CN101311271B - 一种利用重组酿酒酵母生产纤维素乙醇的方法 - Google Patents
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Abstract
本发明公开了一种利用重组酿酒酵母生产纤维素乙醇的方法。其特征在于:该重组酿酒酵母含有引入的能编码葡聚糖内切酶(EG)、纤维二糖水解酶(CHB)、β-葡聚糖苷酶(BG)的基因,可同时分泌三种纤维素酶:葡聚糖内切酶、纤维二糖水解酶和β-葡聚糖苷酶。该酿酒酵母工程菌可将纤维素直接高效转化成葡萄糖,并同时将葡萄糖原位转化成乙醇。本发明的优点在于:无需另外加入纤维素酶,利用该酿酒酵母工程菌即可实现纤维素原料的同步酶水解-发酵从而生产乙醇,可大幅度降低纤维乙醇的生产成本,推动纤维乙醇替代石油燃料的进程。
Description
一、技术领域
本发明涉及发酵工程和基因工程领域,尤其是使用重组酿酒酵母将秸秆等纤维质原料经同步酶水解—发酵转化为纤维素乙醇的的方法,所述的重组酿酒酵母可分泌三种纤维素酶。
二、背景技术
2006年6月,我国出台《可再生能源发展专项资金管理暂行办法》,明确重点扶持发展以秸杆等非粮食资源制取的生物乙醇燃料。开发替代粮食资源生产纤维乙醇,是解决燃料乙醇原料成本高的根本出路。
纤维素酶成本是制约纤维乙醇商业化进程的关键因素。而秸杆等纤维质原料中的纤维素、半纤维素不但被木质素包裹,而且半纤维素部分共价和木质素结合。因此,需要纤维素酶、半纤维素酶和木质素酶三种酶系的协同作用,解除半纤维素和木质素包裹后,才能彻底分解纤维素,将其最大限度的降解为可发酵糖。生产纤维乙醇,通常要进行预处理以除去半纤维素和木质素,以提高纤维素的转化率。因此,降低纤维乙醇成本,有以下措施:(1)寻求有效的方法低成本、大批量生产纤维素酶,降低酶成本;(2)提高纤维素的转化率。
目前,以秸秆等纤维质原料生物法生产燃料乙醇的工艺中,应用较多的是先糖化后发酵(SHF)工艺和同步糖化发酵(SSF)工艺。但无论SHF还是SSF工艺,都需要先生产纤维素酶并进行酶的分离纯化,这势必增加乙醇的生产成本,从而限制了SHF和SSF工艺在纤维乙醇工业化生产中的大规模应用。此外,还有固定化细胞发酵工艺,研究最多的是酵母和运动发酵单胞菌的固定化,虽然此工艺具有细胞可连续使用、最终发酵液乙醇浓度高等优点,但也不能解决纤维素酶成本高的问题。随着生物技术的不断发展,利用基因工程技术改造微生物,得到新的高比活力的重组型纤维素酶,为大量生产纤维素酶提供了可能。纤维素酶基因在大肠杆菌中可以有效表达,然而,细菌分泌的纤维素酶只在胞质中不释放到胞外,使提取、纯化困难。另外,许多真菌和细菌的纤维素酶是糖基化的,使在大肠杆菌中表达更趋复杂。于是,人们把注意力转移到了酵母。酵母为真核表达系统,且不产生毒素,用其表达纤维素酶基因,其产物高度糖基化,表达水平高,而且产物直接分泌至胞外。
酿酒酵母是工业生产乙醇的理想菌株,对乙醇的耐受性强,可以同时表达多基因,而且酵母是真核细胞,能够对表达的外源蛋白进行糖基化。目前,以酿酒酵母作为表达宿主,构建重组型纤维素酶方面,已经成功的实现了两种纤维素酶基因在同一酿酒酵母中的表达。如日本学者Akihiko Kondo成功的将葡聚糖内切酶基因和β-葡萄糖苷酶基因同时转入酿酒酵母,构建了一种能够同时表达两种纤维素酶的酵母工程菌。该工程菌可降解β-葡聚糖生产乙醇,培养基中β-葡聚糖的含量为45g/L时,发酵48h后乙醇的产量为16.5g/L。