitive)strain was significantly up-regulated or down-regulated,and the expression level of the acid-sensitive(acid-resistant)strain remained unchanged.The range was narrowed down to 10 acid-resistant candidate genes based on RT-q PCR results.The expression trend of Gene 10(ATPase gene,OEOE?RS01075),belonged to the the first type,was significantly up-regulated in both the acid-resistant strain b1 and the acid-sensitive strain b2,up-regulated by 13.30 times in b1 and up-regulated by 5.10 times in b2;the expression trend of gene 14(fts H,OEOE?RS00895),belonged to the third type,remained basically unchanged in the acid-resistant strain b1,and was significantly up-regulated in the acid-sensitive strain b2.In addition,some relevant literatures have confirmed fts H has a stress-resistant function.Therefore,Gene 10(ATPase gene)and Gene 14(fts H)were selected as target genes for subsequent bioinformatics analysis and functional verification.2)The results of ATPase amino acid sequence alignment and domain prediction(functional domain and transmembrane domain)showed that ATPase had 5 non-synonymous mutations,of which 3 mutations occured in the functional domain;Fts H has two non-synonymous mutations,one of which is located in the transmembrane domain and has no mutations in the functional domain.Phylogenetic tree result showed that the homology of ATPase in O.oeni and the corresponding protein in L.plantrum was 55%,the homology of Fts H in O.oeni and the corresponding protein in Lactobacillus plantarum was 60%.The above results indicated that there were some differences between the ATPase and Fts H sequences in O.oeni and the L.plantrum.3)The ATPase and Fts H in the acid-resistant strain b1 were named ATPase(acid-resistant)and Fts H(acid-resistant)respectively,and the ATPase and Fts H in the acid-sensitive strain B2 were named ATPase(acid-sensitive)and Fts H(acid-sensitive),respectively.Functional verification test results showed that both ATPase(acid-resistant)and ATPase(acid-sensitive)could improve the acid resistance of recombinant L.plantrum Fts H(acid-resistant)also couldincrease the acid resistance of recombinant L.plantrum.However,Fts H(acid-sensitive)could not improve the acid resistance of the recombinant L.plantrum,which is presumed to mutations of Fts H(acid-sensitive)in the transmembrane domain leading to a loss of acid resistance.