CN107429219B - 高效乙醇发酵菌 - Google Patents
高效乙醇发酵菌 Download PDFInfo
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Abstract
本发明提供一种在不导入外来基因的情况下得到乙醇产生效率高的高效乙醇发酵菌。高效乙醇发酵菌是由五碳糖和六碳糖有效地产生乙醇的发酵菌,其特征在于以保藏号NITE BP‑01963被保藏于专利微生物保藏中心。
Description
技术领域
本发明涉及在使用木质纤维素类生物质生成生物乙醇的过程中用于发酵糖化溶液的微生物。
特别涉及在使用木质纤维素类生物质生成生物乙醇的过程中能够由五碳糖(下文中也称为C5糖)以及六碳糖(下文中也称为C6糖)有效地生成乙醇的微生物。
背景技术
生物乙醇作为由生物质生成的不会发生枯竭的可再生资源而倍受期待。另外,由于燃烧生物乙醇而生成的二氧化碳为碳中性的,因而据信通过促进生物乙醇的利用可抑制作为地球温室化的主要原因的二氧化碳的增加。
生物乙醇是通过将生物质发酵并进行蒸馏提纯乙醇而制得的。为了提高生物乙醇的收率,需要由糖化溶液中生成大多数的醇。在生物乙醇生成的过程中通常使用的酵母无法将木糖、阿拉伯糖等五碳糖转换为醇,因而作为发酵原料,仅使用了六碳糖。
尽管根据原料而不同,但在代表性的生物质中被认为含有35~45%的纤维素、25~40%的半纤维素、15~30%的木质素。因而,在不仅可利用六碳糖聚合而成的纤维素作为底物、而且还可利用主要含有作为五碳糖的木糖等的半纤维素作为底物时,可有效地产生乙醇。
木糖被认为是在生物质中大量含有的糖,其含量仅次于葡萄糖,有效地利用五碳糖是生物乙醇生产中的一大课题。
目前为止公开了少量地利用了木糖的技术,其是通过经基因重组而赋予木糖利用能力、或者利用木糖产生乙醇的微生物的利用等而实现的。
在专利文献1中公开了下文发明:将具有木糖转运体活性的基因导入到宿主细胞中,从而将木糖(C5糖)转换为木酮糖,整合在糖酵解系统的戊糖磷酸途径中,在发酵中加以利用。
在专利文献2中公开了利用被赋予了阿拉伯糖转运体的酵母来生成醇的技术。与专利文献1同样地、将阿拉伯糖(C5糖)经阿拉伯糖醇、木酮糖而整合在糖酵解系统的戊糖磷酸途径中,在发酵中加以利用。
在非专利文献1中公开了通过将源自大肠杆菌的木糖同化基因重组到发酵单胞菌中而赋予木糖同化能力的内容。
在非专利文献2中记载了毕赤属酵母可利用木糖生产乙醇的内容。
现有技术文献
专利文献
专利文献1:日本特开2012-170422号公报
专利文献2:美国专利申请公开第2013/189788号说明书
非专利文献
非专利文献1:Zhang,M.,et al.,Science,1995.Vol.267,pp.240-243.
非专利文献2:Bicho,P.A.,et al.,Appl.Environ.Microbiol.,1988,Vol.54,pp.50-54.
发明内容
发明所要解决的问题
但是,专利文献1的发明是将源自季也蒙假丝酵母(Candiida guilliermondii)的具有木糖转运体活性的蛋白质导入到作为宿主的酿酒酵母(Saccharomyces cerevisiae)中。即导入了外来基因。另外,专利文献2的发明,尽管转运基因不同,但也是向宿主中导入不同种基因的发明。
另外,非专利文献1所记载的技术是导入木糖同化基因的技术,与上述专利文献1和2的技术思想不同,但导入外来基因这一点不变。
因此,上述专利文献1和2、非专利文献1中记载的发明均需要采取用于实施联合国所采纳的“关于生物多样性公约的卡塔赫纳生物安全议定书”的遵循在日本施行的卡塔赫纳法的封锁对策。从而需要用于保证生物安全的设施,因而利用该细菌生成乙醇从成本方面考虑是不利的。
另外,根据非专利文献2记载的技术,关于毕赤属酵母利用,由于野生型的毕赤属酵母的木糖利用性低,因而乙醇生成效率并不那么高。
本发明的课题在于在不导入外来基因的条件下得到乙醇生成效率高的高效率乙醇发酵菌。
用于解决问题的手段
为了解决上述课题,本发明的高效率乙醇发酵菌是一种由五碳糖和六碳糖有效地生成乙醇的发酵菌,其特征在于,其是在以保藏号NITE BP-01962被保藏于专利微生物保藏中心的菌中导入了自克隆得到的转醛醇酶基因、醇脱氢酶基因以及丙酮酸脱羧酶基因而得到的菌,其以保藏号NITE BP-01963被保藏于专利微生物保藏中心。
