耐酸与酸敏酒酒球菌环丙烷脂肪酸合酶基因的差异
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摘要
目的:研究筛选得到的野生耐酸与酸敏酒酒球菌环丙烷脂肪酸基因(cfa)的差异,探索酒酒球菌环丙烷脂肪酸基因与酒酒球菌耐酸性的相关性。方法:35株野生酒酒球菌中,利用酸胁迫环境筛选耐酸能力最强的菌株,并与野生酸敏菌SX-1b及实验室离子注入诱变的耐酸、酸敏突变菌a3、b2共同进行环丙烷脂肪酸基因cfa的克隆、测序和比对;选择存在氨基酸突变的野生耐酸酒酒球菌及野生酸敏菌株的cfa基因在植物乳杆菌ATCC33222中进行外源表达,从而验证耐酸菌株cfa基因与耐酸能力的相关性。结果:(1)筛选出野生型耐酸酒酒球菌CS-7b和ME-5b,确定其耐酸生长极限为p H 2.6;(2)对野生菌与诱变菌的cfa基因进行测序比对发现,耐酸菌株的野生型和诱变型的序列相同,较酒酒球菌PSU-1发生3处碱基突变,而酸敏菌野生型、诱变型的序列则与酒酒球菌PSU-1一致;(3)将SX-1b与CS-7b的cfa基因在植物乳杆菌中表达,构建重组载体pMG36ecfa1和pMG36e-cfa2,并得到对应重组菌L1和L2;(4)在pH 3.6培养基中培养,重组菌L1和L2的生长情况明显优于含空载体的植物乳杆菌L0,且L2的生长优于L1。而在p H 3.4培养基中培养,L0没有生长迹象,只有重组菌L1和L2正常生长,cfa基因的导入突破了植物乳杆菌宿主菌的生长极限。结论:酒酒球菌cfa基因及基因间存在的稳定差异与菌株耐酸表型具有一定相关性。
Objective:To explore the relationship between strains' acid resistance and the cyclopropane fatty acid gene,this research was designed to study the difference in cfa gene between selected wild acid-resistant Oenococcus oeni strains and acid-sensitive strains.Methods:Thirty-five wild Oenococcus oeni were selected in the medium with low pH for the most acid-resistant strain.With acid-sensitive strain-SX-1 b,the cfa genes of wild acid-resistant strains were cloned,sequenced and compared with acid-resistant mutant a3 and acid-sensitive mutant b2.The cfa genes of acid-sensitive strain and wild acid-resistant strain with amino acid mutations were expressed in Lactobacillus plantarum ATCC33222 to verify the correlation between cfa gene mutation of acid-resistant strains and strains' acid resistance.Results:Oenococcus oeni CS-7 b and ME-5 b were selected as wild acid-resistant strains and the acid limit of their growth is pH 2.6;In the comparison of the sequence of cfa genes in wild strains and mutants,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.The cfa genes of SX-1 b and CS-7 b were expressed in Lactobacillus plantarum ATCC33222.The recombinant plasmids pMG36 e-cfa1 and pMG36 e-cfa2 were constructed and corresponding recombinant strains-L1 and L2 were obtained.In the medium at pH3.6,the growth of recombinant strais-L1 and L2 was significantly better than that of Lactobacillus plantarum L0 with empty vector,and the growth of L2 was better than L1.In the p H 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 acid resistance.Conclusion:The cfa gene in O.oeni strains and its stable mutation are related to the acid resistance ability.
Objective:To explore the relationship between strains' acid resistance and the cyclopropane fatty acid gene,this research was designed to study the difference in cfa gene between selected wild acid-resistant Oenococcus oeni strains and acid-sensitive strains.Methods:Thirty-five wild Oenococcus oeni were selected in the medium with low pH for the most acid-resistant strain.With acid-sensitive strain-SX-1 b,the cfa genes of wild acid-resistant strains were cloned,sequenced and compared with acid-resistant mutant a3 and acid-sensitive mutant b2.The cfa genes of acid-sensitive strain and wild acid-resistant strain with amino acid mutations were expressed in Lactobacillus plantarum ATCC33222 to verify the correlation between cfa gene mutation of acid-resistant strains and strains' acid resistance.Results:Oenococcus oeni CS-7 b and ME-5 b were selected as wild acid-resistant strains and the acid limit of their growth is pH 2.6;In the comparison of the sequence of cfa genes in wild strains and mutants,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.The cfa genes of SX-1 b and CS-7 b were expressed in Lactobacillus plantarum ATCC33222.The recombinant plasmids pMG36 e-cfa1 and pMG36 e-cfa2 were constructed and corresponding recombinant strains-L1 and L2 were obtained.In the medium at pH3.6,the growth of recombinant strais-L1 and L2 was significantly better than that of Lactobacillus plantarum L0 with empty vector,and the growth of L2 was better than L1.In the p H 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 acid resistance.Conclusion:The cfa gene in O.oeni strains and its stable mutation are related to the acid resistance ability.
引文
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[19]张哲,樊明涛,董梅,等.植物乳杆菌β-D-葡萄糖苷酶的定位及活性研究[J].食品科学,2014,35(15):161-165.
