CN102229966B - 一种重组酿酒酵母发酵菊芋生产乙醇的方法 - Google Patents
一种重组酿酒酵母发酵菊芋生产乙醇的方法 Download PDFInfo
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Abstract
本发明属于生物技术领域,公开了一种重组酿酒酵母发酵菊芋生产乙醇的方法,该方法是以菊芋粉为培养基,用重组酿酒酵母厌氧发酵生产乙醇;在60h内,乙醇发酵浓度即可达到9%以上。采用本发明所述原料和发酵方法,可以替代我国现有乙醇工业生产中所采用的玉米粉、小麦粉等粮食类原料,为非粮燃料乙醇生产奠定了基础。
Description
技术领域
本发明属于生物技术领域,涉及重组酿酒酵母的构建方法,具体为一种发酵菊芋生产乙醇的重组酿酒酵母的构建方法;本发明还涉及一种发酵乙醇的方法,具体为用重组酿酒酵母发酵乙醇的方法。
背景技术
资源、能源和环境是制约国民经济和社会可持续发展的重大瓶颈问题,积极探索并创立新技术,建立资源和能源可持续利用的环境友好型社会是“十二五”期间科技创新的重要内容之一。利用非耕地大规模种植高产能源植物,发展以生物燃料、生物基产品及生物材料为主体的新兴产业,是实现这一目标的主要出路。
我国“十五”和“十一五”期间试点发展以燃料乙醇为代表的生物能源产业,先后在黑龙江、吉林、河南、安徽和广西四省建设了总量为152万吨的燃料乙醇装置,以玉米、小麦和木薯等淀粉质原料生产燃料乙醇,但我国人口多耕地少的基本国情,严重制约了这一产业的规模化发展。不与人争粮,不与粮争地,不破坏生态环境,是我国生物乙醇产业发展的基本方针!
菊芋(Helianthus tuberous L,俗称洋姜、鬼子姜)是一种可利用边际性土地生长,生态适宜性强[隆小华等。半干旱地区海涂海水灌溉对不同品系菊芋产量构成及离子分布的影响.土壤学报,2007,44:300-306]。鲜菊芋块茎中含水70~80%,碳水化合物15~20%,以菊粉的形式存在。菊粉(Inulin),又称菊糖,是由D-果糖经β-2,1糖苷键连接的聚合度不高的聚果糖,末端为一个葡萄糖残基[Khodzhaeva MA,Kondratenko ES.The structure of the inulin from inula grandis.Chem Nat Compd,1982,3:394-395]。与目前国内外普遍关注的以农作物秸秆为代表的木质纤维素类生物质原料相比,菊芋易被酸或菊粉酶水解而生成果糖和葡萄糖的混合物,是生产乙醇的良好原料[Denoroy P.The crop physiology ofhelianthus tuberosus L:a model orientated view.Biomass and Bioenergy,1996,11:11-32]。
以菊芋块茎为原料生产乙醇的研究工作在国内外均有报道,利用能产生菊粉酶的微生物菌种(例如:克鲁维酵母、黑曲霉等)和乙醇发酵菌株运动发酵单胞杆菌或酿酒酵母进行混菌培养的共发酵[Nakamura T,Ogata Y,Hamada S,etal.Ethanol production from Jerusalem artichoke tubers by Aspergillus niger andSaccharomyces cerevisiae.J Ferment Bioeng,1996,81:564-566];或利用同时具有产菊粉酶和乙醇发酵能力的克鲁维酵母进行乙醇发酵等[袁文杰等一步法发酵菊芋生产乙醇。2008,24:1931-1935;刘建平等鹰嘴豆孢克鲁维酵母利用菊芋原料同步糖化与发酵生产乙醇.生物工程学报,2010,7:982-990.中国专利,CN101265485A]。其中最优的当属克鲁维酵母的一步法发酵工艺,菊芋的糖化和发酵均由克鲁维酵母自身完成,原料不需预先糖化,节约了能耗和额外添加酶的费用,但其缺点是克鲁维酵母属非常规酵母,其乙醇产生机制尚不明了,乙醇耐性较酿酒酵母差,不利于后续的代谢工程改造。并且发酵速率慢,时间长,导致发酵罐设备生产强度很低。
发明内容
本发明的目的是提供一种由本发明获得的重组酿酒酵母发酵菊芋干粉生产燃料乙醇的方法,解决目前现有菌株存在的发酵时间长、乙醇耐性差的问题。