其缺点是由于仅表达了2种纤维素酶,该工程菌只能以葡聚糖为底物而不能直接利用纤维素。国内有关将纤维素酶基因在酿酒酵母菌中表达的报道较少。只有山东大学的汪天虹教授在这一方面进行了开创性的工作,将已构建的携带eg1的质粒pRS415ME转化酿酒酵母H158,获得表达胞外内切葡聚糖酶的重组酵母菌株H1m;随后将携带cbh1的质粒pAJ401-cbh1转入H1m中,构建了同时分泌表达eg1和cbh1的重组酵母菌株HMEPC,HMEPC对纤维素底物滤纸和麦芽汁的降解利用程度均比H1m有所提高。但是通过共转化的方式构建多基因纤维素酶重组酿酒酵母的过程比较烦琐,工作量大,时间长。
上述在酿酒酵母中表达纤维素酶的研究,为“只用一种微生物(而无需添加纤维素酶)直接转化纤维素生产乙醇工艺”展示了良好的前景。但目前的研究局还只是限于将2种纤维素酶基因:葡聚糖内切酶基因和β-葡萄糖苷酶基因进行了表达,由此获得的重组酿酒酵母还不能直接以纤维素为原料生产葡萄糖,并原位生产乙醇。主要原因是纤维素酶是一种多组分的复合酶,包括葡聚糖内切酶(EG),葡聚糖外切酶(或称纤维二糖水解酶)(CHB)和β-葡聚糖苷酶(BG)等3种主要组分。在天然纤维素水解成葡萄糖的过程中,必须依靠3种酶的协同作用才能完成。
因此,将三个纤维素酶基因同时转入酿酒酵母中,构建一种能同时分泌三种纤维素酶的酿酒酵母工程菌,直接将纤维素转化为葡萄糖,并同时将其发酵为乙醇。对于降低纤维乙醇生产成本,促进纤维乙醇的工业化进程具有重要意义。
三、发明内容
本发明的目的在于提供一种用重组酿酒酵母同步酶水解—发酵秸秆等纤维质原料生产纤维素乙醇的方法。所述的重组酿酒酵母可分泌三种纤维素酶:葡聚糖内切酶、纤维二糖水解酶、β-葡聚糖苷酶。
本发明的技术方案如下:
(1)构建重组表达载体
将葡聚糖内切酶基因eg1、纤维二糖水解酶基因cbh1和β-葡聚糖苷酶基因bglc与合适的载体和启动子连接,构建成三基因重组表达载体;
其中所述的质粒载体选自pYEX-BX(7.1kb)、Yeplac195(5.24kb),进一步的各质粒载体所含的选择性标记选自:(1)Ampr,URA3和leu2三个选择标记和(2)Kanr和URA3两个选择标记。
(2)电转化法转化酿酒酵母;
(3)进行阳性转化子的筛选
利用重组子本身带有的抗生素抗性基因或者是氨基酸、尿嘧啶缺陷的选择性培养基进行筛选。其次,进行PCR检测、Southern blot对选择出的菌株进一步鉴定筛选,最终筛选出同时含有三个纤维素酶基因的酿酒酵母阳性转化子。
(4)重组酿酒酵母同步酶水解发酵纤维素生产乙醇
应用上述重组酿酒酵母,以秸杆等纤维质为原料,采用同步酶水解—发酵技术,生产纤维素乙醇。
本发明的优点在于:
(1)本发明的重组酿酒酵母,可同时分泌三种纤维素酶到胞外,即葡聚糖内切酶(EG)、纤维二糖水解酶(CHB)和β-葡聚糖苷酶(BG);
(2)本发明的重组酿酒酵母,可将纤维素直接转化成葡萄糖,同时将葡萄糖原位转化成乙醇;
(3)本发明的一种用重组酿酒酵母直接转化秸秆等纤维质原料生产乙醇的方法,可实现只用一种微生物,无需另外加入纤维素酶就可一步直接将纤维素转化为乙醇;
(4)本发明的一种用重组酿酒酵母直接转化秸秆等纤维质原料生产乙醇的方法,可以大幅度降低纤维素乙醇的生产成本,推动纤维素乙醇替代石油燃料的进程,具有广阔的应用前景。
下面结合具体实施例对本发明作进一步描述。
具体实施方式
实施例1 纤维素酶重组酿酒酵母1
