CN102604848A - 一株高产乙酸酯酿酒酵母工程菌 - Google Patents
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Abstract
一株高产乙酸酯酿酒酵母工程菌。本发明提供的酿酒酵母菌,是选用强启动子PGK1过表达编码醇乙酰基转移酶的ATF2基因,得到高产乙酸酯酿酒酵母工程菌SaccharomycescerevisiaeEY-14,保藏号为CGMCC No.5635。该菌在其它发酵性能不受影响的情况下,转化子菌株与亲本菌株(SaccharomycescerevisiaeCGMCC No2.1525)相比:模拟半固态发酵后,乙酸乙酯含量提高3.6倍,乙酸异戊酯的含量提高到55.2mg/L,乙酸异丁酯含量提高到33.8mg/L;筛选得到的工程菌对发酵设备及条件没有特殊要求,一般酒厂的设备和条件均可使用,因而有广泛的应用前景,能为酒厂的工业化生产带来显著经济效益。
Description
技术领域
本发明属于生物工程技术领域,涉及工业微生物的育种,尤其是一株高产乙酸酯酿酒酵母工程菌。
背景技术
黄酒和白酒是我国的特色酒种,国内普通白酒和黄酒是以纯种培养的酿酒酵母为主进行发酵的,其特点是发酵周期短、原料出酒率高,但由于酿酒酵母产酯香物质的能力极低,致使成品酒品质较差。高档饮料酒(黄酒、白酒)酯香物质含量较高的主要原因是采用自然网罗微生物制曲发酵,通过自然制曲网罗的产酯能力较强的汉逊酵母和假丝酵母等生香微生物来提高酒中酯含量,而这些野生酵母的存在严重影响原料出酒率,其酒精发酵效率不到酿酒酵母的三分之一,因而导致我国高档白酒和黄酒耗粮高、生产周期长、效率低、成本高。
如何提高酒中酯香物质的含量,一直是我国普通白酒、黄酒企业和相关科研单位研究的重要课题。但是,目前国内提高酒中酯香物质含量的方法仍存在很多问题。因此,要从根本上解决酿酒酵母的产酯能力还是需要利用分子生物学育种技术构建高产酯的酿酒酵母工业菌株。
研究表明,乙酸酯类,如乙酸乙酯(溶剂类香气),乙酸异戊酯(香蕉风味)和乙酸异丁酯(成熟水果香),是酒中主要的风味活性酯。这些酯类的形成是在酵母代谢时在酵母内合成的,形成的酯一部分通过细胞扩散到发酵液中,一部分被酵母吸附,留在细胞体内。提高这些乙酸酯的含量不仅可以增进酒的酯香,同时能有效地扩张、松驰神经,可减少喝酒引起的副作用。
参与乙酸酯合成的酶主要是醇乙酰基转移酶(AATase),该酶催化醇和乙酰辅酶A形成乙酸酯。该酶是一种巯基酶,有三种不同的类型:AATase I、Lg-AATase I和AATase II,分别由ATF1、Lg-ATF1和ATF2编码。而国内还没有醇乙酰基转移酶及其编码基因对白酒和黄酒风味和品质影响的相关研究。
发明内容
本发明的目的是解决酿酒酵母自身产酯能力较低的问题,提供一株高产乙酸酯酿酒酵母工程菌株。
本发明提供的高产乙酸酯酿酒酵母(Saccharomyces cerevisiae),具体为EY-14,已于2011年12月22日保藏于中国微生物保藏管理委员会普通微生物中心,地址:中国北京市朝阳区大屯路甲3号,保藏号为CGMCC No 5635。
该酿酒酵母在其它发酵性能不受影响的情况下,模拟半固态发酵后,转化子菌株与亲本菌株(Saccharomyces cerevisiae CGMCC No 2.1525)相比,乙酸乙酯含量提高3.6倍,乙酸异戊酯的含量提高到55.2mg/L,乙酸异丁酯含量提高到33.8mg/L。
本发明高产乙酸酯酿酒酵母工程菌株的构建方法包括:
1)将PGK1启动子和终止子与pUC19质粒连接得到pUC-P;
2)将来源于酿酒酵母的同源片段IAH连接到第1)步得到的pUC-PGK1上,得到pUC-PGK1-IAH;
3)将编码醇乙酰基转移酶ATF2基因插入到第2)步得到的pUC-PGK1-IAH中的PGK1启动子和终止子之间,得到pUC-PGK1-IAH-ATF2;
4)将kan基因连接到第3)步得到的pUC-PGK1-IAH-ATF2上,得到质粒pUC-PGK1-IAH-ATF2-kan(以下简称pUC-PIA2K);
5)将第4)步构建的过表达质粒pUC-PIA2K用Bpu1102I酶切,用醋酸锂转化法分别将其插入酿酒酵母a型和α型单倍体,得到同源重组后的酿酒酵母基因工程单倍体菌株,
6)将纯化后的酿酒酵母a型和α型重组单倍体进行融合,通过抗性平板和生孢实验筛选得到高产乙酸酯酿酒酵母工程菌(双倍体)。
本发明同时提供了一种专门用于鉴定所述高产乙酸酯的酿酒酵母工程菌的基因序列,该基因序列是以ATF-U和ATF-D为引物,以所述高产乙酸酯的酿酒酵母工程菌菌株基因组为模板,扩增片段测序为一特异性序列,如序列表1所示。
模拟半固态发酵:将酵母菌接入5mL麦芽汁培养液中,30℃过夜培养12h;将菌液全部转至20mL新鲜麦芽汁培养液中,30℃培养24h。取100g粳米置于25~30℃的水中,浸渍72h;取出后洗净,常压蒸煮30min。将米摊凉,投入500mL三角瓶中,加入10g熟麦曲和105mL水。最后,接入25mL酵母麦芽汁培养液,28℃发酵5天。对发酵液进行分析,包括失重、酒度、残糖和GC测定香气成分含量。
GC分析:发酵液经蒸馏萃取后采用气相色谱法测定。内标物为乙酸正戊酯。气相色谱仪为Agilent 7890C;色谱柱HPINNOWAX Polyethylene Glyco 260℃:30m×320μm×0.5μm,配FID检测器。载气为高纯氮,流速2.0mL/min。起始柱温为52℃,以2℃/min的升温速度升至70℃,再以4℃/min的升温速度升至90℃,最后以10℃/min的升温速度升至200℃。检测器温度为250℃,进样口温度为230℃,进样量为1.0μL。分流方式为分流,分流比为25∶1。
本发明的优点和积极效果:
本发明选用强启动子PGK1过表达编码醇乙酰基转移酶的ATF2基因,获得了高产乙酸酯酿酒酵母工程菌EY-14(CGMCC No 5635)。
本发明所获得的高产乙酸酯酿酒酵母工程菌Saccjaromyces cerevisiae EY-14(保藏号CGMCC No 5635)与初始的酿酒酵母菌(受体菌Saccharomyces cerevisiae CGMCC No2.1525)相比:模拟半固态发酵后,乙酸乙酯含量提高3.6倍,乙酸异戊酯的含量提高到55.2mg/L,乙酸异丁酯含量提高到33.8mg/L,为酿酒工业生产提供了优良菌株。
附图说明
图1.pUC-PIAK质粒的验证电泳图。
图2.阳性重组酿酒酵母单倍体的验证,其中(a)为a型阳性重组单倍体验证结果;(b)为α型阳性重组单倍体验证结果。
图3.高产乙酸酯酿酒酵母工程菌的构建路线图。
本发明所述的高产乙酸酯酿酒酵母工程菌(Saccharomyces cerevisiae)具体为EY-14,已于2011年12月22日保藏于中国微生物保藏管理委员会普通微生物中心(简称CGMCC,地址为:中国北京市朝阳区大屯路甲3号),保藏号为CGMCC No 5635。
具体实施方式
本发明所使用的酿酒酵母双倍体菌体是可以采用任何来源的酿酒酵母双倍体菌株。
下述实施例中的方法,如无特别说明,均为常规方法。
实施例1:高产酯酿酒酵母基因工程菌的构建
(1)基因工程菌株的构建
1)将PGK1启动子和终止子与pUC19质粒连接得到pUC-PGK1;
2)将来源于酿酒酵母的同源片段IAH连接到第1)步得到的pUC-PGK1上,得到pUC-PGK1-IAH;
3)将编码醇乙酰基转移酶ATF2基因插入到第2)步得到的pUC-PGK1-IAH中的PGK1启动子和终止子之间,得到pUC-PGK1-IAH-ATF2;
4)将kan基因连接到第3)步得到的pUC-PGK1-IAH-ATF2上,得到质粒pUC-PGK1-IAH-ATF2-kan(以下简称pUC-PIA2K);
图1为pUC-PIA2K质粒的验证电泳图:其中泳道1为5000bp DNALadder Marker;泳道2为受体菌基因组PCR扩增同源片断IAH;泳道3为pUC-PIA2K质粒PCR扩增同源片断IAH;泳道4为受体菌基因组PCR扩增ATF2;泳道5为pUC-PIA2K质粒PCR扩增ATF2;泳道6为pUG6质粒PCR扩增Kan;泳道7为pUC-PIA2K质粒PCR扩增Kan;泳道8为pUC-PIA2K质粒,PGK1上游引物+下游引物PCR扩增结果;泳道9为1Kb DNA LadderMarker;泳道10为载体pUC19单酶切线性结果;泳道11为pUC-PIA2K质粒单酶切线性结果。
5)Bpu1102I(Blp I)酶切质粒pUC-PIA2K;用醋酸锂转化法分别将其插入酿酒酵母(Saccharomyces cerevisiae CGMCC No 2.1525)a型和α型单倍体,得到同源重组后的酿酒酵母基因工程单倍体菌株。图2中(a)为a型阳性重组单倍体PCR验证结果;(b)为α型阳性重组单倍体PCR验证结果。其中泳道1为5000bp DNALadder Marker,泳道2为受体菌a/α型单倍体上游PCR阴性对照;泳道3为a/α型重组单倍体上游PCR产物;泳道4为受体菌a/α型单倍体下游PCR阴性对照;泳道5为a/α型重组单倍体下游PCR产物。
6)将纯化后的酿酒酵母a型和α型重组单倍体进行融合,通过抗性平板和生孢实验筛选得到高产乙酸酯酿酒酵母工程菌EY-14(双倍体)。图3为高产乙酸酯酿酒酵母工程菌的构建过程。
(2)基因工程菌株的特异性序列
获得的基因工程菌株EY-14染色体中含有一段特异性序列,可通过PCR扩增测序后进行菌株鉴定。
特异性片段扩增的引物序列为:
ATF-U:5’-TCGGCCTAAACGTTTGCTCCACA-3’
ATF-D:5’-TGGTTTGGAGGAGAAGATAACGACG-3’
该特异性片段的基因序列见序列表1。
实施例2:高产酯酿酒酵母工程菌与出发菌株发酵性能的研究
将实施例1得到的工程菌和受体菌分别接入5mL麦芽汁培养液中,30℃过夜培养12h;将菌液全部转至20mL新鲜麦芽汁培养液中,30℃培养24h。取100g粳米置于25~30℃的水中,浸渍72h;取出后洗净,常压蒸煮30min。将米摊凉,投入500mL三角瓶中,加入10g熟麦曲和105mL水。最后,接入25mL酵母麦芽汁培养液,28℃发酵5天。发酵期间每隔12h振荡并称重,记录失重;发酵结束后,停止培养并称重;测定发酵液的残糖浓度、酒精体积分数以及主要香气成分含量,以发酵能力、残糖浓度和产物生成量表征其综合性能,结果见表1。
表1酿酒酵母受体菌和工程菌的发酵性能
注:所示数据为三个平行试验结果的平均值。
实施例3:高产酯酿酒酵母工程单倍体与出发菌株单倍体发酵性能的研究
将工程单倍体(a/α型)和受体菌单倍体(a/α型)分别接入5mL麦芽汁培养液中,30℃过夜培养12h;将菌液全部转至20mL新鲜麦芽汁培养液中,30℃培养24h。取100g粳米置于25~30℃的水中,浸渍72h;取出后洗净,常压蒸煮30min。将米摊凉,投入500mL三角瓶中,加入10g熟麦曲和105mL水。最后,接入25mL酵母麦芽汁培养液,28℃发酵5天。发酵期间每隔12h振荡并称重,记录失重;发酵结束后,停止培养并称重;测定发酵液的残糖浓度、酒精体积分数以及主要香气成分含量,以发酵能力、残糖浓度和产物生成量表征其综合性能,结果见表2。
表2酿酒酵母受体菌单倍体和工程单倍体的发酵性能
注:所示数据为三个平行试验结果的平均值。
Claims (2)
1.一株高产乙酸酯酿酒酵母(Saccharomyces cerevisiae),具体为EY-14,保藏号为CGMCC No 5635。
2.根据权利要求1所述的酿酒酵母,其特征在于,在其它发酵性能不受影响的情况下,模拟半固态发酵后,转化子菌株与亲本菌株(Saccharomyces cerevisiae CGMCC No 2.1525)相比,乙酸乙酯含量提高3.6倍,乙酸异戊酯的含量提高到55.2mg/L,乙酸异丁酯含量提高到33.8mg/L。
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