| Malate-lactic acid fermentation is an essential biological process in the production of high-quality wine.Through this process,acidity can be reduced,microbial stability can be improved,and aroma and flavour complexity can be increased.Oenococcus oeni is the main initiator and executor of malate-lactic fermentation in wine.The conditions for malate-lactic acid fermentation are complex and not conducive to microbial survival(high ethanol and SO2 levels,low temperature and low p H).Acid stress is the most common adverse environment,which can cause irreversible damage to the cells of the strain.Oenococcus oeni,as the dominant strain of male-lactic acid fermentation,has a series of regulatory mechanisms to confront acid stress pressure.By analyzing the response mechanism of Oenococcus oeni under acid stress,we can further understand the effect of acid stress on the growth and metabolism of the strain and which could provide an essential reference for the directional modification of key metabolic pathways in the future.Currently,researches on the stress response mechanism of Oenococcus oeni mainly concentrated on changes of single or several genes,proteins,metabolites,or changes in metabolic pathways.However,the feedbacks of bacteria are always in a global manner at different levels under acid stress conditions,involving with transcriptional regulations and metabolism regulations,genes and proteins,metabolism of reactants and products.In this study,O.oeni SD-2a was used as the research object.Firstly,O.oeni SD-2a was judged to have good stress response ability through the biological function verification of its small heat shock protein Hsp20.Then,acid stress assays were designed for metabonomic analysis.Combined with transcriptome data,binary network was used to identify the relationship between genes and metabolites.Then,based on the genomic data,a genome-scale metabolic network model(GSMM)of O.oeni SD-2a was constructed(i QY500).Comparison analyses were carried out to evaluate the conservation of the genomes and the genome-scale metabolic network model in different O.oeni.The accuracy of i QY500 was verified by comparing the assay results with the computer simulation results.Finally,the metabolome and transcriptome data were integrated into i QY500 to explore the acid stress response mechanism of O.oeni SD-2a.The main research results were as follows:(1)Sequence alignment showed that the Hsp20 gene of O.oeni SD-2a had a conserved motif"A-X-X-X-X-G-X-L",which was a characteristic sequence of the small heat shock protein family.Hsp20 of O.oeni SD-2a were successfully cloned,expressed and purified,and the Hsp20 recombinant E.coli strains were by heterologous expression(BL21(DE3)/Hsp20).Recombinant E.coli with vacant plasmid was used as control strain(BL21(DE3)/Ctrl).The two types of strains were evaluated with a series of stressful conditions(heat stress,acid and alkali stress,oxidative stress,salt stress).The overexpression of Hsp20 was induced by IPTG and the results showed that the Hsp20recombinant E.coli showed stronger resistances ability than the control strain under different stress conditions.(2)The UPLC-Q-TOF/MS method for metabolomics analysis of O.oeni SD-2a was successfully established by liquid-mass spectrometry.The metabolites of the samples treated with 1 h and 3 h acid stress,and samples without acid stress were evaluated.Results showed that there were significant differences in metabolites between the bacteria under acid stress(p H 3.0)and non-acid stress(p H 4.8).However,different treatment time(1 h and3 h)had little effect on the differences in metabolites between the bacteria,and 86 and 84metabolites with significant changes were screened out under acid stress for 1 h and 3 h,respectively.However,different from the metabolic response,the transcriptional response was time-specific,that is,it was significantly affected by different acid stress duration.62genes were upregulated only at 1 h stress,and 94 genes were upregulated only at 3 h stress.Ninety-eight genes were down-regulated only under 1 h stress,and 45 genes were down-regulated only under 3 h stress.(3)It was proposed that O.oeni SD-2a could adopt Stringent Response(SR)strategy under acid stress conditions.The significant synthesis of Guanosine Tetraphosphate and Pentaphosphate((p)pp Gpp)is the primary indicator SR in bacteria.Synthesis of(p)pp Gpp was associated with GTP pyrophosphate kinase(1?792).Transcriptome analysis showed that the expression of 1?792 gene in O.oeni SD-2a was increased by more than two times under p H 3.0 acid stress.What's more,bipartite network analysis showed that pyrimidine and purine nucleotide and genes related with post-translation process(such as 1?1096,30S ribosomal assembly GTP enzyme genes)showed significant negative regulation relationship.In addition,the abundance of nucleotide acid under acid stress were decreased.Therefore,the results can also be used as an evidence that O.oeni SD-2a could adopt SR strategy under acid stress condition.(4)Based on the complete genome sequence of O.oeni SD-2a,a crude model was constructed using Carveme software,then the GSMM was furtherly curated by on the genomic annotation information,literature mining and database comparison,the final GSMM of O.oeni SD-2a was named as i QY500.i QY500 included 500 open reading frames,509 metabolites,and 652 reactions.Then,comparative genomic analysis and comparative GSMMs analyses were performed on five completely sequenced Oenococcus strains(SD-2a,PSU-1,19,UBOCC-A-315001,CRBO?1381),respectively.Results showed that the core genes of the five Oenococcus strains were 1492.Most of the accessory and unique genes could be attributed to the five strains to adapt to their own living environment.GSMMs comparison of the five strains showed that there were 508 reactions in total(excluding the reaction of artificial addition),and only 16 reactions did not fall in the core region,indicating that different Oenococcus strains had a relatively conservative metabolic basis.(5)Carbon and nitrogen utilization were used as qualitative verification assays,the relative growth rate essays were used as quantitative assays,then,the accuracy of i QY500model was evaluated by comparing the computer simulation results with the data of experimental observation.The results showed that i QY500 has a good predictive ability.Finally,transcriptome and metabolome data were integrated into i QY500 to explore the redistribution of the metabolic fluxes under acid stress,thus revealing the metabolic response strategy of O.oeni.The results showed that the significant metabolic strategies adopted by O.oeni included redistribution of pyruvate,decreased glycolysis and increased utilization of carbon sources other than glucose,the enhanced response of nucleotide salvage pathway,and increased metabolic fluxes of arginine deiminase pathway-related pathways.Simultaneously,the increased fluxes in pyruvate oxidase involved reactions and the increased fluxes of glutathione oxidoreductase and glutathione peroxidase-related reactions suggested that acid stress might induce oxidative stress in the cell. |
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