[20]李娅妮.植物乳杆菌P-8 lai同源重组敲除载体的构建及电转化条件的研究[D].呼和浩特:内蒙古农业大学,2014.
[21]BELTRAMO C,DESROCHE N,TOURDOT-MAR魪CHAL R,et al.Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium[J].Res Microbiol,2006,157(3):267-274.
[22]LIU Y P,TANG H Z,LIN Z L,et al.Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation[J].Biotechnol Adv,2015,33(7):1484-1492.
[2]BARTOWSKY E J,BORNEMAN A R.Genomic variations of Oenococcus oeni strains and the potential to impact on malolactic fermentation and aroma compounds in wine[J].Appl Microbiol Biotechnol,2011,92(3):441-447.
[3]BETTERIDGE A,GRBIN P,JIRANEK V.Improving Oenococcus oeni to overcome challenges of wine malolactic fermentation[J].Trends Biotechnol,2015,33(9):547-553.
[4]TOURDOT-MARECHAL R,GABORIAU D,BENEY L,et al.Membrane fluidity of stressed cells of Oenococcus oeni[J].International Journal of Food Microbiology,2000,55(1/2/3):269-273.
[5]BENEY L,GERVAIS P.Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses[J].Appl Microbiol Biotechnol,2001,57(1/2):34-42.
[6]CHU-KY S,TOURDOT-MARECHAL R,MARECHALP A,et al.Combined cold,acid,ethanol shocks in Oenococcus oeni:Effects on membrane fluidity and cell viability[J].Biochim Biophys Acta,2005,1717(2):118-124.
[7]GRANDVALET C,ASSAD-GARCIA J S,CHU-KYS,et al.Changes in membrane lipid composition in ethanol-and acid-adapted Oenococcus oeni cells:Characterization of the cfa gene by heterologous complementation[J].Microbiology-Sgm,2008,154(Pt9):2611-2619.
[8]BUDIN-VERNEUIL A,PICHEREAU V,AUFFRAYY,et al.Proteome phenotyping of acid stress-resistant mutants of Lactococcus lactis MG1363[J].Proteomics,2007,7(12):2038-2046.
[9]DA SILVEIRA M G,GOLOVINA E A,HOEK-STRA F A,et al.Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells[J].Appl Environ Microbiol,2003,69(10):5826-5832.
[10]ZHANG G Q,FAN M T,LV Q,et al.The effect of cold,acid and ethanol shocks on synthesis of membrane fatty acid,freeze-drying survival and malolactic activity of Oenococcus oeni[J].Annals of Microbiology,2013,63(2):477-485.
[11]HUA L,ZHAO W Y,WANG H,et al.Influence of culture pH on freeze-drying viability of Oenococcus oeni and its relationship with fatty acid composition[J].Food and Bioproducts Processing,2009,87(1):56-61.
[12]MONTANARI C,KAMDEM S L S,SER-RAZANETTI D I,et al.Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses[J].Food Microbiology,2010,27(4):493-502.
[13]TEIXEIRA H,GONCALVES M G,ROZES N,et al.Lactobacillic acid accumulation in the plasma membrane of Oenococcus oeni:A response to ethanol stress?[J].Microbial Ecology,2002,43(1):146-153.
[14]ZHAO W Y,LI H,WANG H,et al.The effect of acid stress treatment on viability and membrane fatty acid composition of Oenococcus oeni SD-2a[J].Agricultural Sciences in China,2009,8(3):311-316.
[15]BASTIANINI A,GRANCHI L,GUERRINI S,et al Fatty acid composition of malolactic Oenococcus oeni strains exposed to pH and ethanol stress[J].Italian Journal of Food Science,2000,12(3):333-342.
[16]TO T M H,GRANDVALET C,ALEXANDRE H,et al.Cyclopropane fatty acid synthase from Oenococcus oeni:Expression in Lactococcus lactis subsp cremoris and biochemical characterization[J].Archives of Microbiology,2015,197(9):1063-1074.
[17]陈其玲,任晓宁,王玲,等.酒酒球菌β-葡萄糖苷酶活性与耐酸胁迫能力的相关性分析[J].食品科学,2017,38(2):115-120.
[18]李亚辉,董梅,崔禾苗,等.酒酒球菌SD-2a的β-D-葡萄糖苷酶活性研究[J].食品科技,2013,38(8):48-52.
[19]张哲,樊明涛,董梅,等.植物乳杆菌β-D-葡萄糖苷酶的定位及活性研究[J].食品科学,2014,35(15):161-165.
[20]李娅妮.植物乳杆菌P-8 lai同源重组敲除载体的构建及电转化条件的研究[D].呼和浩特:内蒙古农业大学,2014.
[21]BELTRAMO C,DESROCHE N,TOURDOT-MAR魪CHAL R,et al.Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium[J].Res Microbiol,2006,157(3):267-274.
[22]LIU Y P,TANG H Z,LIN Z L,et al.Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation[J].Biotechnol Adv,2015,33(7):1484-1492.