载体pYEX-BX(7.1kb)(含有Ampr,URA3和leu2三个选择标记)、3-磷酸甘油醛脱氢酶基因启动子(GAPDHP)和终止子(GAPDHT)、信号肽编码序列采用T.reesei中的木聚糖酶基因的信号肽序列(XYNSEC),构建重组表达载体I,其表达序列框为GAPDHP-XYNSEC-cbh1-GAPDHT-GAPDHP-XYNSEC-eg1-GAPDHT-GAPDHP-GLUSEC-bglc-GAPDHT,命名为pYEX-BX-GAPDH-EchBl。采用电击转化法将构建好的重组表达载体转化到酿酒酵母S.cerevisiae MT8-1中;根据所转入重组表达载体上带有的选择性标记,将转化后的酵母细胞涂布在含有氨苄霉素或卡那霉素的培养基上或者是在营养缺陷型的固体培养基平板上,如果转化成功,酵母细胞将获得抗生素抗性或者是营养缺陷型抗性,在相应的选择性培养基上将会长出含有重组子的单菌落。然后,提质粒进行PCR扩增,双酶切进行检测转化子中是否同时含有eg1,cbh1和bglc三个纤维素酶基因,最终筛选出同时含有三种纤维素酶基因的酿酒酵母阳性转化子。
实施例2 纤维酶重组酿酒酵母2
载体pYEX-BX(7.1kb)(含有Ampr,URA3和1eu2三个选择标记)、磷酸甘油酸激酶基因启动子(PGKP)和终止子(PGKT)、信号肽编码序列采用T.reesei中的木聚糖酶基因的信号肽序列(XYNSEC),构建重组表达载体II,其表达序列框为PGKP-XYNSEC-cbh1-PGKT-PGKP-XYNSEC-eg1-PGKT-PGKP-PGKP-GLUSEC-bglc-PGKT,命名为pYEX-BX-PGK-EchB1;采用电击转化法将构建好的重组表达载体转化到酿酒酵母S.cerevisiae MT8-1中;根据所转入重组表达载体上带有的选择性标记,将转化后的酵母细胞涂布在含有氨苄霉素或卡那霉素的培养基上或者是在营养缺陷型的固体培养基平板上,如果转化成功,酵母细胞将获得抗生素抗性或者是营养缺陷型抗性,在相应的选择性培养基上将会长出含有重组子的单菌落。然后,提质粒进行PCR扩增,双酶切进行检测转化子中是否同时含有eg1,cbh1和bglc三个纤维素酶基因,最终筛选出同时含有三种纤维素酶基因的酿酒酵母阳性转化子。
实施例3 纤维素酶重组酿酒酵母3
载体Yeplac195(5.24kb)(含有Kanr和URA3两个选择标记)、3-磷酸甘油醛脱氢酶基因启动子(GAPDHP)和终止子(GAPDHT)、Rhizopusoryzae的葡萄糖淀粉酶基因信号肽(GLUSEC),构建重组表达载体III,其表达序列框为GAPDHP-XYNSEC-cbh1-GAPDHT-GAPDHP-XYNSEC-eg1-GAPDHT-GAPDHP-GLUSEC-bglc-GAPDHT,命名为Yeplac-GAPDH-EchBl。采用电击转化法将构建好的重组表达载体转化到酿酒酵母S.cerevisiae MT8-1中;根据所转入重组表达载体上带有的选择性标记,将转化后的酵母细胞涂布在含有氨苄霉素或卡那霉素的培养基上或者是在营养缺陷型的固体培养基平板上,如果转化成功,酵母细胞将获得抗生素抗性或者是营养缺陷型抗性,在相应的选择性培养基上将会长出含有重组子的单菌落。然后,提质粒进行PCR扩增,双酶切进行检测转化子中是否同时含有eg1,cbh1和bglc三个纤维素酶基因,最终筛选出同时含有三种纤维素酶基因的酿酒酵母阳性转化子。
实施例4重组酿酒酵母转化玉米秸杆生产乙醇
(1)秸杆预处理
