基于生物量产率的低产乙醇酿酒酵母的筛选
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摘要
目的:建立一种从酿酒酵母突变库中快速筛选低产乙醇酵母菌株的方法。方法:以转录因子spt15随机突变酿酒酵母文库为研究模型,以高生物量产率作为低产乙醇酿酒酵母的筛选标记,利用24孔板对spt15突变酵母文库进行初筛。对初筛获得的目标菌株,以乙醇产量作为筛选标记进行复筛,最终获得乙醇合成能力较低且生物量较高的酿酒酵母菌株。结果:(1)利用易错PCR技术构建了spt15随机突变文库,并将其转化至酿酒酵母YS59,获得酿酒酵母YS59 spt15突变文库;(2)经初筛,获得14株生物量产率≥70%的酿酒酵母YS59 spt15突变菌株;(3)通过复筛,筛选出编号712的低产乙醇突变菌株,其乙醇合成量降低了28.1%;(4)通过序列比对,发现spt15-712上有9个碱基发生突变,分别是A161G,T426C,T462C,T493A,A506G,T546C,T622C,T658C,A750T9。结论:成功建立了一种高通量筛选低产乙醇酿酒酵母的方法 ,并利用这种方法筛选出乙醇合成量降低28.1%的低产乙醇突变菌株。
Objective: Establish a rapid method of screening low-ethanol yeast strain from S. cerevisiae mutation library. Method: The S. cerevisiae random mutation library of transcription factor spt15 was used as a research model, and the high biomass yield was used as screening target of the low-ethanol S.cerevisiae strains for the primary screening. The24-hole plate was used for screening high biomass yield strains from the spt15 mutant yeast library. Then rescreening low-ethanol strains, used low ethanol production as the target, from the strains obtained from primary screening. Result:(1) We successfully constructed a S. cerevisiae YS59 spt15 mutant library. The spt15 random mutation library was established by error-prone PCR technology firstly, and then it was transformed into S. cerevisiae YS59;(2) Compared with the control strain, we obtained 14 S. cerevisiae YS59 spt15 mutant strains with the biomass yields which were more than 70% through primary screening;(3) By rescreening, a low-ethanol mutant strain NO. 712 with the amount of ethanol producyion decreased by 28.1% was got;(4) Comparing with the spt15(Gene Accession No.: NC_001137),there were 9 bases mutated in spt15-712, which were A161 G, T426 C, T462 C, T493 A, A506 G, T546 C, T622 C,T658 C, A750 T, respectively. Conclusion: We established a high-throughput method of screening low-ethanol S. cerevisiae successfully, and the low-ethanol mutant strain with ethanol production was decreased by 28.1% was selected by this method.
Objective: Establish a rapid method of screening low-ethanol yeast strain from S. cerevisiae mutation library. Method: The S. cerevisiae random mutation library of transcription factor spt15 was used as a research model, and the high biomass yield was used as screening target of the low-ethanol S.cerevisiae strains for the primary screening. The24-hole plate was used for screening high biomass yield strains from the spt15 mutant yeast library. Then rescreening low-ethanol strains, used low ethanol production as the target, from the strains obtained from primary screening. Result:(1) We successfully constructed a S. cerevisiae YS59 spt15 mutant library. The spt15 random mutation library was established by error-prone PCR technology firstly, and then it was transformed into S. cerevisiae YS59;(2) Compared with the control strain, we obtained 14 S. cerevisiae YS59 spt15 mutant strains with the biomass yields which were more than 70% through primary screening;(3) By rescreening, a low-ethanol mutant strain NO. 712 with the amount of ethanol producyion decreased by 28.1% was got;(4) Comparing with the spt15(Gene Accession No.: NC_001137),there were 9 bases mutated in spt15-712, which were A161 G, T426 C, T462 C, T493 A, A506 G, T546 C, T622 C,T658 C, A750 T, respectively. Conclusion: We established a high-throughput method of screening low-ethanol S. cerevisiae successfully, and the low-ethanol mutant strain with ethanol production was decreased by 28.1% was selected by this method.
引文
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[10]WANG M L,CHOONG Y M,SU N W,et al.A rapid method for determination of ethanol in alcoholic beverages using capillary gas chromatography[J].Journal of Food and Drug Analysis,2003,11(2):133-140.
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[14]杜祎,李敬龙,张金玲,等.一种快速检测露酒中乙醇含量的新技术[J].酿酒科技,2015(12):1-2.
[15]杨瑛.酶电极法快速测定黄原胶中乙醇含量[J].食品工业科技,2014,35(16):82-85.
[16]冯东,王丙莲,梁晓辉,等.生物传感器法快速测定酒中的乙醇含量[J].酿酒科技,2012(2):83-86.
[17]MATTOS I L,FERNANDEZ-ROMERO J M,LUQUE DE CASTRO M D,et al.Simultaneous spectrofluorimetric determination of glycerol and ethanol in wine by flow injection using immobilized enzymes[J].Analyst,1995,120(1):179-181.
