BAT基因改造对酿酒酵母高级醇生成量的影响

酿酒酵母BAT基因编码支链氨基酸转氨酶,其中BAT1和BAT2基因分别编码线粒体和细胞质氨基酸转氨酶,位于细胞不同的位置导致二者的生理功能有所差异,BAT1基因在线粒体中倾向催化α-酮酸合成氨基酸,细胞质中的BAT2基因将氨基酸转化为α-酮酸,通过敲除BAT2以减少α-酮酸合成,过表达BAT1以增加α-酮酸消耗达到降低酿酒酵母高级醇的合成的目的。本研究以酿酒酵母AY15单倍体α5为出发菌株,结合融合PCR技术构建重组质粒p UC-BABPB1K,获得BA-PGK-BAT1-BB重组盒,并利用醋酸锂转化法和同源重组技术筛选出缺失BAT2基因同时过表达BAT1基因的突变株B-8,将其和亲本菌株α5、BAT2基因缺失菌株α5ΔBAT2进行酒精发酵实验,发酵结束后进行发酵性能和高级醇的测定。实验结果表明,与亲本菌株相比,异丁醇降低了25%,异戊醇降低了15%,活性戊醇降低了30%;与α5ΔBAT2菌株相比,异丁醇提高了0.5倍,异戊醇增加了0.1倍,活性戊醇增加了0.3倍。     

BAT2基因缺失葡萄酒酵母的构建及其对葡萄酒高级醇的影响

该研究以葡萄酒酵母BV-0为出发菌,利用融合聚合酶链式反应（PCR）构建BAT2基因敲除组件,通过电转化导入菌株BV-0进行同源重组,获得BAT2单等位基因敲除菌株BV-bk;再由菌株BV-bk产孢分离出两种不同配型的单倍体BAT2基因敲除菌进行杂交融合,获得BAT2双等位基因敲除菌株BV-BK;最后利用Cre/loxP重组系统去除菌株BV-bk、BV-BK的KanMX抗性基因得到菌株BV-b、BV-B。采用菌株BV-0、BV-b及BV-B分别发酵葡萄酒,并测定葡萄酒的基本理化指标及高级醇含量。结果表明,与出发菌株BV-0相比,菌株BV-b和BV-B发酵的葡萄酒的基本理化指标基本一致,异丁醇含量分别下降14.9%、67.0%,异戊醇含量分别下降17.87%、31.10%,且菌株BV-B发酵葡萄酒中正丙醇含量上升36.55%,说明两株BAT2基因敲除菌株可应用于葡萄酒发酵中以降低异丁醇和异戊醇含量,从而降低葡萄酒的苦味和醉度,具有潜在的工业应用价值。     

酿酒酵母苏氨酸合成酶缺失对高级醇生成量的影响

通过构建酿酒酵母苏氨酸合成酶基因(THR4)缺失的工程菌株,研究该基因对酿酒酵母高级醇生成量的影响。以质粒pUC-19为载体,KanMX抗性基因为筛选标记,构建了重组质粒pUC-TABK,经PCR扩增得到YAKanMX-YB重组盒,并以酿酒酵母AY15单倍体α5为出发菌株,通过醋酸锂转化和G418抗性筛选,获得THR4基因缺失的突变株α5-T7。将转化子和亲本菌株分别进行模拟酒精发酵及酒精浓醪发酵,发酵结束后进行发酵性能和高级醇生成量的测定。结果显示,与亲本菌株相比,突变株正丙醇生成量分别提高了1.61倍和2.6倍,异戊醇生成量分别提高了0.27倍和0.24倍,而异丁醇生成量没有明显差异,表明THR4基因缺失会使酿酒酵母高级醇特别是正丙醇生成量提高。     

LEU1基因缺失对酿酒酵母高级醇生成量的影响

LEU1基因编码异丙基苹果酸合成酶,为了研究该基因对酿酒酵母高级醇生成量的影响,以酿酒酵母AY15单倍体A8为出发菌株,通过构建重组质粒p UC-LABK获得LA-Kan MX-LB重组盒,并利用醋酸锂转化法和同源重组技术,筛选出LEU1基因缺失的突变株A-L9。将突变株和亲本菌株分别进行酒精发酵实验,发酵结束后进行发酵性能和高级醇生成量的测定。两种发酵条件下的实验结果表明,与亲本菌株相比,突变株的正丙醇生成量分别提高了0.18倍和0.47倍,异丁醇的生成量分别提高了0.52倍和1.58倍,而异戊醇的生成量则分别降低了0.29倍和0.51倍。     