本发明的技术方案具体如下:
一种重组酿酒酵母发酵菊芋生产乙醇的方法,其特征在于所述方法包括:
a提供菊芋原料:鲜菊芋原料清洗、捣碎或切片、晾干或烘干后研磨成粉;
b菊芋粗粉加水配成的发酵培养基(菊芋粗粉与水的质量比为1∶4),未灭菌,直接将OD600为10的重组酿酒酵母按10%体积比接种发酵培养基中,30-33℃进行厌氧发酵24-60h,产生乙醇;
优选方案:在步骤b中,30-33℃进行厌氧发酵24h后,添加干菊芋粉,搅拌均匀使添加后菊芋粉浓度达到280g/L,使得发酵液中总糖浓度达到180g/L,继续厌氧发酵24-36h;
所述重组酿酒酵母包含p-inu基因;
所述包含p-inu基因的重组酿酒酵母的构建包括:
①以马克斯克鲁维酵母(Kluyveromyces marxianus CGMCC2.1549)基因组为模板,PCR扩增得到p-inu基因,并连接到克隆载体;
②BamH I和BssH II酶分别切割载体质粒和含p-inu基因的克隆载体;
③将p-inu基因插入载体质粒;
④用所构建的重组质粒转入酿酒酵母(Saccharomyces cerevisiaeCGMCC2.1882)中,得到重组酿酒酵母;
步骤①中扩增引物序列如下:
p-inu forward primer:GCCggatccGAATTCTCAAACCGAA(BamH I);
p-inu reverse primer:CGGgcgcgcAGATCAGATCAAACG(BssH II);
扩增和保存质粒用大肠杆菌E.coli DH5α;
所述克隆载体为pMD19-T。
本发明是以菊芋粉为培养基,用重组酿酒酵母厌氧发酵生产乙醇,具有乙醇发酵速率快、残糖低、糖醇转化率高等特点,采用本发明所述原料和发酵方法,在60h内,乙醇发酵浓度即可达到9%以上,可以替代我国现有乙醇工业生产中所采用的玉米粉、小麦粉等粮食类原料,为非粮燃料乙醇生产奠定了基础。
附图说明
本发明共有附图四张:
图1为用于转化酿酒酵母、含有菊粉酶基因的表达载体pOH/inu结构示意图;
图2为基因工程菌生长及产菊粉酶随时间变化图;
图3为基因工程菌菊芋粉乙醇发酵随时间变化图;
图4为基因工程菌菊芋粉批式补料乙醇发酵随时间变化图;
图1中:INU为菊粉酶编码基因;HO-L为核酸内切酶基因的上游序列;HO-R为核酸内切酶基因的下游序列;Kam遗传霉素编码基因。
具体实施方式
实施例1 菊粉酶整合表达载体的构建
根据菊粉酶(GenBank:X57202)序列,设计引物p-inu forward primer:GCCggatccGAATTCTCAAACCGAA(BamH I)和p-inu reverse primer:CGGgcgcgcAGATCAGATCAAACG(BssH II),以K.marxianus马克斯克鲁维酵母(CGMCC2.1549)基因组为模板扩增p-inu基因。PCR体系:模板1μl,F/R引物各1μl,酶1μl,dNTP 4μl,缓冲液5μl,ddH2O 37μl;PCR程序:94℃预变性5min;94℃ 1min,63.6℃ 1min,72℃ 3min,30个循环;72℃延伸10min。PCR产物经胶回收纯化后,与载体pMD19-T连接。阳性克隆提取质粒后测序,测序分析结果证明该菊粉酶基因与NCBI报道的菊粉酶基因一致。pMD19T/p-inu经BamH I和BssH II酶切后与经BamH I和BssH II酶切的HO-2(HO-poly-KanMX4-HO,Warren P V,James D R,Janet M S and Stillman D J.Yeastvectors for integration at the HO locus.Nucleic Acids Research,2001,Vol,29,No.12e59)进行连接。连接产物转化E.coli DH5α感受态细胞,在LB选择培养基上培养,挑取单菌落,小量提取质粒酶切鉴定并进行DNA序列测定,得到整合载体HO/p-inu(图1)。菊粉酶基因的表达是在K.marxianus酵母的菊粉酶启动子、分泌信号肽的控制下组成型表达。
实施例2 S.cerevisiae酿酒酵母(CGMCC2.1882)的转化及转化子验证整合载体HO/p-inu经Not I酶切后,进行1%琼脂糖电泳,回收目的DNA片段,采用电击法转化S.cerevisiae酿酒酵母(CGMCC2.1882)感受态细胞,在含有300μg/mL G418的YPD(无水葡萄糖20,酵母浸粉10,蛋白胨20和1.5%琼脂粉)平板上。采用电击法转化,可以参照BIORAD MicroPulserTM电转仪使用说明书。30℃培养2-3天,挑选转化子,提取HI6/6的基因组DNA。采用PCR法进行验证。HI6/6的验证所使用的引物序列为HI6/6 forward primer:AGCTTAATTATCCTGGGCACGAGT和HI6/6 reverse primer:GCGAGCTCAGCTCGTTTTCGACACTGGA。PCR程序:94℃预变性5min;94℃1min,60℃ 1min,72℃ 3min,30个循环;72℃延伸10min。1%琼脂糖电泳显示获得大小正确片段,证明获得阳性转化子。
实施例3 转化子产酶情况考察
取S.cerevisiae 6525和转化子HI6/6各100μL,接至50mL的YPD培养基中,30℃培养24h后,按10%接种到种子培养基,100/250mL摇瓶,30℃培养每24h取样。发酵液经适当稀释后,测定菌体密度(OD600);发酵液5000rp离心10min后,上清液用于测定菊粉酶活性。转化子HI6/6生物量几乎与出发菌株生长一致,达到9g/L;转化子HI6/6的菊粉酶活力在144h达到最高酶活86U/mL,是出发菌株的4.5倍。
实施例4 转化子发酵性能考察
取S.cerevisiae 6525和转化子HI6/6各100μL,接至50mL的YPD液体培养基中,30℃培养24h后,测OD600为10,按10%体积比接种到菊芋粗粉发酵培养基中(料水比为1∶4),无其他成分添加,未灭菌,100/250mL摇瓶,30℃厌氧发酵,每12h取样测定菊粉酶的活性、还原糖、总糖及乙醇含量。乙醇对理论值的收率=乙醇/(总糖-剩余总糖)/0.511。
转化子HI6/6的最终乙醇浓度为55g/L,高于出发菌株49g/L,较出发菌株最终乙醇浓度提高了12.24%,糖醇转化率为0.495,为理论值的96.9%。
实施例5 基因工程菌在批式补料工艺的乙醇发酵
取S.cerevisiae 6525和转化子HI6/6各100μL,接至50mL的YPD液体培养基中,30℃培养24h,测OD600为10,按10%体积比接种到菊芋粗粉发酵培养基中(菊芋粗粉与水的质量比为1∶4),1.5L发酵罐中装料量为1L,无其他成分添加,未灭菌,30℃进行厌氧发酵,24h后,直接添加干菊芋粉80g,搅拌均匀使添加后菊芋粉浓度达到280g/L,使得发酵液中总糖浓度达到180g/L,继续厌氧发酵36h。
转化子HI6/6的最终乙醇浓度为72.5g/L,高于出发菌株66.5g/L,较出发菌株最终乙醇浓度提高了9%。
Claims (1)
1.一种重组酿酒酵母发酵菊芋生产乙醇的方法,其特征在于所述方法包括:
a提供菊芋原料:鲜菊芋原料清洗、捣碎或切片、晾干或烘干后研磨成粗粉;
b菊芋粗粉加水配成的发酵培养基,将OD600数值为10的重组酿酒酵母按10%体积比接种于发酵培养基中,30-33℃进行厌氧发酵24h后,添加干菊芋粉,搅拌均匀使添加后菊芋粉浓度达到280g/L,使得发酵液中总糖浓度达到180g/L,继续厌氧发酵24-36h,产生乙醇;
所述发酵培养基中菊芋粗粉与水的质量比为1﹕4;
所述重组酿酒酵母包含p-inu基因;
所述包含p-inu基因的重组酿酒酵母的构建包括:
①以马克斯克鲁维酵母基因组为模板,PCR扩增得到p-inu基因,并连接到克隆载体;
②BamH I和BssH II酶分别切割载体质粒和含p-inu基因的克隆载体;
③将p-inu基因插入载体质粒;
④用所构建的重组质粒转入酿酒酵母中,得到重组酿酒酵母;
所述步骤①中扩增引物序列如下:
p-inu forward primer:GCCggatccGAATTCTCAAACCGAA;
p-inu reverse primer:CGGgcgcgcAGATCAGATCAAACG;
所述扩增和保存质粒用大肠杆菌E.coli DH5α;
所述克隆载体为pMD19-T
所述载体质粒为HO-2。
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