[18]王庆华,高丽丽,梁会超,等.利用反义RNA技术抑制酿酒酵母羊毛甾醇合酶基因的表达[J].药学学报,2015,50(01):118-120.
[19]李应华.Mle A基因在酿酒酵母中的整合型表达[J].中国农业科学,2009,42(4):1372-1375.
[20]张瑾.可同化氮素对酵母酒精发酵影响的研究[D].杨凌:西北农林科技大学,2009.
[21]HU R F,LIN L,LIU T J,et al.Reducing sugar content in hemicellulose hydrolysate by DNS method:a revisit[J].Journal of Biobased Materials and Bioenergy,2008,2(2):156-159.
[22]中华人民共和国质量监督检验检疫总局.GB/T 15038-2006葡萄酒、果酒通用分析方法[S].北京:中国标准出版社,2006.
[23]中华人民共和国质量监督检验检疫总局.GB15037-2006葡萄酒[S].北京:中国标准出版社,2006.
[2]MORALES P,ROJAS V,QUIROS M,et al.The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture[J].Applied Microbiology and Biotechnology,2015,99(9):3993-3994.
[3]SCHMIDTKE L M,BLACKMAN J W,AGBOOLA S O.Production technologies for reduced alcoholic wines[J].Journal of Food and Science,2012,77(1):25-28.
[4]CONTRERAS A,HIDALGO C,HENSCHKE PA,et al.Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine[J].Applied and Environmental Microbiology,2014,80(5):1670-1671.
[5]CONTRERAS A,HIDALGO C,SCHMIDT S,et al.The application of non-Saccharomyces yeast in fermentations with limited aeration as a strategy for the production of wine with reduced alcohol content[J].International Journal of Food Microbiology,2015,205:7-9.
[6]ROSSOUW D,HEYNS E H,SETATI M E,et al.Adjustment of trehalose metabolism in wine[J].Applied and Environmental Microbiology,2013,79(17):5197-5200.
[7]VARELA C D,KUTYNA R,SOLOMON M R,et al.Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts[J].Applied and Environmental Microbiology,2012,78(16):6068-6070.
[8]KUTYNA D R,VARELA C,HENSCHKE P A,et al.Microbiological approaches to lowering ethanol concentration in wine[J].Trends in Food Science&Technology,2010,21(6):293-300.
[9]ZHANG C Y,LIN N B,CHAI X S,et al.A rapid method for simultaneously determining ethanol and methanol content in wines by full evaporation headspace gas chromatography[J].Food Chemistry,2015,183:169-172.
[10]WANG M L,CHOONG Y M,SU N W,et al.A rapid method for determination of ethanol in alcoholic beverages using capillary gas chromatography[J].Journal of Food and Drug Analysis,2003,11(2):133-140.
[11]陈霞.葡萄酒中乙醇含量的气相色谱法测定[J].中国酿造,2010(9):152-154.
[12]WU X J,CHOI M M F.Spongiform immobilization architecture of ionotropy polymer hydrogel coentrapping alcohol oxidase and horseradish peroxidase with octadecylsilica for optical biosensing alcohol in organic solvent[J].Analytical Chemistry,2004,76(15):4279-4282.
[13]袁文杰,孔亮,孜力汗,等.高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分[J].分析化学,2009,37(6):850-852.
[14]杜祎,李敬龙,张金玲,等.一种快速检测露酒中乙醇含量的新技术[J].酿酒科技,2015(12):1-2.
[15]杨瑛.酶电极法快速测定黄原胶中乙醇含量[J].食品工业科技,2014,35(16):82-85.
[16]冯东,王丙莲,梁晓辉,等.生物传感器法快速测定酒中的乙醇含量[J].酿酒科技,2012(2):83-86.
[17]MATTOS I L,FERNANDEZ-ROMERO J M,LUQUE DE CASTRO M D,et al.Simultaneous spectrofluorimetric determination of glycerol and ethanol in wine by flow injection using immobilized enzymes[J].Analyst,1995,120(1):179-181.
[18]王庆华,高丽丽,梁会超,等.利用反义RNA技术抑制酿酒酵母羊毛甾醇合酶基因的表达[J].药学学报,2015,50(01):118-120.
[19]李应华.Mle A基因在酿酒酵母中的整合型表达[J].中国农业科学,2009,42(4):1372-1375.
[20]张瑾.可同化氮素对酵母酒精发酵影响的研究[D].杨凌:西北农林科技大学,2009.
[21]HU R F,LIN L,LIU T J,et al.Reducing sugar content in hemicellulose hydrolysate by DNS method:a revisit[J].Journal of Biobased Materials and Bioenergy,2008,2(2):156-159.
[22]中华人民共和国质量监督检验检疫总局.GB/T 15038-2006葡萄酒、果酒通用分析方法[S].北京:中国标准出版社,2006.
[23]中华人民共和国质量监督检验检疫总局.GB15037-2006葡萄酒[S].北京:中国标准出版社,2006.