Construction Of Oenococcus Oeni Expression System And Research On Function Of Esterase

Recently researchers focus on Oenococcus oeni,because of the main role in wine making.The main researches are involved in the culture,isolation,identification of O.oeni and the genes which related to the start of malolactic fermentation as well as the gene of resistance to extreme environment.Due to the lack of an efficient transformation system of O.oeni,these genes are now commonly heterologously expressed in Saccharomyces cerevisiae,which has significant drawbacks in studying the correlation between genes and traits of O.oeni.Therefore,based on the replication origin of Oenococcus genome,the replication origin of Escherichia coli,the high efficienent expression promoter and terminator of O.oeni gene and the erythromycin resistance gene,a high efficient expression vector p HH40 suitable for O.oeni was constructed in this paper,and the esterase gene function was studied by using the engineering bacteria O.oeni with expressing the esterase.Furthermore,the stability of fermentation performance was determined.At the same time,the function of esterase gene was investigated.The results were as following:1.Optimization of culture conditions of O.oeni.Different media,culture temperature and oxygen conditions were selected to optimize the culture conditions.In terms of medium,ATB,MRSM and m FT80 were used to culture O.oeni.The results showed that the growth rate of O.oeni in m FT80 was the highest,and the OD₆₀₀reached 2.37 in the stable period.Then,O.oeni was cultured in m FT80 medium at 20?,25?,28?and 32?.It was found that the time of reaching the index phase at 25?was 50 h earlier than that at other temperatures,and the OD₆₀₀ in the stable phase reached 2.83.In addition,the growth rate of O.oeni in anaerobic condition was better than that in oxygen condition,and the OD₆₀₀ could reach 3.04 in anaerobic condition at stable period.In conclusion,O.oeni achieved the best growth state in m FT80,25?and anaerobic conditions.2.The expression system of O.oeni was constructed.The gene expression vector p HH40 of O.oeni was successfully constructed by recombining the origin of Oenococcus genome replication,the origin of Escherichia coli replication,the promoter and terminator of high efficient expression of O.oeni and the erythromycin resistance gene by homologous recombination.The erythromycin tolerance assay showed that the growth of the original O.oeni was completely inhibited when the concentration of erythromycin reached 20?g/m L,so 20?g/m L was used as the screening concentration for recombinant O.oeni after transformation.Then,the competent state and transformation parameters of O.oeni were studied.After that,the expression vector p HH40 was transferred into O.oeni,and the transformed strain O.oeni/p HH40 was obtained.As the result,the expression system of O.oeni was constructed successfully.3.The esterase expressing bacteria of O.oeni was constructed and the function of esterase was studied.The esterase gene was inserted into the p HH40 to construct an esterase over-expressing vector,and then the recombinant strain O.oeni/p HH40-esterase was obtained.The characteristics of recombinant esterase were studyed respectively after fermenting in the simulated wine and Zexiang wine,which the original strain was the control,O.oeni/p HH40 and O.oeni/p HH40-esterase were used as experimental groups.The basic characteristics such as residual sugar,alcohol content and malic acid were measured for all wines.The results showed that there was no significant difference in the above basic characteristics between original strains and two genetically modified strains.Therefore,it suggested that the recombient O.oeni does not affect its main fermentation performance.Moreover,GC-MS was used to detect the type and contents of esters in fermented wines.The results showed that there was no significant difference in the types and contents of esters between the original strain and recombinant O.oeni.However,the total esters contents of O.oeni/p HH40-esterase in simulated wine and Zexiang wine were reduced by 23.6%and 54.9%,respectively,compared with the other two of O.oeni strains.Among them,the contents of 6 ethyl esters in the simulated wine decreased significantly,including ethyl acetate 34.6%,ethyl caproate14.4%,ethyl octanoate 24.8%,ethyl capric acid33.6%,ethyl laurate 49.4%and ethyl palmitate 41.7%,the contents of 8 substances in Zexiang wine were significantly reduced.In addition to the 6 ethyl esters mentioned above,the contents of isoamyl acetate and hexyl acetate were also significantly reduced by 83.4%and 85.5%,respectively.The above 8 kinds of esters mostly have fruit aroma.Therefore,it can be inferred that this esterase could remain active in wine environment,and could hydrolyze many intermediate esters,and might balance the content of esters in wine.In conclusion,the expression vector p HH40 and transformation system of O.oeni were successfully constructed in this study.The esterase gene from Lactobacillus plantarum was inserted into p HH40,and the transformed O.oeni/p HH40-esterase was obtained.The recombinat O.oeni/p HH40-esterase did not affect its main fermentation performance when fermented in the simulated wine and Zexiang wine.In addition,the esterase in this study can adapt to the harsh wine environment,and it has a wide range of substrates,furthermore,it can decompose of esters in wine,which has potential use value for regulating the content of esters in wine.