一次敲除BAT2同时过表达Lg-ATF1对啤酒酵母产醇酯的影响
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摘要
乙酸乙酯和高级醇是啤酒中的重要风味物质,为了探究BAT2基因和Lg-ATF1基因对啤酒酵母产醇酯能力的影响,进而解决啤酒中存在的醇高酯低的问题。本研究通过酶切连接法构建重组质粒p UC-PLABBK,采用醋酸锂转化法和胞内同源重组技术,以多倍体啤酒酵母菌株S5为出发菌株,Kan MX基因作为筛选标记,最终获得过量表达Lg-ATF1基因同时敲除BAT2基因的重组菌株S5-Lg。通过啤酒发酵实验和数字PCR探究重组酵母菌株S5-Lg与出发菌株S5醇酯含量与相关基因表达量的变化。结果显示,与出发菌株相比,S5-Lg的总高级醇生成量降低9.12%,其中异丁醇和异戊醇的生成量分别降低了10.63%和9.55%,乙酸乙酯生成量提升了26.81%,BAT2基因表达量降低45.72%,Lg-ATF1基因表达量大幅提升。BAT2基因和Lg-ATF1基因可以影响啤酒酵母产生高级醇和乙酸乙酯的含量,对改善啤酒风味有重要参考意义。
Ethyl acetate and higher alcohols are important flavor substances in beer. The effect of BAT2 gene and Lg-ATF1 gene in beer yeast on producing alcohol esters was explored to solve the problem of more higher alcohols and less ethyl acetate in beer. The expression vector p UC-PLABBK was constructed by using the method of enzyme digestion and connection. Saccharomyces cerevisiae multiploid S5 was used as the original strain and the Kan MX gene was used as a selection marker in this study. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain S5-Lg that overexpressing Lg-ATF1 and at the same time deleting BAT2. The variation between the content of higher alcohols, ethyl acetate and expression of related genes in the engineered yeast strain S5-Lg was investigated by fermentation experiments and digital PCR. The results showed that compared with the original strain S5, the ethyl acetate production of the S5-Lg strain was increased by 26.81%, total higher alcohol production decreased by 45.72%, of which the contents of isobutanol and isoamyl alcohol were reduced by 10.63% and 9.55%, respectively. The expression of Lg-ATF1 gene was greatly increased, and the expression level of BAT2 gene was decreased by 45.72%. It effectively changed the ability of brewer's yeast to produce ethyl acetate and higher alcohols and has important significance for improving beer flavor.
Ethyl acetate and higher alcohols are important flavor substances in beer. The effect of BAT2 gene and Lg-ATF1 gene in beer yeast on producing alcohol esters was explored to solve the problem of more higher alcohols and less ethyl acetate in beer. The expression vector p UC-PLABBK was constructed by using the method of enzyme digestion and connection. Saccharomyces cerevisiae multiploid S5 was used as the original strain and the Kan MX gene was used as a selection marker in this study. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain S5-Lg that overexpressing Lg-ATF1 and at the same time deleting BAT2. The variation between the content of higher alcohols, ethyl acetate and expression of related genes in the engineered yeast strain S5-Lg was investigated by fermentation experiments and digital PCR. The results showed that compared with the original strain S5, the ethyl acetate production of the S5-Lg strain was increased by 26.81%, total higher alcohol production decreased by 45.72%, of which the contents of isobutanol and isoamyl alcohol were reduced by 10.63% and 9.55%, respectively. The expression of Lg-ATF1 gene was greatly increased, and the expression level of BAT2 gene was decreased by 45.72%. It effectively changed the ability of brewer's yeast to produce ethyl acetate and higher alcohols and has important significance for improving beer flavor.
