| In wine production,Oenococcus oeni is the main microorganism that carries out malolactic fermentation because of its strong resistance to stress.In the process of adapting to the environment,the self-regulation of membrane components is an important mechanism.The cell mainly changes the mobility and rigidity of the membrane to deal with the external stress conditions and maintain the normal physiological function of the cells.In this mechanism,the synthesis of cyclopropane fatty acid(CFA)in the cell membrane plays important roles in adapting to the rapid changes of environment in bacteria and in the mechanisms to deal with stressful environmental mechanisms of Oenococcus oeni.The study about relationship between the function of the cyclopropan fatty acid synthase gene(cfa)and the tolerance of the strain can understand the mechanism of the changes of the membrane composition of Oenococcus oeni better,and Lactobacillus plantarum as another important strain for the initiation of malic acid-lactic acid fermentation,this study provides a broad stream of ideas and foundations for its ability of initiating of malic acid-lactic acid fermentation.In this study,pH was chosen as the stress factor of strain screening,selected the wild acid-resistant and acid-sensitive Oenococcus oeni strains,and determined the growth limit of acid-resistant strain.Then,in the acid stress environment,the recombinant plant Lactobacillus found that the content of cyclopropane fatty acid increased sharply,and the introduction of exogenous cfa gene increased.Cyclopropan fatty acid synthase gene was sequenced for acid-resistant and acid-sensitive wild and mutant strains.On the basis of this,the cfa genes of wild acid-resistant and acid-sensitive strains were introduced into Escherichia coli to induce and express the purified product.The same gene were introduced into Lactobacillus plantarum.The viability of recombinant Lactobacillus plantarum,degradation of malic acid and membrane fatty acid composition changes were detected.The main results of the study are as follows:(1)35 strains of Oenococcus oeni were screened under pH 2.8 and five strains were selected.Then the five strains were cultured under pH 2.6 and 2.8,and viability(OD600)was determined.The final result suggested Oenococcus oeni CS-7b and ME-5b were selected as wild acid-resistant strains and the acid limit of their growth is pH 2.6.(2)The cfa genes of the wild acid-resistant strains CS-7b,ME-5b and the acid-sensitive strain SX-1b were sequenced and compared with the mutant strain a3(acid resistant)and b1(acid sensitivity)in the laboratory.The sequence of wild and mutagenesis acid-resistant strains was identical and had mutations in three sites compared with Oenococcus oeni PSU-1.The sequences of wild and mutagenesis acid-sensitive strains were consistent with Oenococcus oeni PSU-1.(3)The cfa genes of SX-1b and CS-7b were successfully transferred into the Escherichia coli and the induction condition were determined,16 ℃,150 rpm,19 h induction,and the protein was purified by Ni-NTA column and obtained.(4)The cfa genes of SX-1b and CS-7b were expressed in Lactobacillus plantarum ATCC3332.The recombinant plasmids pMG36e-cfa1 and pMG36e-cfa2 were constructed and corresponding recombinant strains--L1 and L2 were obtained.In the medium at pH 3.6,the growth of recombinant strains---L1 and L2 was significantly better than that of Lactobacillus plantarum L0 with empty vector,and the growth of L2 was better than that of L1.In the pH 3.4 medium,only the L1 and L2 grew up normally while L0 did not grow,which suggests the introduction of cfa gene broke the growth limit of Lactobacillus plantarum.In the acid stress environment,the content of cyclopropane fatty acid increased rapidly in the recombinant strains,and the introduction of exogenous cfa gene promoted the increase.Results of the L-malic acid consumption test shown that,all recombinant strains preserved MLF ability. |
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