一次敲除BAT2同时过表达Lg-ATF1对啤酒酵母产醇酯的影响

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。     

THI3/BAT2基因缺失酿酒酵母菌构建及其在黄酒酿造中的应用

为探究酿酒酵母支链氨基酸转氨酶编码基因（BAT2基因）和类丙酮酸脱羧酶编码基因（THI3基因）的缺失对黄酒中高级醇的影响,以酿酒酵母（Saccharomyces cerevisiae）菌XF1的单倍体XF1a7/XF1α6和THI3基因缺失菌XF1-T的单倍体XF1a7-T/XF1α6-T为原始菌,采用Cre/loxP重组系统构建了BAT2单基因缺失和THI3/BAT2双基因缺失的双倍体酵母重组菌XF1-B和XF1-TB。将XF1、XF1-B和XF1-TB进行黄酒发酵,实验结果表明,重组菌XF1-B、XF1-TB与原始菌XF1的生长性能相似、发酵性能无差异,重组菌XF1-B、XF1-TB酿造黄酒的异戊醇分别降低了18.12%、26.06%,异丁醇分别降低了35.21%、18.83%,正丙醇分别提高了15.42%、32.75%,总高级醇分别为269.59 mg/L、270.77 mg/L,分别降低了15.65%、15.28%。XF1-B和XF1-TB酿造黄酒中异戊醇/异丁醇比值分别为3.24和2.34,降低了27.78%。综上,重组菌XF1-B和XF1-TB都有效降低了黄酒中总高级醇含量,而且XF1-TB与XF1-B相比异戊醇/异丁醇比值显著降低,这能够降低黄酒“上头”的醉度,有效提高黄酒品质。     

ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究

以啤酒酵母工业菌株S5为对照,对过表达醇乙酰基转移酶编码基因ATF1同时敲除氨基酸转氨酶编码基因BAT2酿酒酵母工程菌株S5-1进行发酵性能的研究。结果表明,与出发菌株相比,突变株生长状况、发酵速度、酒精度等基本发酵性能没有明显变化,而突变株发酵后的乙酸酯总量有较大程度的提升,为85.44mg/L,提高了6.96倍,高级醇总量有较大程度的下降,为79.25mg/L,降低了27.28%。研究结果为啤酒风味的改善奠定了良好的基础。     

低产高级醇酿酒酵母菌株筛选及其单倍体制备

通过比较7株酿酒酵母菌株的发酵性能和高级醇生成量,筛选出发酵周期短、发酵速度快、酒精度最高和总高级醇含量较低的菌株AY-15.以AY-15为出发菌株制作单倍体,经过杜氏管发酵实验和酒精浓醪发酵实验,最终获得a-8和α-22 2株单倍体菌株,其发酵性能与AY-15基本相当,酒精度分别为15.6 %vol和15.5 %vol;高级醇含量均比AY-15高级醇生成量低,分别为302.268 mg/L和298.446 mg/L.a-8和α-22可为后续分子育种降低高级醇生成量奠定良好基础.     

乙醛脱氢酶基因过表达酿酒酵母在黄酒中降高级醇作用

为降低黄酒中高级醇产量,以酿酒酵母（Saccharomyces cerevisiae）单倍体菌株AY12α为出发菌,利用同源重组技术构建乙醛脱氢酶基因过表达菌株,并在不同黄酒发酵工艺下探究其对酿酒酵母产高级醇的影响。结果表明,成功构建了5株乙醛脱氢酶基因（ALD2、ALD3、ALD4、ALD5和ALD6）过表达菌株,在大米加麦曲的传统黄酒发酵工艺下,所有改造菌株均可以促进乙酸、降低高级醇的生成,其中改造菌株α-ALD6效果最显著（P＜0.01）,乙酸产量是出发菌AY12α的2.92倍,总高级醇产量下降72.04%。在不同发酵工艺中,改造菌株α-ALD5与α-ALD6均可显著促进乙酸、降低高级醇生成（P＜0.01）;在纯种根霉曲做糖化剂的发酵工艺中,改造菌株在培菌糖化24 h后接入降高级醇效果更好。与纯种根霉曲相比,改造菌株以麦曲作糖化剂时降高级醇效果更好,且改造菌株α-ALD6在大米加麦曲的传统黄酒发酵工艺下降高级醇效果最好。     

酿酒酵母单倍体的分离及其产高级醇和乙酯类化合物的特性

为了获得低产高级醇和高产乙酯类化合物的单倍体酿酒酵母。对5株野生二倍体酿酒酵母菌株进行随机孢子分离获得单倍体后代,并通过Triple M模拟汁发酵,对其单倍体的高级醇和乙酯类物质产量进行研究。结果表明:5株野生酿酒酵母在KAc产孢培养基上的产孢率均高于50%。与二倍体亲本相比,单倍体菌株的高级醇和乙酯类化合物的产量发生显著改变,产高级醇和乙酯类化合物特性在单倍体后代中发生性状分离。单倍体酿酒酵母中,31y3-15高级醇产量最低,为135.3 mg/L,31y3-29和174y1-26的乙酯类物质生成量均高于650mg/L。研究获得的单倍体酿酒酵母,为研究酿酒酵母产高级醇和产酯特性的遗传基础,以及杂交育种提供了重要材料。     

Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains

Esters and higher alcohols produced by yeast during the fermentation of wort have the greatest impact on the smell and taste of beer. Alcohol acetyltransferase, which is mainly encoded by the ATF1 gene, is one of the most important enzymes for acetate ester synthesis. Cytosolic branched-chain amino acid aminotransferase, on the other hand, which is encoded by the BAT2 gene, plays an important role in the production of branched-chain alcohols. The objective of this study is to construct engineered brewer’s yeast strains that produce more acetate esters and less higher alcohols. Industrial brewer’s yeast strain S5 was used as the parental strain to construct ATF1 overexpression and BAT2 deletion mutants. The engineered strains S5-2 and S5-4, which feature partial BAT2 allelic genes replaced by the constructed ATF1 overexpression cassette, were obtained. The ester production of the engineered strains was observed to increase significantly compared with that of the parental cells. The concentrations of ethyl acetate produced by the engineered strains S5-2 and S5-4 increased to 78.88 and 117.40 mg L^?1, respectively, or about 7.7-fold and 11.5-fold higher than that produced by parental S5 cells. The isoamyl acetate produced by S5-2 and S5-4 also increased to 5.14 and 9.25 mg L^?1, respectively; by contrast, no isoamyl acetate was detected in the fermentation sample of the parental strain S5. Moreover, S5-2 and S5-4, respectively, produced about 65 and 51 % of higher alcohols produced by the parental strain. The increase in acetate ester content and decrease in higher alcohol concentration shown by the engineered brewer’s yeast strains at the end of fermentation process indicate that the new strains are useful in future developments in the wheat beer industry.     

芽孢杆菌强化发酵降低小曲白酒中高级醇的含量

为探索芽孢杆菌的添加对小曲酒高级醇含量的影响,以13株大曲来源的芽孢杆菌为出发菌株,通过固态发酵和液态发酵,得到2株可降低小曲酒高级醇含量的芽孢杆菌;经过形态、生理生化和16S rDNA测序分析,鉴定菌株B8为地衣芽孢杆菌（Bacillus licheniformis）,菌株B13为贝莱斯芽孢杆菌（Bacillus velezensis）;将2株功能芽孢杆菌制成芽孢杆菌麸曲,进行小试和中试小曲白酒固态酿造试验,验证芽孢杆菌对小曲白酒风味物质的影响。结果表明,B8与B13强化发酵可明显降低高级醇含量,主要变现为异戊醇与异丁醇的降低,以及微量正戊醇的产生,中试最高降低率可达29.87%,且低于某市售小曲清香型白酒成品酒中高级醇含量。该研究为解决小曲白酒中高级醇含量偏高问题提供了方法,以期提高小曲白酒质量。     

低产高级醇啤酒酵母在啤酒中试中的应用发酵分析

对选育到的低产高级醇啤酒酵母MS-11进行应用发酵研究分析。结果表明,对比出发菌株SC-2和工业生产菌株DAB,MS-11菌株在保持原啤酒风味的基础上,不仅提高发酵速度,缩短发酵时间5d,降低能耗近10％,增加设备周转率20％左右,降低生产成本;而且生产的成品啤酒防老化效果好。     

构建ADH2和THI3基因敲除酿酒酵母用于降低糯米酒中高级醇的研究

为了降低糯米酒高级醇含量,以酿酒酵母（Saccharomyces cerevisiae）菌株XF1的单倍体XF1a7和XF1α6为原始菌,采用Cre/loxP同源重组系统构建乙醇脱氢酶基因ADH2和类丙酮酸脱羧酶基因THI3缺失的单倍体酵母,再通过单倍体的杂交构建ADH2单基因缺失双倍体酵母XF1-A和ADH2与THI3双基因缺失的双倍体酵母XF1-AT。结果表明,重组菌XF1-A、XF1-AT与原始菌XF1的生长性能相似,菌株XF1-A和XF1-AT的基本发酵性能与菌株XF1无显著差异,菌株XF1-A酿造糯米酒中高级醇含量为522.16 mg/L,比菌株XF1低11.16%;菌株XF1-AT的高级醇含量为462.03 mg/L,比菌株XF1低21.39%。综上,ADH2和THI3基因敲除酿酒酵母能够有效降低糯米酒中高级醇生成量。     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

酿酒酵母高级醇合成路径及关键基因

高级醇是在酒精发酵过程中由酿酒酵母代谢产生,在酿酒酵母细胞质和线粒体中由基因编码相关的酶催化α-酮酸脱羧还原而成,是酒的重要香气成分,对酒的香气和口感有重要影响。该文综述了高级醇代谢的相关途径及部分关键基因及功能,以期深入分析了解高级醇并为通过分子生物学方式构建适用于工业生产的低产高级醇的酵母菌株提供理论参考。     

Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation

The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre–loxP system and a removable G418^r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop‐out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling