CN117384774A - 重组酿酒酵母及其构建方法以及其在发酵产乙醇中的应用 - Google Patents
重组酿酒酵母及其构建方法以及其在发酵产乙醇中的应用 Download PDFInfo
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Abstract
本发明涉及微生物发酵领域,公开了重组酿酒酵母及其构建方法以及其在发酵产乙醇中的应用,所述重组酿酒酵母含有过表达的木糖异构酶XI基因、过表达的木糖转运蛋白TrXlt1基因和过表达的木酮糖激酶XKS1基因。所述重组酿酒酵母优选包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。本发明所述的重组酿酒酵母能够高效利用纤维素原料发酵生产乙醇,能够耐受高浓醪和抑制物的胁迫,具有发酵速度快、高糖耗和高乙醇产量的优点,解决纤维素乙醇工业中木糖消耗速度慢、乙醇产量低、耐胁迫能力差的问题。
Description
技术领域
本发明涉及微生物发酵领域,具体涉及一种重组酿酒酵母,一种重组酿酒酵母的构建方法,一株酿酒酵母(Saccharomyces cerevisiae),一种提高酿酒酵母对胁迫环境耐受性的方法,一种菌剂,所述重组酿酒酵母、所述酿酒酵母或所述菌剂在发酵产乙醇中的应用,以及一种发酵产乙醇的方法。
背景技术
纤维素制造乙醇是世界性难题,其中能够高效利用纤维素酶解液(含葡萄糖和木糖)生产乙醇的菌株成为纤维素乙醇工业的重中之重。酿酒酵母(Saccharomycescerevisiae)由于具有生物安全性(Generally regarded as safe,GRAS)、遗传背景清晰、遗传操作成熟及具有较好的环境胁迫耐受性等优点已成为燃料乙醇工业重要的细胞工厂。近年来,合成生物学、生物技术与生物信息学等不断发展,具有特定功能的调控元件在显示出巨大的应用潜力。合成生物学组件也被称为“合成生物学的基石”,这其中,启动子和终止子又是最核心的合成生物学组件之一,它们也是构建基因表达载体系统和开发合成生物学工具的基本要素。人工合成的启动子和终止子可以通过调节代谢途径生产生物和化学产品,用于工业上大规模的生产过程。利用合成生物方法将木糖代谢的关键基因整合到酿酒酵母基因组中,赋予酿酒酵母木糖发酵能力。
目前,天然利用木糖的微生物将木糖转化为乙醇的代谢途径共两条,一条代谢途径是木糖还原酶和木糖醇脱氢酶途径(XR-XDH途径),另外一条是木糖异构酶途径(XI途径)。CN105199976A发明公开了一株共发酵葡萄糖和木糖的重组酿酒酵母菌株及其应用,该酵母是将含有异源木糖异构酶基因Ru-xyl A表达框的DNA片段整合到染色体的PHO13基因座位,并将含有非氧化磷酸戊糖途径四个基因(RPE1,RKI1,TAL1以及TKL1)串联表达框的两个相应DNA片段整合到上染色体GRE3基因座位的一部分,并将木糖专一性转运蛋白基因Mgt05196(N360F)表达框的DNA片段整合到上述操作获得的菌株GRE3基因座位剩余的另一部分,将重组菌在木糖和预处理玉米秸秆沥出液中进行长期驯化培养。在纤维素乙醇工业中,水解液不需要处理直接进行发酵,菌株受到的胁迫性较强,但专利中重组酿酒酵母在木糖和玉米秸秆沥出液中进行驯化,胁迫环境较弱,不能适用于纤维素乙醇生产环境。CN102199554A发明公开了具有多重胁迫抗性的酿酒酵母菌株及其在纤维素乙醇发酵中的应用,发明提供的重组菌能够适应较宽的发酵温度范围(30-40℃),可以降低发酵过程中的冷却成本,对纤维素酶解液中抑制物具有较高的耐受性,但是重组菌对木糖利用较弱,不能最有效利用纤维素酶解液发酵生产乙醇。
发明内容
本发明的目的是为了克服现有技术存在的酿酒酵母无法同时高效利用纤维素酶解液和对胁迫环境具有高耐受性的问题,提供一种重组酿酒酵母,一种重组酿酒酵母的构建方法,一株酿酒酵母(Saccharomyces cerevisiae),一种提高酿酒酵母对胁迫环境耐受性的方法,一种菌剂,所述重组酿酒酵母、所述酿酒酵母或所述菌剂在发酵产乙醇中的应用,以及一种发酵产乙醇的方法,该重组酿酒酵母能够高效利用纤维素酶解液生产乙醇,且对胁迫环境具有高耐受性。
为了实现上述目的,本发明第一方面提供一种重组酿酒酵母,所述重组酿酒酵母含有过表达的木糖异构酶XI基因、过表达的木糖转运蛋白TrXlt1基因和过表达的木酮糖激酶XKS1基因。
优选地,木糖异构酶XI基因、木糖转运蛋白TrXlt1基因和木酮糖激酶XKS1基因各自独立地通过SEQ ID No.1所示的启动子pUAS-TDH3和SEQ ID No.2所示的终止子t-100调控。
优选地,所述重组酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
本发明第二方面提供一种重组酿酒酵母的构建方法,该方法包括:利用CRISPR/Cas9系统向出发酿酒酵母菌株中导入能够过表达的木糖异构酶XI基因重组片段、能够过表达的木糖转运蛋白TrXlt1基因重组片段和能够过表达的木酮糖激酶XKS1基因重组片段,得到重组酿酒酵母。
本发明第三方面提供一株酿酒酵母(Saccharomyces cerevisiae),所述酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
本发明第四方面提供一种提高酿酒酵母对胁迫环境耐受性的方法,该方法包括对酿酒酵母进行以下至少一种处理,
(1)将CCW14基因位点NC_001144.5:904354的碱基由A突变为C;
(2)将COX14基因位点NC_001145.3:14693的碱基由C突变为T;
(3)将ATG33基因位点NC_001144.5:840758的碱基由G突变为C;
(4)将ATP10基因位点NC_001144.5:907570的碱基由G突变为C;
(5)将CCC1基因位点NC_001144.5:576996的碱基由T突变为G。
本发明第五方面提供一株酿酒酵母(Saccharomyces cerevisiae),该酿酒酵母的保藏编号为GDMCC No.62491。
本发明第六方面提供一株酿酒酵母(Saccharomyces cerevisiae),相比于如第五方面所述的酿酒酵母,该酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
本发明第七方面提供一种菌剂,所述菌剂包含如上所述的重组酿酒酵母和如上所述的酿酒酵母中的至少一种。
本发明第八方面提供如上所述的重组酿酒酵母、如上所述的酿酒酵母或如上所述的菌剂在发酵产乙醇中的应用。
本发明第九方面提供一种发酵产乙醇的方法,该方法包括:将酿酒酵母接种至发酵培养基中进行发酵,生产乙醇;
其中,所述酿酒酵母选自如上所述的重组酿酒酵母、如上所述的酿酒酵母和如上所述的菌剂中的至少一种;
所述发酵培养基包含葡萄糖和木糖。
本发明通过对出发的酿酒酵母菌株进行基因改造,得到本发明所述的重组酿酒酵母,其能够利用纤维素酶解液发酵生产乙醇,且木糖利用率提高,葡萄糖和木糖的代谢速度加快,乙醇产量提高;在经过优选的基因突变处理后,能够进一步提高菌株的代谢速度,缩短发酵时间,提高乙醇产量,菌株对高糖浓度和高乙醇浓度具有更强的耐受性。
本发明中通过对酿酒酵母的Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因中的至少一种进行非同义突变,使得得到的酿酒酵母能够耐受更严苛的发酵环境,能够更快的利用纤维素酶解液中的木糖和葡萄糖进行发酵生产更多的乙醇,有利于工业生产。
本发明所述的酿酒酵母能够利用纤维素酶解液发酵生产乙醇,可以大幅度缩短发酵时间,从而减少能量消耗,降低乙醇生产成本,其在大规模工业生产中具有很大的潜力。
生物保藏
本发明提供的菌株酿酒酵母Saccharomyces cerevisiae SDIC Sc E01的分类命名为Saccharomyces cerevisiae,于2022年5月25日保藏于广东省微生物菌种保藏中心(缩写为GDMCC),其保藏编号为GDMCC No.62491,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1是本发明实施例5中SDIC Sc、SDIC Sc 1、SDIC Sc 2、SDIC Sc E、SDIC Sc C和SDIC Sc E01在混合糖培养基中进行摇瓶发酵的过程中葡萄糖、木糖和乙醇的含量变化图;
图2是本发明实施例6中菌株SDIC Sc E01在玉米秸秆纤维素酶解液中进行摇瓶发酵的过程中葡萄糖、木糖、乙醇、甘油和乙酸的含量变化图。
图3是本发明实施例6中菌株SDIC Sc E在玉米秸秆纤维素酶解液中进行摇瓶发酵的过程中葡萄糖、木糖、乙醇、甘油和乙酸的含量变化图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
本发明第一方面提供一种重组酿酒酵母,所述重组酿酒酵母含有过表达的木糖异构酶XI基因、过表达的木糖转运蛋白TrXlt1基因和过表达的木酮糖激酶XKS1基因。
在本发明中,通过启动子和终止子控制基因的表达,优选地,木糖异构酶XI基因、木糖转运蛋白TrXlt1基因和木酮糖激酶XKS1基因各自独立地通过SEQ ID No.1所示的启动子pUAS-TDH3和SEQ ID No.2所示的终止子t-100调控。在所述优选的情况下,能够使得重组酿酒酵母能够更高效的利用纤维素酶解液生产乙醇。
所述木糖异构酶XI基因可以为本领域常规使用的XI基因,比如可以为CN111235138A公开的XI基因。
优选地,针对木糖异构酶XI基因,所述重组酿酒酵母含有UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)重组片段,更优选含有SEQ ID No.3所示的序列。
其中,UP(PHO13)同源臂(300bp)、Down(PHO13)同源臂(311bp)以酿酒酵母基因组为模板进行扩增,pUAS-TDH3(785bp)、XI-t-100(1476bp)片段合成于北京擎科生物科技有限公司。
在本发明中,可以采用本领域常规的技术手段构建重组片段并进行扩增,比如可以采用按照FastPfu DNA Polymerase说明,以表1中引物扩增相应的片段,获得目的片段,利用融合PCR技术扩增重组片段。本领域技术人员可以参照本领域常规的步骤进行操作,在此不再赘述。
优选地,所述木糖异构酶XI基因位于PHO13基因座位,更优选位于IV染色体的32584和32764位点之间。
木糖转运蛋白TrXlt1基因可以为本领域常规使用的TrXlt1基因,比如可以来源于里氏木霉Trichoderma reesei(GenBank:AAX21096.1)。
优选地,针对木糖转运蛋白TrXlt1基因,所述重组酿酒酵母含有UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)重组片段,更优选含有SEQ ID No.4所示的序列。
其中,UP(ALD6)同源臂(525bp)、Down(ALD6)同源臂(508bp)以酿酒酵母基因组为模板进行扩增。
优选地,所述木糖转运蛋白TrXlt1基因位于ALD6基因座位,更优选位于XVI染色体的433122和4333268位点之间。
木酮糖激酶XKS1基因可以为本领域常规使用的XKS1基因,比如可以来源于酿酒酵母(Saccharomyces cerev isiae)或嗜热细菌(Thermus thermophilus)。
优选地,针对木酮糖激酶XKS1基因,所述重组酿酒酵母含有UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)重组片段,更优选含有SEQ ID No.5所示的序列。
其中,UP(XKS1)同源臂(463bp)、Down(XKS1)同源臂(500bp)以酿酒酵母基因组为模板进行扩增。
优选地,所述木酮糖激酶XKS1基因位于酿酒酵母ARS737上游(优选为200bp),更优选位于VII染色体的888363和888412位点之间。
在本发明中,所述重组酿酒酵母还可以含有基因的突变,优选地,所述重组酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
其中,Ccw14(GenBank:QHB10530.1)基因为编码共价连接细胞壁蛋白(Ccw14p)的基因,Cox14(GenBank:QHB10610.1)基因为编码细胞色素c氧化酶(Cox14p)的基因,Atg33(GenBank:KZV09604.1)基因为编码线粒体分裂特异蛋白(Atg33p)的基因,Atp10(GenBank:QHB10532.1)基因为编码ATP合成酶(Atp10p)的基因,Ccc1(GenBank:QHB10370.1)基因为编码液泡Fe2+/Mn2+转运蛋白(Ccc1p)的基因。
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由A突变为C。
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由T突变为C。
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由C突变为G。
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由C突变为G。
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由G突变为T。
Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的突变可以任意地为纯合突变或杂合突变。优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变。
上述基因的突变使得相应编码的蛋白也发生突变,优选地,所述重组酿酒酵母中,共价连接细胞壁蛋白(Ccw14p)的突变为CCW14 I211L;优选地,所述重组酿酒酵母中,所述细胞色素c氧化酶(Cox14p)的突变为COX14 T21A;优选地,所述重组酿酒酵母中,所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33D146E;优选地,所述重组酿酒酵母中,所述ATP合成酶(Atp10p)的突变为ATP10 D164E;优选地,所述重组酿酒酵母中,所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1G58C。
上述重组酿酒酵母的构建方法可以为本领域常规的构建方法,比如可以为本发明第二方面所述的方法。
本发明第二方面提供一种重组酿酒酵母的构建方法,该方法包括:利用CRISPR/Cas9系统向出发酿酒酵母菌株中导入能够过表达的木糖异构酶XI基因重组片段、能够过表达的木糖转运蛋白TrXlt1基因重组片段和能够过表达的木酮糖激酶XKS1基因重组片段,得到重组酿酒酵母。
优选地,木糖异构酶XI基因、木糖转运蛋白TrXlt1基因和木酮糖激酶XKS1基因各自独立地通过SEQ ID No.1所示的启动子pUAS-TDH3和SEQ ID No.2所示的终止子t-100调控。
优选地,能够过表达的木糖异构酶XI基因重组片段为UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)重组片段,更优选含有SEQ ID No.3所示的序列。
在本发明中,可以采用本领域常规的重组质粒实现相关基于的导入,重组质粒的构建可以按照本领域常规的方法进行。用于制备所述重组质粒的骨架可以为本领域常规使用的质粒,优选采用pRS423-KanMX-gRNA-GAL1p-Cas9为骨架。
实现XI基因导入的重组质粒可以为本领域常规的重组质粒,优选地,相应的重组质粒为pRS423-KanMX-gRNA(XI)-GAL1p-Cas9重组质粒,更优选具有SEQ ID No.6所示的序列。
优选地,能够过表达的木糖转运蛋白TrXlt1基因重组片段为UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)重组片段,更优选含有SEQ ID No.4所示的序列。
实现TrXlt1基因导入的重组质粒可以为本领域常规的重组质粒,优选地,相应的重组质粒为pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9重组质粒,更优选具有SEQ ID No.7所示的序列。
优选地,能够过表达的木酮糖激酶XKS1基因重组片段为UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)重组片段,更优选含有SEQ ID No.5所示的序列。
实现TrXlt1基因导入的重组质粒可以为本领域常规的重组质粒,优选地,相应的重组质粒为pRS423-KanMX-gRNA(XKS1)-GAL1p-Cas9重组质粒,更优选具有SEQ ID No.8所示的序列。
重组质粒的制备方法比如可以为:利用CHOPCHOP网站(https://chopchop.cbu.uib.no/)检索靶位点PHO13基因、ADL6基因、ARS737上游-200bp区域的特异性的gRNA序列。据此,设计携带有N20序列的Cas9靶位点,设计正向引物并在引物5′端插入N20(5′→3′),设计反向引物并在引物5′端插入N20(5′→3′)前15bp反向互补序列。利用质粒pRS423-KanMX-gRNA-GAL1p-Cas9为骨架,反向扩增pRS423-KanMX-gRNA-GAL1p-Cas9特异gRNA序列(N20)的线性质粒,进一步将线性质粒纯化和DpnI进行37℃消化处理2h,纯化后的线性质粒通过15bp同源序列实现质粒环化(利用试剂盒Clone ExpressTM II One StepCloning Kit完成),构建pRS423-KanMX-gRNA(XI)-GAL1p-Cas9、pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9和pRS423-KanMX-gRNA(XKS1)-GAL1p-Cas9。消化反应可以按照DpnI使用说明书进行。
利用CRISPR/Cas9系统向出发酿酒酵母菌株中导入基因重组片段的方法可以为本领域常规的方法,比如可以制备酿酒酵母感受态,将构建好的融合基因片段和相应的重组质粒转化进入感受态中培养,然后利用酵母菌落PCR方法以融合基因片段中相应的同源臂为引物扩增目的片段进行测序分析,测序正确的菌株可以进行丢质粒处理,并进行验证,得到的丟质粒的菌株即为目标菌株。丟质粒的方法可以为本领域常规方法,比如可以将菌株在YPX中传代培养,每天转接一次,丢掉质粒。多次传代后,可以在含G418的YPX平板中验证,不生长菌株即为丟质粒的菌株。
优选地,该方法还包括对重组酿酒酵母进行基因突变处理,所述突变使得所述重组酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。相应的突变位点和突变方式可以参见第一方面的描述,在此不再说明。
优选地,所述突变使得所述重组酿酒酵母中,共价连接细胞壁蛋白(Ccw14p)的突变为CCW14 I211L。
优选地,所述突变使得所述细胞色素c氧化酶(Cox14p)的突变为COX14 T21A;所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33D146E。
优选地,所述突变使得所述ATP合成酶(Atp10p)的突变为ATP10 D164E。
优选地,所述突变使得所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1G58C。
所述突变的方法可以为本领域常规的能够导致上述突变的方法,比如可以通过随机诱变(比如驯化)或着定点诱变(比如可以为胞苷脱氨酶碱基编辑系统等)的方法。
本发明第三方面提供一株酿酒酵母(Saccharomyces cerevisiae),所述酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
相应的突变位点和突变方式可以参见第一方面的描述,在此不再说明。
本发明第四方面提供一种提高酿酒酵母对胁迫环境耐受性的方法,该方法包括对酿酒酵母进行以下至少一种处理,
(1)将CCW14基因位点NC_001144.5:904354的碱基由A突变为C;
(2)将COX14基因位点NC_001145.3:14693的碱基由C突变为T;
(3)将ATG33基因位点NC_001144.5:840758的碱基由G突变为C;
(4)将ATP10基因位点NC_001144.5:907570的碱基由G突变为C;
(5)将CCC1基因位点NC_001144.5:576996的碱基由T突变为G。
优选地,(1)至(5)中任意一项所述的突变为杂合突变或纯合突变,更优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的突变为纯合突变,Ccc1基因上的突变为杂合突变。
相应的突变位点和突变方式可以参见第一方面的描述,可以通过第二方面所述的突变方法进行处理,在此不再说明。
优选地,所述处理使得酿酒酵母耐受浓度为50g/L以上的乙醇。
优选地,所述处理使得酿酒酵母耐受浓度为80g/L以上的葡萄糖和/或30g/L以上的木糖。
优选地,所述处理使得酿酒酵母耐受4g/L以上的乙酸和/或1g/L以上的糠醛和/或0.5g/L以上的5-羟甲基糠醛。
本发明第五方面提供一株酿酒酵母(Saccharomyces cerevisiae),该酿酒酵母的保藏编号为GDMCC No.62491。
本发明提供的菌株酿酒酵母Saccharomyces cerevisiae SDIC Sc E01的分类命名为Saccharomyces cerevisiae,于2022年5月25日保藏于广东省微生物菌种保藏中心(缩写为GDMCC),其保藏编号为GDMCC No.62491,保藏地址为广州市先烈中路100号大院59号楼5楼。
本发明中,利用CRISPR/Cas9系统向出发酿酒酵母菌株SDIC Sc中导入能够过表达的木糖异构酶XI基因重组片段、能够过表达的木糖转运蛋白TrXlt1基因重组片段和能够过表达的木酮糖激酶XKS1基因重组片段,得到重组酿酒酵母SDIC Sc E,使用纤维素酶解液对SDIC Sc E进行多次驯化,得到能够适应高抑制物的酿酒酵母菌株SDIC Sc E01,为本方面所述的酿酒酵母GDMCC No.62491。
本发明第六方面提供一株酿酒酵母(Saccharomyces cerevisiae),相比于如第五方面所述的酿酒酵母,该酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变。
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由C突变为A。
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由C突变为T。
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由G突变为C。
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由G突变为C。
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由T突变为G。
Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的突变可以任意地为纯合突变或杂合突变。优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变。
优选地,相比于第五方面所述的酿酒酵母,所述酿酒酵母中共价连接细胞壁蛋白(Ccw14p)的突变为CCW14 L211I;优选地,相比于第五方面所述的酿酒酵母,所述酿酒酵母中所述细胞色素c氧化酶(Cox14p)的突变为COX14 A21T;优选地,相比于第五方面所述的酿酒酵母,所述酿酒酵母中所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33E146D;优选地,相比于第五方面所述的酿酒酵母,所述酿酒酵母中所述ATP合成酶(Atp10p)的突变为ATP10E164D;优选地,相比于第五方面所述的酿酒酵母,所述酿酒酵母中所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1C58G。
相比于如第五方面所述的酿酒酵母,酿酒酵母具有位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的非同义突变,CCW14基因的突变位点为NC_001144.5:904354,碱基由C突变为A,COX14基因的突变位点为NC_001145.3:14693,碱基由C突变为T,ATG33基因的突变位点为NC_001144.5:840758,碱基由G突变为C,CCC1基因的突变位点为NC_001144.5:576996,碱基由T突变为G;Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变,此时的酵母为重组酿酒酵母SDIC Sc E。
本发明第七方面提供一种菌剂,所述菌剂包含如上所述的重组酿酒酵母和如上所述的酿酒酵母中的至少一种。
所述菌剂的形式可以不受特别的限制,比如所述菌剂可以为液体菌剂、半液体菌剂或固体菌剂。
优选地,所述菌剂中所述酿酒酵母的活菌浓度为108CFU/g以上。
其制备方法可以是本领域常规的制备方法。
本发明第八方面提供如上所述的重组酿酒酵母、如上所述的酿酒酵母或如上所述的菌剂在发酵产乙醇中的应用。
优选地,用于发酵产乙醇的原料包含纤维素原料。
关于纤维素原料的描述请参见第九方面。
本发明第九方面提供一种发酵产乙醇的方法,该方法包括:将酿酒酵母接种至发酵培养基中进行发酵,生产乙醇;
其中,所述酿酒酵母选自如上所述的重组酿酒酵母、如上所述的酿酒酵母和如上所述的菌剂中的至少一种;
所述发酵培养基包含葡萄糖和木糖。
优选地,所述发酵培养基中含有60g/L以上的葡萄糖和20g/L以上的木糖,更优选含有60-90g/L的葡萄糖和20-50g/L的木糖。
所述发酵培养基中可以包含纤维素酶解液,用于提供葡萄糖和木糖。所述纤维素酶解液的制备方法可以为本领域常规的制备方法,比如可以对纤维素原料进行粉碎、水解和酶解处理,得到纤维素酶解液。所述纤维素原料可为玉米秸秆、小麦粉秸秆和玉米芯等本领域常规使用的纤维素原料。其中,粉碎、水解和酶解的方式可以为本领域常规的处理方式,在此不再具体说明。优选地,所述纤维素酶解液含有80g/L以上的葡萄糖和30g/L以上的木糖。
在本发明中,所述酿酒酵母在接种前可以先进行至少一级扩培,所述扩培可以在摇瓶和/或扩培罐中进行,得到中资源。比如,可以在摇瓶中进行一级扩培,在扩培罐中进行二级扩培,得到种子液。其中,每级扩培过程中的pH值分别可以为3-6;每级扩培温度分别可以为28-32℃;每级扩培的时间分别可以为12-30小时。用于扩培的培养基可以为本领域常规使用的培养基,比如可以含有酵母所需氮源(比如蛋白胨和/或酵母膏)、葡萄糖和/或木糖,还可以含有无机盐、微量元素和维生素等。比如可以种子培养基,其包含1-5重量%的葡萄糖、1-5重量%的蛋白胨和0.5-2重量%的酵母膏,pH为5-6;或者可以为混合糖培养基,其包含5-8重量%的葡萄糖、1-5重量%的木糖、1-5重量%的蛋白胨和0.5-2重量%的蛋白胨,pH为5-6。可以使用氨水对pH进行调节。
所述种子液的接种量可以在较宽的范围选择,优选为1-10体积%。
所述发酵可以在摇瓶或者发酵罐中进行,本领域技术人员可以根据实验或生产规模选择合适的发酵容器。本领域技术人员可以根据实际情况调控发酵过程中的各参数。
优选地,所述发酵的条件包括:所述发酵的pH值为3-6;温度为28-32℃;时间为32-48小时,优选48-80小时。
应当理解的是,还可以对发酵得到的物料进行后处理,比如进行蒸馏,以获得含有不同浓度乙醇的物料。具体的方法可以为本领域常规的方法,本领域技术人员可以根据需要选择。
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
以下将通过实施例对本发明进行详细描述。下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明实施例中培养基、酶与试剂、相关微生物及分子生物学技术
(1)培养基
LB培养基:10g/L蛋白胨,5g/L酵母提取物,10g/L NaCl;固体培养基添加20g/L琼脂粉;灭菌条件:115℃,30min;使用时,添加氨苄青霉素(Amp)至100μg/m L用于筛选E.coli转化子。
YP培养基:20g/L蛋白胨,10g/L酵母提取物,调节pH至5.5;固体培养基添加20g/L琼脂粉;灭菌条件:115℃,30min。使用时,添加不同浓度的葡萄糖或木糖作为碳源分别制成YPD或YPX培养基,或同时添加葡萄糖和木糖做为碳源制成混合糖培养基,用于菌株生长及发酵性能的检测。必要时添加800μg/mL G418用于相应重组酿酒酵母菌株筛选及培养。
玉米秸秆纤维素酶解液制备:
汽爆玉米秸秆含水量是61.4%,酶解液底物浓度为20%,100g的酶解液需要称取汽爆秸秆51.8g,加入自来水46.6g并用氨水调节pH值5.0,加入8%(w/w底物干物质)1.6gCellic Ctec3(公司:诺维信),在50℃、100rpm条件下酶解72h,利用HPLC检测,葡萄糖含量为86.1g/L,木糖35.8g/L,乙酸4g/L,糠醛1g/L,5-羟甲基糠醛0.5g/L。
(2)酶与试剂
Clone ExpressTM II One Step Cloning Kit:公司:Vazyme/诺唯赞,货号C112-02;
FastPfu DNA Polymerase:公司:全式金生物,货号:AP221-01;
Dpn1:公司Thermo ScientificTM,货号:FD1703。
葡萄糖、木糖、乙酸、乙醇、糠醛、羟甲基糠醛、蛋白胨、酵母提取物、琼脂粉、浓硫酸等均购买与Solarbio/索莱宝。
(3)分子技术操作
大肠杆菌感受态细胞转化
按照:Biomed/博迈德的大肠杆菌感受态细胞(NEB10-beta感受态细胞,公司:Biomed/博迈德,货号:BC113-01)的转化说明书进行操作。
醋酸锂转化法
挑取单菌落接种至酵母培养基中过夜培养(30℃,250rpm/min),18h后吸取种子液转接至酵母培养基(50mL YPD的250mL的三角瓶),起始OD600控制在0.1,培养6-8h后至OD600=0.8-1.6;取50mL细胞液,3000g离心3min,收集细胞;30mL无菌ddH2O水洗,再同上离心;弃去水,把细胞悬浮1mL的无菌水中,混匀后按每管100μL分装于1.5mL EP管,5000×g离心1min后去上清,备用。取1.5mL的EP管,加入100μL的100mmol/L醋酸锂混匀,5000×g离心5s,沉淀细胞,弃去醋酸锂;向EP管中依次加入240μL的PEG MW 3350(50%,w/v),36μL 1.0M醋酸锂,25μL 2mg/mL单链载体DNA(Single-strandedcarrier DNA),加入线性质粒片段4μL,剩余加入无菌水至总体积360μL,重悬菌体,充分混匀。30℃培养箱中保温30min;然后42℃水浴锅中热激22min;4000g离心1min,弃去上清,用适量无菌水重悬,涂布于筛选平板,30℃培养,48h后可观察到单菌落。
菌落PCR验证
酵母菌落PCR验证:挑取适量单克隆于PCR管中,加入15-20μL的20mmol/L NaOH溶液,吹吸混匀;100℃加热10min;-80℃冷冻10min;100℃加热10min。吸取1-2μL作为模板,进行PCR扩增验证。
大肠杆菌菌落PCR验证:挑取适量单克隆于PCR管中,加入酶扩增体系,吹吸混匀,PCR扩增验证。
丢质粒方法
重组酿酒酵母中含有质粒,将测序正确的重组菌在YPX中传代培养,每天转接一次,丢掉质粒,传代10天后将菌株涂布与G418的YPX平板中验证,不生长菌株即丢掉质粒,得到重组菌。
(4)HPLC检测:
检测柱:Bio-Rad Aminex HPX-87H,300×7.8mm
检测条件:0.5mM硫酸、0.6mL/min,柱温63℃,示差检测器,温度35℃,时间为30min。
(5)引物和基因
本发明中的引物和基因均在北京擎科生物科技有限公司合成。
实施例1
本实施例用于说明重组片段和重组质粒的构建
(1)重组片段的构建:以酿酒酵母基因组为模板,按照FastPfu DNAPolymerase说明,以表1中引物扩增相应的片段,获得片段UP(PHO13)同源臂(300bp)、Down(PHO13)同源臂(311bp)、UP(ALD6)同源臂(525bp)、Down(ALD6)同源臂(508bp)、UP(XKS1)同源臂(463bp)、Down(XKS1)同源臂(500bp)。
片段pUAS-TDH3(785bp)、XI-t-100(1476bp)、TrXlt1-t-100(1761bp)、XKS1-t-100(1959bp)为北京擎科生物科技有限公司合成。
以表1中引物,采用融合PCR技术扩增重组片段UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)(SEQ ID NO.3)、UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)(SEQ IDNO.4)、UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)(SEQ ID NO.5)。
(2)重组质粒构建:利用CHOPCHOP网站(https://chopchop.cbu.uib.no/)检索靶位点PHO13基因、ADL6基因、ARS737上游-200bp区域的特异性的gRNA序列。据此,设计携带有N20序列的Cas9靶位点,设计正向引物并在引物5′端插入N20(5′→3′),设计反向引物并在引物5′端插入N20(5′→3′)前15bp反向互补序列,反向扩增pRS423-KanMX-gRNA-GAL1p-Cas9特异gRNA序列(N20)的线性质粒,进一步将线性质粒纯化和DpnI进行37℃消化处理2h,消化反应按照说明书。纯化后的线性质粒通过15bp同源序列实现质粒环化(利用试剂盒Clone ExpressTM II One Step Cloning Kit完成),构建pRS423-KanMX-gRNA(XI)-GAL1p-Cas9(SEQ ID NO.6)、pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9(SEQ ID NO.7)和pRS423-KanMX-gRNA(XKS1)-GAL1p-Cas9(SEQ ID NO.8)。质粒转化NEB10-beta感受态细胞,并送至北京擎科生物科技有限公司测序分析。
表1引物
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实施例2
本实施例用于说明重组酿酒酵母的构建。
1、重组菌SDIC Sc 1的构建
制备酿酒酵母SDIC Sc(实验室保存菌)菌株感受态,将构建好的融合基因片段UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)和pRS423-KanMX-Grna(XI)-GAL1p-Cas9转化进入酿酒酵母的感受态中,涂布与G418 YPX平板中,30℃培养2-3天。利用酵母菌落PCR方法,以UP(PHO13)-F和Down(PHO13)-R为引物扩增目的片段,并送至北京擎科生物科技有限公司测序分析。将上述测序正确的菌株在YPX中传代培养,每天转接一次,丢掉质粒,传代10天后将菌株涂布与G418的YPX平板中验证,不生长菌株即丢掉质粒,得到重组菌SDIC Sc 1。
2、重组菌SDIC Sc 2的构建
制备酿酒酵母SDIC Sc 1感受态,将构建好的融合基因片段UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)和pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9转化进入酿酒酵母SDIC Sc 1菌株的感受态中,涂布与G418 YPX平板中,30℃培养2-3天。利用酵母菌落PCR方法,以UP(ALD6)-F和Down(ALD6)-R为引物扩增目的片段,并送至北京擎科生物科技有限公司测序分析。将上述测序正确的菌株在YPX中传代培养,每天转接一次,丢掉质粒,传代10天后将菌株涂布与G418的YPX平板中验证,不生长菌株即丢掉质粒,得到重组菌SDIC Sc 2。
3、重组菌SDIC Sc E的构建
制备酿酒酵母SDIC Sc 2感受态,将构建好的融合基因片段UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)和pRS423-KanMX-Grna(XKS1)-GAL1p-Cas9转化进入酿酒酵母SDICSc 2菌株的感受态中,涂布与G418 YPX平板中,30℃培养2-3天。利用酵母菌落PCR方法,以UP(XKS1)-F和Down(XKS1)-R为引物扩增目的片段,并送至北京擎科生物科技有限公司测序分析。将上述测序正确的菌株在YPX中传代培养,每天转接一次,丢掉质粒,传代10天后将菌株涂布与G418的YPX平板中验证,不生长菌株即丢掉质粒,得到重组菌SDIC Sc E。
4、重组菌SDIC Sc C的构建
以启动子pADH1(417bp,SEQ ID NO.9)替换启动子pUAS-TDH、终止子tTEF1(198bp,SEQ ID NO.10)替换t100,调控XI、TrXlt1、XKS1,按照重组菌SDIC Sc E的构建方法构建重组菌SDIC Sc C。
实施例3
本实施例用于说明酿酒酵母SDIC Sc E01的获取。
将菌株SDIC Sc E接种于装有20mL种子培养基(木糖2%,蛋白胨2%,酵母膏1%,pH5.5)的50mL摇瓶中,30℃、200r/min的条件下恒温培养24小时后,转接到含有50mL纤维素酶解液的250mL三角瓶中,接种量使得培养基的OD600为1,在30℃、200r/min条件下发酵48小时,得到一代培养液。将2.5mL一代培养液接种到装有47.5mL纤维素酶解液的250mL三角瓶中,在30℃、200r/min条件下继续发酵48小时,得到二代培养液,然后以相同的方法传代培养。以此类推,驯化进展150次传代培养,获得能够适应高抑制物的酿酒酵母菌株SDIC ScE01,将其保存,备用。
该菌株即为本发明提供的菌株酿酒酵母Saccharomyces cerevisiae SDIC ScE01,其分类命名为Saccharomyces cerevisiae,于2022年5月25日保藏于广东省微生物菌种保藏中心(缩写为GDMCC),其保藏编号为GDMCC No.62491,保藏地址为广州市先烈中路100号大院59号楼5楼。
实施例4
本实施例用于说明与酵母菌株SDIC Sc E01胁迫抗性性能关联的遗传变异
通过对酵母菌株SDIC Sc E、SDIC Sc E01进行全基因组重测序,利用比较基因组学分析手段,挖掘与两菌株之间的积累非同义突变,相比于SDIC Sc E,SDIC Sc E01中积累的非同义突变结果如表2所示。
菌株SDIC Sc E、SDIC Sc E01的基因组DNA和测序文库由杭州联川生物技术有限公司提取和构建,采用Illumina HiSeq 4000高通量测序平台的150bp双端测序的方法测序。以酿酒酵母菌株S288c为参考基因组(RefSeq assembly accession:GCF_000146045.2),获得17.1百万Clean reads,测序深度为147x和98.8%的测序覆盖率。
采用McKenna等人报道的GATK v3.5基因组分析流程,检测两菌株中的单核苷酸多态性(single nucleotide polymorphism,SNP)突变和插入/缺失(insertion/deletion,InDels)突变(McKenna,A.,et al.,The Genome Analysis Toolkit:a MapReduceframework for analyzing next-generation DNA sequencing data.Genome Res,2010.20(9):p.1297-303.)。然后,对发生在蛋白编码区的非同义突变利用SIFT进行突变对蛋白功能影响的预测分析。(Kimon Frousios.et al.,Predicting the functionalconsequences of non-synonymous DNA sequence variants--evaluation ofbioinformatics tools and development of a consensusstrategy.Genomics.2013.102(4):223-228)
表2 SDIC Sc E01中积累的非同义突变Gene
注:WT:野生型;MT:突变;Homo:纯合;Het:杂合。蛋白功能损伤预测值越小,说明该突变对蛋白结构和功能的影响越大。
基因组重测序分析锁定候选的非同义突变,分别以SDIC Sc E和SDIC Sc E01的基因组DNA为模板,经PCR扩增突变所在的DNA片段,并在北京擎科生物技术有限公司进行Sanger测序,结果如表2所示。与SDIC Sc E相比,在SDIC Sc E01中发现四个纯合的非同义突变,即CCW14I211L、COX14T21A、ATG33D146E、ATP10D164E,发现了一个杂合突变,即CCC1G58C。这些突变所影响的生理功能包括:共价连接细胞壁蛋白(Ccw14p)、细胞色素c氧化酶(Cox14p)、线粒体分裂特异蛋白(Atg33p)、ATP合成酶(Atp10p)、液泡Fe2+/Mn2+转运蛋白(Ccc1p)。
实施例5
本实施例用于说明酿酒酵母混合糖发酵性能的比较。
将酵母菌株SDIC Sc、SDIC Sc 1、SDIC Sc 2、SDIC Sc E、SDIC Sc C、SDIC Sc E01分别接种于盛有20mL种子培养基(葡萄糖2%,蛋白胨2%,酵母膏1%,pH5.5)的50mL摇瓶中,在30℃、200r/min条件下培养24小时,得到种子液。
分别取5mL种子液转接到装有200mL混合糖发酵培养基(葡萄糖7.5%,木糖2.5%,蛋白胨2%,酵母膏1%,pH5.5)的500mL摇瓶中,在30℃、200r/min条件下发酵60h,利用HPLC检测发酵液中葡萄糖、木糖消耗、乙醇含量。发酵实验重复三次,数据取平均值进行计算。
发酵过程代谢物的变化如图1所示,其中,SDIC Sc发酵过程中各组分含量的变化如图1-A所示,SDIC Sc 1发酵过程中各组分含量的变化如图1-B所示,SDIC Sc 2发酵过程中各组分含量的变化如图1-C所示,SDIC Sc E发酵过程中各组分含量的变化如图1-D所示,SDIC Sc C发酵过程中各组分含量的变化如图1-E所示,SDIC Sc E01发酵过程中各组分含量的变化如图1-F所示。
从图1中可以看出,SDIC Sc E01的乙醇产量、葡萄糖和木糖消耗速度菌快于SDICSc、SDIC Sc 1、SDIC Sc 2、SDIC Sc E、SDIC Sc C,其中SDIC Sc、SDIC Sc 1、SDIC Sc 2、SDIC Sc E、SDIC Sc C和SDIC Sc E01的最高乙醇产量分别为29.6g/L、34.5g/L、37.3g/L、41.3g/L、32.5g/L和46.5g/L。SDIC Sc、SDIC Sc 1、SDIC Sc 2、SDIC Sc E、SDIC Sc C、SDICSc E01混合糖醇转化率分别为0.38g/g、0.41g/g、0.41g/g、0.42g/g、0.39g/g和0.47g/g,与SDIC Sc相比转化率分别提高了7.9%、7.9%、10.5%、4.3%、23.7%。菌株SDIC Sc E01的葡萄糖24h消耗完全,但SDIC Sc E的葡萄糖需要48h消耗完全;通过启动子调控,SDIC Sc E的乙醇产量高于SDIC Sc C,说明通过本发明所述的启动子调控,有助于糖利用效率和乙醇产量提高;菌株SDIC Sc E01的木糖消耗速度显著快于菌株SDIC Sc E,且发酵结束时,菌株SDIC Sc E仍有5g/L木糖残留通过驯化,提高了菌株SDIC Sc E01的葡萄糖和木糖消耗速度,且提高乙醇产量。
通过图1的结果可知,与出发菌株SDIC Sc以及SDIC Sc1和SDIC Sc2相比,SDIC ScE提高了葡萄糖和木糖的代谢速度、木糖利用效率和乙醇产量;而通过工业玉米秸秆纤维素酶解液驯化SDIC Sc E得到的SDIC Sc E01,相比于SDIC Sc E,进一步提高了糖消耗速度和乙醇产量。
实施例6
本实施例用于说明酿酒酵母SDIC Sc E和SDIC Sc E01纤维素酶解液发酵的结果。
将酵母菌株SDIC Sc E和SDIC Sc E01分别接种于盛有20mL种子培养基(2%葡萄糖,蛋白胨2%,酵母膏1%,pH5.5)的50mL摇瓶中,在30℃、200r/min条件下培养24小时,得到种子液。
分别取5mL种子液转接到装有200mL纤维素酶解液培养基(用氨水调节pH值为5.0)的500mL摇瓶中,在30℃、200r/min条件下发酵72小时,利用HPLC检测发酵液中乙醇含量。发酵实验重复三次,数据取平均值进行计算。
SDIC Sc E01发酵过程中各组分含量的变化如图2所示,从图2中可以看出,纤维素酶解液中初始葡萄糖、木糖含量分别为86.1g/L和35.8g/L,48h时葡萄糖消耗完全,72h时木糖消耗完全;乙醇最高产量达到53.5g/L,糖醇转化率为0.44g/g;副产物甘油含量为4.5g/L,乙酸含量为3.2g/L。
SDIC Sc E发酵过程中各组分含量的变化如图3所示,从图3中可以看出,纤维素酶解液中,60h时葡萄糖消耗完全,72h时木糖剩余10g/L;乙醇最高产量达到42.4g/L,糖醇转化率为0.40g/g;副产物甘油含量为4.9g/L,乙酸含量为4.3g/L。
从上述结果也能够看出,相比于SDIC Sc E,SDIC Sc E01对更高糖浓度的培养基和高浓度的乙醇产物具有更高的耐受性,且葡萄糖和木糖的代谢速度更快,木糖利用效率更高,获得了更高的乙醇产量,同时副产物产量更低。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
SEQ ID No.4
UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)
CTACACTTTGACACTGCTGAACCAGTCAAGATCACACTTCCAAATGGTTTGACATACGAGCAACCAACCGGTCTATTCATTAACAACAAGTTTATGAAAGCTCAAGACGGTAAGACCTATCCCGTCGAAGATCCTTCCACTGAAAACACCGTTTGTGAGGTCTCTTCTGCCACCACTGAAGATGTTGAATATGCTATCGAATGTGCCGACCGTGCTTTCCACGACACTGAATGGGCTACCCAAGACCCAAGAGAAAGAGGCCGTCTACTAAGTAAGTTGGCTGACGAATTGGAAAGCCAAATTGACTTGGTTTCTTCCATTGAAGCTTTGGACAATGGTAAAACTTTGGCCTTAGCCCGTGGGGATGTTACCATTGCAATCAACTGTCTAAGAGATGCTGCTGCCTATGCCGACAAAGTCAACGGTAGAACAATCAACACCGGTGACGGCTACATGAACTTCACCACCTTAGAGCCAATCGGTGTCTGTGGTCAAATTATTCCATGGAACTTTCCAATAATGATGCGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGTCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTATCGGATTTGGAACATATATGTGCTTGCCGCCTTTGGCACCATCGGCGGCATGATCTTCGGCTTTGAGATCAGCTCCATGAGCGCCTGGATTGGCTCCGAACAATACCTCGAATACTTCAACCACCCGGACTCAACTGAGCAAGGCGGCATCACGGCAGCCATGTCCGCCGGCTCTTTGGTTGGCTCTCTCCTAGCTGGATGGCTCGCTGATCGTCTGGGACGCCGTTTAGCTATACAAATCGCCTCTGTGGACTGGATTGTCGGAGCGGTGCTGCAGTGCTCTTCCCAGAATGTTGCACATCTCGTCGTTGGTCGCATCGTCTCCGGACTTGCGATTGGCATTACGTCCTCCCAATGCATTGTCTACCTATCTGAACTTGCACCTTCTCGCATCCGTGGCCGAGTCGTTGGTATACAGCAGTGGTCCATTGACTGGGGCATCTTGATCATGTACCTGATCTCGTACGGCTGCTCGGTTTCAATTCATCGACCGGCGGCATTCCGTATTGCTTGGGGCTTGCAGGCTGTTCCCGGAGCTGTCCTGTTCTTCTCGCTCTTCTTCTTCCCTGAATCACCACGTTGGCTCGCAACCAAAGATCGCTGGGAGGAATGCCACGAAGTGCTTGCCAACCTGCATGCCAAAGGCGATCGAAACAATATCGAGGTGTTGGCCGAGCTGGAAGAAGTCAGAGAAGCAGCGAGGATCGCCGCGGAGTCAAAGGAGATCGGCTACCTGGGACTGTTCGCTCCCAAGATGTGGAAACGTACACTTGTCGGCGTCAGTGCACAGATTTGGCAGCAACTCCTCGGTGGTAATGTGATGCTTTACTACCTTGTCTACATCTTCAACATGGCTGGCATGTCCGGAAATACCGCGCTCACGTCTTCAATTATCCAATATGTCATCTTTCTCGTCACGACTGGAGGAGTTCTCTTTGTCGTTGATCGCATTGGTCGCCGGTGGTTGCTTATTGTTGGGGCCATCATTTGCGGCGTCATCCATTTCATCGTCGGTGCCGTCATGGCAGTCTACGGGCACCACGTCGACAGTGTGGATGGAAACGATATTCTGAGGTGGCAGATTGGTGGCCCTCCTGCCAAGGCCATCATTGCGCTTTGTTACATCTTTGTTGGAGTCTATGGAGTCACATGGGCCCACGGTGCATGGATTTACTGCGGAGAAGTCTTCCCCTTGAAGTACCGAGCCAAGGGCGTTGGCCTGGCTGCTGCAGGCAATTGGGCTTTCAACCTCGCCTTGGCCTTTTTTGTCCCCCCAGCTTTTACGAACATCCAGTGGAAAGCGTACATGATTTTCGGGACATTCTGCATTGCCATGGTGTTCCATATCTACTTCATGTACCCCGAAACGGTGAAGAAGTCACTCGAAGAGATTGACGTGCTATTCGAAGGAGATATTCCCGCATGGCGCAGCGCGAGTGCCGTGAGCACATTTGATGAGAAAGTAGCGCGAGCAAAGGAAGCGGGAGGCTTGGAAGAGTTTTCAAAACAAGCCGACATCAAGCACGAAGAAAAGGTCTAAGCTGGGATTACACATGGCATGGATGAACTATACAAATAATATATATATATATATATATATATATCCCTTTTCTAAAAAAGGGGGGAAAGGGCCCCCCTTTCAAAGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTTCCTGGTAGAACTGTTGGTGCTGCTTTGACCAACGACCCAAGAATCAGAAAGCTGGCTTTTACCGGTTCTACAGAAGTCGGTAAGAGTGTTGCTGTCGACTCTTCTGAATCTAACTTGAAGAAAATCACTTTGGAACTAGGTGGTAAGTCCGCCCATTTGGTCTTTGACGATGCTAACATTAAGAAGACTTTACCAAATCTAGTAAACGGTATTTTCAAGAACGCTGGTCAAATTTGTTCCTCTGGTTCTAGAATTTACGTTCAAGAAGGTATTTACGACGAACTATTGGCTGCTTTCAAGGCTTACTTGGAAACCGAAATCAAAGTTGGTAATCCATTTGACAAGGCTAACTTCCAAGGTGCTATCACTAACCGTCAACAATTCGACACAATTATGAACTACATCGATATCGGTAAGAAAGAAGGCGCCAAGATCTTAACTGGTGGCGAAAAAGTTGGTGACAAGGGTTACTTCATCAGACCAACCGTTTTCTACGATGTTAATGAA
SEQ ID No.5
UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)
TCCTGCTGTAGTTATGGCACTAAAGTTTTTTTGTAAATCTTTTTATATGTTAATAGATTTTACCAGTGGGACCCTGGAAAGAAAATATGTGTAAATGATGTTTTCTTTTGGATGATGTTCATTGCACAATGGTTCGCAATGTCATTCATTCATCCATTTCTTCTAGCTTTTCAATTATTAATGCCTTAAAATTTTTCAATTTCACCGCGTTACCCGAAGATGTAAATGTACCTACTCAAATAAAAGGTTCCTTGCTGGTTGGCTTTCGTAGCTAAATTGAGTGCACGTTACAAGATTATAAATGCGTTTATATATATATATTCCAGTGAATGATCTACTTTTGCTTAAGCGGCAGAATTGCAAATCTTAATTTTTCTTTTTTGTTTATGTTTTATTTCCTTGTGACAAATAAAATTTTTTTCTCTTCTGAATTTTTAAAACTAAAGAACAAAATATTAAAATACGGATTAGAAGCCGCCGAGCGGGcGACAGCCCTCCGAcGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGTCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTTGTGTTCAGTAATTCAGAGACAGACAAGAGAGGTTTCCAACACAATGTCTTTAGACTCATACTATCTTGGGTTTGATCTTTCGACCCAACAACTGAAATGTCTCGCCATTAACCAGGACCTAAAAATTGTCCATTCAGAAACAGTGGAATTTGAAAAGGATCTTCCGCATTATCACACAAAGAAGGGTGTCTATATACACGGCGACACTATCGAATGTCCCGTAGCCATGTGGTTAGAGGCTCTAGATCTGGTTCTCTCGAAATATCGCGAGGCTAAATTTCCATTGAACAAAGTTATGGCCGTCTCAGGGTCCTGCCAGCAGCACGGGTCTGTCTACTGGTCCTCCCAAGCCGAATCTCTGTTAGAGCAATTGAATAAGAAACCGGAAAAAGATTTATTGCACTACGTGAGCTCTGTAGCATTTGCAAGGCAAACCGCCCCCAATTGGCAAGACCACAGTACTGCAAAGCAATGTCAAGAGTTTGAAGAGTGCATAGGTGGGCCTGAAAAAATGGCTCAATTAACAGGGTCCAGAGCCCATTTTAGATTTACTGGTCCTCAAATTCTGAAAATTGCACAATTAGAACCAGAAGCTTACGAAAAAACAAAGACCATTTCTTTAGTGTCTAATTTTTTGACTTCTATCTTAGTGGGCCATCTTGTTGAATTAGAGGAGGCAGATGCCTGTGGTATGAACCTTTATGATATACGTGAAAGAAAATTCAGTGATGAGCTACTACATCTAATTGATAGTTCTTCTAAGGATAAAACTATCAGACAAAAATTAATGAGAGCACCCATGAAAAATTTGATAGCGGGTACCATCTGTAAATATTTTATTGAGAAGTACGGTTTCAATACAAACTGCAAGGTCTCTCCCATGACTGGGGATAATTTAGCCACTATATGTTCTTTACCCCTGCGGAAGAATGACGTTCTCGTTTCCCTAGGAACAAGTACTACAGTTCTTCTGGTCACCGATAAGTATCACCCCTCTCCGAACTATCATCTTTTCATTCATCCAACTCTGCCAAACCATTATATGGGTATGATTTGTTATTGTAATGGTTCTTTGGCAAGGGAGAGGATAAGAGACGAGTTAAACAAAGAACGGGAAAATAATTATGAGAAGACTAACGATTGGACTCTTTTTAATCAAGCTGTGCTAGATGACTCAGAAAGTAGTGAAAATGAATTAGGTGTATATTTTCCTCTGGGGGAGATCGTTCCTAGCGTAAAAGCCATAAACAAAAGGGTTATCTTCAATCCAAAAACGGGTATGATTGAAAGAGAGGTGGCCAAGTTCAAAGACAAGAGGCACGATGCCAAAAATATTGTAGAATCACAGGCTTTAAGTTGCAGGGTAAGAATATCTCCCCTGCTTTCGGATTCAAACGCAAGCTCACAACAGAGACTGAACGAAGATACAATCGTGAAGTTTGATTACGATGAATCTCCGCTGCGGGACTACCTAAATAAAAGGCCAGAAAGGACTTTTTTTGTAGGTGGGGCTTCTAAAAACGATGCTATTGTGAAGAAGTTTGCTCAAGTCATTGGTGCTACAAAGGGTAATTTTAGGCTAGAAACACCAAACTCATGTGCCCTTGGTGGTTGTTATAAGGCCATGTGGTCATTGTTATATGACTCTAATAAAATTGCAGTTCCTTTTGATAAATTTCTGAATGACAATTTTCCATGGCATGTAATGGAAAGCATATCCGATGTGGATAATGAAAATTGGGATCGCTATAATTCCAAGATTGTCCCCTTAAGCGAACTGGAAAAGACTCTCATCTAAGCTGGGATTACACATGGCATGGATGAACTATACAAATAATATATATATATATATATATATATAGTACGGACTTAAAAAAGGGGGGAAAGGGCCCCCCTTTCAAAGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTAATGTCCTAGACCCTTAATAAGCAAAACACTCATCATTATAACGGGAAAAAATTTGAAGCAAGCTCAAAAGAGATATATATATCATCCAGCATAGACTACTATTAATTTCTATCATAATTGCAATAAAAAAGGAAATATTTAGGGGATCAAGACCATTATTCCATCAGAATGGAAAAAAGTTTAAAAGATCACGGAGATTTTGTTCTTCTGAGCTTCTGCTGTCCTTGAAAACAAATTATTCCGCTGGCCGCCCCAAACAAAAACAACCCCGATTTAATAACATTGTCACAGTATTAGAAATTTTCTTTTTACAAATTACCATTTCCAGCTTACTACTTCCTATAATCCTCAATCTTCAGCAAGCGACGCAGGGAATAGCCGCTGAGGTGCATAACTGTCACTTTTCAATTCGGCCAATGCAATCTCAGGCGGACGAATAAGGGGGCCCTCTCGAGAAAAACAAAAGGAGGATGAGATTAGTACTTTAATGTTGTGTTCA
SEQ ID No.6
pRS423-KanMX-gRNA(XI)-GAL1p-Cas9
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAATTCCCGTTTTAAGAGCTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTTCCCCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGACGGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGATCCCCCCGGGGTGCTTCAGTATTACATTTTTTGCCTTCAACGCCTTGATTGTTCTATTTTTGCTAATAATAAATCTATTTCATCGGACTAAAAGTCCATTAGTTGTAAGCGGATTTAGCTCAGTTGGGAGAGCGCCAGACTGAAGAAAAACTTCGGTCAAGTCATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCAGATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGGGACTTTATGGGGTACGAATCTCTAGGAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTATTTTTTGTCACTATTGTTATGTAAAATGCCACCTCTGACAGTATGGAACGCAAACTTCTGTCTAGTGGATAACAGAATTTTTCTATGGCCAATTTATTCTTTTTTTTTCCCTTTTCTTAATGTGCGTATCATTGCCGGGTTTGAAATTTCGAAATTAACTACTAAAAATTACCGCTTTAATGTATGGAATATGTGCCCGGGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTTGGATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTTATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTACCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACGGTGGAGGGCCAAAAAAGAAAAGAAAAGTTGAAGATGCTTCTGCTCATCATCACCATCACCATCACCATTAAATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGTCGGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
SEQ ID No.7
pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAATTCCCGTTTTAAGAGCTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTTCCCCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGACGGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGATCCCCCCGGGGTGCTTCAGTATTACATTTTTTGCCTTCAACGCCTTGATTGTTCTATTTTTGCTAATAATAAATCTATTTCATCGGACTAAAAGTCCATTAGTTGTAAGCGGATTTAGCTCAGTTGGGAGAGCGCCAGACTGAAGAAAAACTTCGGTCAAGTCATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCAGATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGGGACTTTCTTTGCTTCTTTATGTAAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTATTTTTTGTCACTATTGTTATGTAAAATGCCACCTCTGACAGTATGGAACGCAAACTTCTGTCTAGTGGATAACAGAATTTTTCTATGGCCAATTTATTCTTTTTTTTTCCCTTTTCTTAATGTGCGTATCATTGCCGGGTTTGAAATTTCGAAATTAACTACTAAAAATTACCGCTTTAATGTATGGAATATGTGCCCGGGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTTGGATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTTATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTACCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACGGTGGAGGGCCAAAAAAGAAAAGAAAAGTTGAAGATGCTTCTGCTCATCATCACCATCACCATCACCATTAAATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGTCGGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
SEQ ID No.8
pRS423-KanMX-gRNA(XKS1)-GAL1p-Cas9
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAATTCCCGTTTTAAGAGCTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTTCCCCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGACGGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGATCCCCCCGGGGTGCTTCAGTATTACATTTTTTGCCTTCAACGCCTTGATTGTTCTATTTTTGCTAATAATAAATCTATTTCATCGGACTAAAAGTCCATTAGTTGTAAGCGGATTTAGCTCAGTTGGGAGAGCGCCAGACTGAAGAAAAACTTCGGTCAAGTCATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCAGATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGGGACTTTCTTTTTCTTCTTTTGGTATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTATTTTTTGTCACTATTGTTATGTAAAATGCCACCTCTGACAGTATGGAACGCAAACTTCTGTCTAGTGGATAACAGAATTTTTCTATGGCCAATTTATTCTTTTTTTTTCCCTTTTCTTAATGTGCGTATCATTGCCGGGTTTGAAATTTCGAAATTAACTACTAAAAATTACCGCTTTAATGTATGGAATATGTGCCCGGGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTTGGATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTTATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTACCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACGGTGGAGGGCCAAAAAAGAAAAGAAAAGTTGAAGATGCTTCTGCTCATCATCACCATCACCATCACCATTAAATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGTCGGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
SEQ ID No.9
pADH1
ATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTATCTCA
SEQ ID No.10
tTEF1
ACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTG
Claims (12)
1.一种重组酿酒酵母,其特征在于,所述重组酿酒酵母含有过表达的木糖异构酶XI基因、过表达的木糖转运蛋白TrXlt1基因和过表达的木酮糖激酶XKS1基因。
2.根据权利要求1所述的重组酿酒酵母,其中,木糖异构酶XI基因、木糖转运蛋白TrXlt1基因和木酮糖激酶XKS1基因各自独立地通过SEQ ID No.1所示的启动子pUAS-TDH3和SEQ ID No.2所示的终止子t-100调控;
优选地,针对木糖异构酶XI基因,所述重组酿酒酵母含有UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)重组片段,更优选含有SEQ ID No.3所示的序列;
优选地,针对木糖转运蛋白TrXlt1基因,所述重组酿酒酵母含有UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)重组片段,更优选含有SEQ ID No.4所示的序列;
优选地,针对木酮糖激酶XKS1基因,所述重组酿酒酵母含有UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)重组片段,更优选含有SEQ ID No.5所示的序列;
优选地,所述木糖异构酶XI基因位于PHO13基因座位,更优选位于IV染色体的32584和32764位点之间;
优选地,所述木糖转运蛋白TrXlt1基因位于ALD6基因座位,更优选位于XVI染色体的433122和4333268位点之间;
优选地,所述木酮糖激酶XKS1基因位于酿酒酵母ARS737上游,更优选位于VII染色体的888363和888412位点之间。
3.根据权利要求1或2所述的重组酿酒酵母,其中,所述重组酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变;
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由A突变为C;
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由T突变为C;
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由C突变为G;
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由C突变为G;
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由G突变为T;
优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变;
更优选地,所述重组酿酒酵母中,共价连接细胞壁蛋白(Ccw14p)的突变为CCW14I211L;所述细胞色素c氧化酶(Cox14p)的突变为COX14T21A;所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33D146E;所述ATP合成酶(Atp10p)的突变为ATP10D164E;所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1G58C。
4.一种重组酿酒酵母的构建方法,其特征在于,该方法包括:利用CRISPR/Cas9系统向出发酿酒酵母菌株中导入能够过表达的木糖异构酶XI基因重组片段、能够过表达的木糖转运蛋白TrXlt1基因重组片段和能够过表达的木酮糖激酶XKS1基因重组片段,得到重组酿酒酵母;
优选地,木糖异构酶XI基因、木糖转运蛋白TrXlt1基因和木酮糖激酶XKS1基因各自独立地通过SEQ ID No.1所示的启动子pUAS-TDH3和SEQ ID No.2所示的终止子t-100调控;
优选地,能够过表达的木糖异构酶XI基因重组片段为UP(PHO13)-pUAS-TDH-XI-t100-Down(PHO13)重组片段,更优选含有SEQ ID No.3所示的序列;更优选地,相应的重组质粒为pRS423-KanMX-gRNA(XI)-GAL1p-Cas9重组质粒,进一步优选具有SEQ ID No.6所示的序列;
优选地,能够过表达的木糖转运蛋白TrXlt1基因重组片段为UP(ALD6)-pUAS-TDH-TrXlt1-t100-Down(ALD6)重组片段,更优选含有SEQ ID No.4所示的序列;更优选地,相应的重组质粒为pRS423-KanMX-gRNA(TrXlt1)-GAL1p-Cas9重组质粒,进一步优选具有SEQ IDNo.7所示的序列;
优选地,能够过表达的木酮糖激酶XKS1基因重组片段为UP(XKS1)-pUAS-TDH-XKS1-t100-Down(XKS1)重组片段,更优选含有SEQ ID No.5所示的序列;更优选地,相应的重组质粒为pRS423-KanMX-gRNA(XKS1)-GAL1p-Cas9重组质粒,进一步优选具有SEQ ID No.8所示的序列。
5.根据权利要求4所述的方法,其中,该方法还包括对重组酿酒酵母进行基因突变处理,所述突变使得所述重组酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变;
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由A突变为C;
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由T突变为C;
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由C突变为G;
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由C突变为G;
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由G突变为T;
优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变;
更优选地,所述突变使得所述重组酿酒酵母中,共价连接细胞壁蛋白(Ccw14p)的突变为CCW14I211L;所述细胞色素c氧化酶(Cox14p)的突变为COX14T21A;所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33D146E;所述ATP合成酶(Atp10p)的突变为ATP10D164E;所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1G58C。
6.一株酿酒酵母(Saccharomyces cerevisiae),其特征在于,所述酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变;
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由A突变为C;
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由T突变为C;
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由C突变为G;
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由C突变为G;
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由G突变为T;
优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变;
更优选地,所述重组酿酒酵母中,共价连接细胞壁蛋白(Ccw14p)的突变为CCW14I211L;所述细胞色素c氧化酶(Cox14p)的突变为COX14T21A;所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33D146E;所述ATP合成酶(Atp10p)的突变为ATP10D164E;所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1G58C。
7.一种提高酿酒酵母对胁迫环境耐受性的方法,其特征在于,该方法包括对酿酒酵母进行以下至少一种处理,
(1)将CCW14基因位点NC_001144.5:904354的碱基由A突变为C;
(2)将COX14基因位点NC_001145.3:14693的碱基由C突变为T;
(3)将ATG33基因位点NC_001144.5:840758的碱基由G突变为C;
(4)将ATP10基因位点NC_001144.5:907570的碱基由G突变为C;
(5)将CCC1基因位点NC_001144.5:576996的碱基由T突变为G;
优选地,所述处理使得酿酒酵母耐受浓度为50g/L以上的乙醇;
优选地,所述处理使得酿酒酵母耐受浓度为80g/L以上的葡萄糖和/或30g/L以上的木糖;
优选地,(1)至(5)中任意一项所述的突变为杂合突变或纯合突变,更优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的突变为纯合突变,Ccc1基因上的突变为杂合突变。
8.一株酿酒酵母(Saccharomyces cerevisiae),其特征在于,该酿酒酵母的保藏编号为GDMCC No.62491。
9.一株酿酒酵母(Saccharomyces cerevisiae),其特征在于,相比于权利要求8所述的酿酒酵母,该酿酒酵母包含位于Ccw14基因、Cox14基因、Atg33基因、Atp10基因和Ccc1基因上的至少一个非同义突变;
优选地,CCW14基因的突变位点为NC_001144.5:904354,碱基由C突变为A;
优选地,COX14基因的突变位点为NC_001145.3:14693,碱基由C突变为T;
优选地,ATG33基因的突变位点为NC_001144.5:840758,碱基由G突变为C;
优选地,ATP10基因的突变位点为NC_001144.5:907570,碱基由G突变为C;
优选地,CCC1基因的突变位点为NC_001144.5:576996,碱基由T突变为G;
优选地,Ccw14基因、Cox14基因、Atg33基因和Atp10基因上的非同义突变为纯合突变,Ccc1基因上的非同义突变为杂合突变;
更优选地,相比于权利要求8所述的酿酒酵母,所述酿酒酵母中共价连接细胞壁蛋白(Ccw14p)的突变为CCW14L211I;所述细胞色素c氧化酶(Cox14p)的突变为COX14A21T;所述线粒体分裂特异蛋白(Atg33p)的突变为ATG33E146D;所述ATP合成酶(Atp10p)的突变为ATP10E164D;所述液泡Fe2+/Mn2+转运蛋白(Ccc1p)的突变为CCC1C58G。
10.一种菌剂,其特征在于,所述菌剂包含权利要求1-3中所述的重组酿酒酵母和权利要求6、8和9中所述的酿酒酵母中的至少一种。
11.权利要求1-3中任意一项所述的重组酿酒酵母、权利要求6、8和9中任意一项所述的酿酒酵母或权利要求10所述的菌剂在发酵产乙醇中的应用;
优选地,用于发酵产乙醇的原料包含纤维素原料。
12.一种发酵产乙醇的方法,其特征在于,该方法包括:将酿酒酵母接种至发酵培养基中进行发酵,生产乙醇;
其中,所述酿酒酵母选自权利要求1-3中所述的重组酿酒酵母、权利要求6、8和9中所述的酿酒酵母和权利要求10所述的菌剂中的至少一种;
所述发酵培养基包含葡萄糖和木糖;
优选地,所述发酵培养基包含纤维素酶解液。
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