取玉米秸杆200g,粉碎,过20目筛,加入0.5%硫酸1.2L,在150℃处理20h,再转入5L反应器,蒸汽加热1.5min至压强达15bar,维持10min,冷却,至压力为2bar。取出秸杆原料,用50℃水洗涤5次,过滤,得120g处理后的玉米秸杆,备用。
(2)菌种培养
斜面培养基组成(g/L)及培养条件:葡萄糖,20;酵母浸膏,3;琼脂,20;麦芽汁,3;pH5.5,温度30±1℃
种子培养基组成(g/L)及培养条件:葡萄糖,50;酵母浸膏,5;(NH4)SO4,7.5;K2HPO4,3.5;MgSO4·7H2O,0.75;CaCl2·2H2O,1M柠檬酸缓冲液,pH5.5±0.1,35±0.5℃,培养30h,转速150rpm
接种量:按每100ml转化液接种1.0g菌体(以干菌体计)
(3)秸杆转化为乙醇
发酵液组成(g/l):酵母浸膏,5;(NH4)2SO4,7.5;K2HPO4,3.5;MgSO4·7H2O,0.75;CaCl2·2H2O,1;预处理的秸杆,50,0.05M柠檬酸缓冲液,置250ml摇瓶中,用2M NaOH调pH5.0±0.1。
发酵液灭菌后,加入重组酿酒酵母菌种1.0g(以干菌体计)、0.1g Tween80(1g/l),发酵液总体积为100ml,在38℃,通氮气,厌氧发酵48-60h,乙醇浓度达10.2g/L。
实施例5重组酿酒酵母转化稻草秸杆生产乙醇
(1)秸杆预处理
取稻草150g,粉碎,过20目筛,加入0.5%硫酸1.2L,在150℃处理20h,再转入5L反应器,蒸汽加热1.5min至压强达15bar,维持10min,冷却,至压力为2bar。取出秸杆原料,用50℃水洗涤5次,过滤,得105g处理后的稻草秸杆,备用。
(2)菌种培养
斜面培养基组成(g/L)及培养条件:葡萄糖,20;酵母浸膏,3;琼脂,20;麦芽汁,3;pH5.5,温度30±1℃
种子培养基组成(g/L)及培养条件:葡萄糖,50;酵母浸膏,5;(NH4)SO4,7.5;K2HPO4,3.5;MgSO4·7H2O,0.75;CaCl2·2H2O,1M柠檬酸缓冲液,pH5.5±0.1,35±0.5℃,培养30h,转速150rpm
接种量:按每100ml转化液接种1.0g菌体(以干菌体计)
(3)秸杆转化为乙醇
发酵液组成(g/l):酵母浸膏,5;(NH4)2SO4,7.5;K2HPO4,3.5;MgSO4·7H2O,0.75;CaCl2·2H2O,1;预处理的稻草秸杆,50,0.05M柠檬酸缓冲液,置250ml摇瓶中,用2M NaOH调pH5.0±0.1。
发酵液灭菌后,加入重组酿酒酵母菌种1.0g(以干菌体计)、0.1gTween80(1g/l),发酵液总体积为100ml,在38℃,有氧发酵24-48h,乙醇浓度达7.82g/L。
Claims (1)
1.一种利用重组酿酒酵母生产纤维素乙醇的方法,其特征在于该重组酿酒酵母可将纤维质原料直接酶解为葡萄糖,并同时由该重组酿酒酵母将葡萄糖原位发酵转化为乙醇;所述的重组酿酒酵母可同时分泌三种纤维素酶:葡聚糖内切酶、纤维二糖水解酶和β-葡聚糖苷酶;构建该重组酿酒酵母的方法是:将葡聚糖内切酶基因、纤维二糖水解酶基因、β-葡聚糖苷酶基因与质粒载体及启动子连接,构建成3基因重组表达载体,转化酿酒酵母;所述质粒载体含有Ampr,URA3和leu2三个选择性标记或Kanr和URA3两个选择性标记。
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