引文
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[17]Gietz R D,Woods R A.Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method[J].Methods in Enzymology,2002,350:87-96
[18]Lilly M,Bauer F F,Lambrechts M G,et al.The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates[J].Yeast,2006,23:641-659
[19]李潇,董胜胜,付肖蒙,等.转录因子MSN2基因过表达对酿酒酵母耐受性的影响[J].现代食品科技,2018,34(9):95-102LI Xiao,DONG Sheng-sheng,FU Xiao-meng,et al.Investigation on stress tolerance of saccharomyces cerevisiae of overexpression of transcription factor MSN2 gene[J].Modern Food Science and Technology,2018,34(9):95-102
[20]Zhang C Y,Liu Y L,Qi Y N,et al.Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains[J].European Food Research and Technology,2013,236(6):1009-1014
[21]Pfisterer E,Hancock I,Garrison I.Effects of fermentation environment on yeast lipid synthesis[J].Journal of the American Society of Brewing Chemists,1977,35(2):49-54
[22]Zhang C Y,Liu Y L,Qi Y N,et al.Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains[J].European Food Research and Technology,2013,236(6):1009-1014
[2]张吉娜,何秀萍,郭雪娜,等.低双乙酰抗老化啤酒酵母工程菌的构建[J].生物工程学报,2005,21(6):942-946ZHANG Ji-na,HE Xiu-ping,GUO Xue-na,et al.Genetically modified industrial brewing yeast with high glutathione and low-diacetyl production[J].Chinese Journal of Biotechnology,2005,21(6):942-946
[3]郝俊光,尹花,乔万昌,等.国外对啤酒风味活性酯类形成与调控的认识[J].酿酒科技,2011,9:76-80HAO Jun-guang,YIN Hua,QIAO Wan-chang,et al.Overseas cognition of the formation and the control of beer flavoring active esters[J].Liquor-Making Science&Technology,2011,9:76-80
[4]Verstrepen K J,Derdelinckx G,Dufour J P,et al.Flavor-active esters:Adding fruitiness to beer[J].Journal of Bioscience and Bioengineering,2003,96(2):110-118
[5]肖冬光,陈叶福,张翠英.啤酒生产中高级醇的形成与低产高级醇酵母菌种的选育[J].齐鲁工业大学学报,2013,27(3):15-19XIAO Dong-guang,CHEN Ye-fu,ZHANG Cui-ying.Synthesis of higher alcohols during beer fermentation andbrewing yeast improving for low higher alcohols producing[J].Journal of Qilu University of Technology,2013,27(3):15-19
[6]Pires E J,Teixeira J A,BRA N T,et al.Yeast:The soul of beer's aroma-a review of flavour-active esters and higher alcohols produced by the brewing yeast[J].Applied Microbiology&Biotechnology,2014,98(5):1937-1949
[7]何祖荣.啤酒酿造过程高级醇形成的控制[J].酿酒科技,2001,1:64-65HE Zu-rong.Control of formation of higher alcohols in beer brewing[J].Liquor-Making Science&Technology,2001,1:64-65
[8]顾国贤.论啤酒风味类型[J].酿酒科技,2005,2:26-28GU Guo-xian.Discussion on beer flavor styles[J].Liquor-making Science&Technology,2005,2:26-28
[9]Lilly M,Lambrechts M G,Pretorius I S.Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates[J].Appl Environ Microbiol,2000,66(2):744-753
[10]Dong J,Xu H,Zhao L,et al.Enhanced acetate ester production of Chinese liquor yeast by overexpressing ATF1,through precise and seamless insertion of PGK1,promoter[J].Journal of Industrial Microbiology&Biotechnology,2014,41(12):1823-1828
[11]Cui-Ying Zhang,Yu-Lan Liu,Ya-Nan Qi,et al.Increased esters and decreased higher alcohols production by engineered;brewer's yeast strains[J].European Food Research&Technology,2013,236(6):1009-1014
[12]Verstrepen K J,Laere S D M V,Vanderhaegen B M P,et al.Expression levels of the yeast alcohol acetyltransferase genes ATF1,Lg-ATF1,and ATF2 control the formation of a broad range of volatile esters[J].Applied&Environmental Microbiology,2003,69(9):5228-5237
[13]Zhang J,Zhang C,Wang J,et al.Expression of the Gene Lg-ATF1 Encoding Alcohol Acetyltransferases from Brewery Lager Yeast in Chinese Rice Wine Yeast[C]//Proceedings of the 2012 International Conference on Applied Biotechnology.Tianjin,China:Springer-Verlag Berlin Heidelberg,2014:43-51
[14]Dickinson J R,Salgado L E,Hewlins M J.The catabolism of amino acids to long chain and complex alcohols in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2003,278(10):8028-34
[15]Dickinson J R,Norte V.A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae[J].Febs Letters,1993,326(1-3):29-32
[16]Fukuda K,Yamamoto N,Kiyokawa I Y,et al.Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate[J].Applied&Environmental Microbiology,1998,64(10):4076
[17]Gietz R D,Woods R A.Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method[J].Methods in Enzymology,2002,350:87-96
[18]Lilly M,Bauer F F,Lambrechts M G,et al.The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates[J].Yeast,2006,23:641-659
[19]李潇,董胜胜,付肖蒙,等.转录因子MSN2基因过表达对酿酒酵母耐受性的影响[J].现代食品科技,2018,34(9):95-102LI Xiao,DONG Sheng-sheng,FU Xiao-meng,et al.Investigation on stress tolerance of saccharomyces cerevisiae of overexpression of transcription factor MSN2 gene[J].Modern Food Science and Technology,2018,34(9):95-102
[20]Zhang C Y,Liu Y L,Qi Y N,et al.Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains[J].European Food Research and Technology,2013,236(6):1009-1014
[21]Pfisterer E,Hancock I,Garrison I.Effects of fermentation environment on yeast lipid synthesis[J].Journal of the American Society of Brewing Chemists,1977,35(2):49-54
[22]Zhang C Y,Liu Y L,Qi Y N,et al.Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains[J].European Food Research and Technology,2013,236(6):1009-1014