野生型季也蒙毕赤酵母(Meyerozyma guilliermondii)虽说具备葡萄糖同化能力以及木糖同化能力,但是并不是说其具备对于生产生物乙醇为充分的木糖利用能力。与此相对地,本发明的高效乙醇发酵菌(下文也称作BP-01963菌株。)是以下一种菌:通过对季也蒙毕赤酵母(Meyerozyma guilliermondii)的亲代菌株进行菌株育种,选择出木糖利用效率高的菌,从而得到发酵菌(保藏号NITE BP-01962,下文也称作BP-01962菌株),并在该发酵菌中导入自克隆得到的转醛醇酶基因、醇脱氢酶基因以及丙酮酸脱羧酶基因而得到的菌。
所述转醛醇酶基因、醇脱氢酶基因以及丙酮酸脱羧酶基因均是季也蒙毕赤酵母(Meyerozyma guilliermondii)的基因。
所述转醛醇酶基因是戊糖磷酸途径中的酶,可以预见通过强化上述酶可以变得更容易利用木糖。另外,所述醇脱氢酶基因使乙醛生成乙醇。此外,丙酮酸脱羧酶基因使丙酮酸脱碳酸生成乙醛和CO2。
其结果,本发明的高效率乙醇发酵菌在不导入外来基因的情况下,能够得到比亲代菌株高的乙醇产生效率。
附图说明
图1是示出涉及BP-01963菌株、BP-01962菌株、以及N菌株在源自稀硫酸处理的玉米秸秆的酶糖化液中的糖浓度和发酵收率的关系的图表。
图2是示出涉及源自氨处理的稻秆的酶糖化液的BP-01963菌株的葡萄糖与木糖的消化量,以及乙醇的生成量的经时性变化的图表。
具体实施方式
以下,参照附图对本发明的实施方式进行更详细的说明。
作为子囊菌系酵母的季也蒙毕赤酵母(Meyerozyma guilliermondii)的野生菌株,不仅具备葡萄糖同化能力而且还具备木糖利用能力,但其木糖利用能力对于生物乙醇生产而言并不能称为充分。因此,本实施方式的高效乙醇发酵菌是通过下述方式而得到的发酵菌:将作为子囊菌系酵母的季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株作为亲代菌株并在向源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基中对该亲代菌株进行驯化培养,选择出在该培养基中培育的菌从而得到的发酵菌(保藏号:NITE BP-01962),在该发酵菌中导入自克隆得到的转醛醇酶基因(下文缩写为TAL基因)、乙醇脱氢酶基因(下文缩写为ADH基因)、以及丙酮酸脱氢酶基因(下文,缩写为PDC基因)。
所述源自氨处理的稻秆的酶糖化液,例如可以是使用下述方式得到的酶糖化液。首先,将日本埼玉县熊谷市产的稻秆浸渍在等量的25质量%氨水中,于80℃浸渍3小时后,将氨解吸出,由此进行前处理。接着,对已施以所述前处理的稻秆进行pH调节后,添加糖化酶(Meiji Seika Pharma株式会社制,商品名:支顶孢属纤维素酶),在50℃温度下保存72小时进行酶糖化,得到含有酶糖化液的浆液。接着,通过压滤对所述浆液进行固液分离,将所回收的液体成分作为所述源自氨处理的稻秆的酶糖化液。所述源自氨处理的稻秆的酶糖化液例如含有3~15质量%的葡萄糖和1~10质量%的木糖。
作为所述诱变剂,可以使用例如N-乙基-N-亚硝基脲(ENU)、甲磺酸乙酯(EMS)等乙基化剂、5-溴-2'-脱氧尿嘧啶核苷(BrdU)等碱基类似化合物、硝基胺、亚硝基胍等亚硝基化合物等。
BP-01962菌株是以下一种变异菌株:在向所述源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基中对亲代菌株进行驯化培养,并反复地选择出在该培养基中培育的菌,从而获得变异菌株。因此,BP-01962菌株与季也蒙毕赤酵母(Meyerozymaguilliermondii)的野生菌株或N菌株相比,在不导入外来基因的情况下就能够提高木糖同化性能和乙醇发酵性能。
向BP-01962菌株中进一步地导入自克隆得到的TAL基因、ADH基因、以及PDC基因从而得到的本实施方式的高效乙醇发酵菌,由本申请人保藏于独立行政法人日本国家技术与评价研究所专利微生物保藏中心(日本国〒292-0818千叶县木更津市上总镰足2-5-8 122号室)。保藏日为2014年11月19日,保藏号为NITE BP-01963。
上述TAL基因、ADH基因以及PDC基因中的任一个均为季也蒙毕赤酵母(Meyerozymaguilliermondii)的基因,因此即便将自克隆得到的上述基因导入BP-01962菌株,也不属于导入外来基因。
其结果,BP-01963菌株与BP-01962菌株或N菌株相比,在不导入外来基因的情况下,进一步提高了木糖同化性能和乙醇发酵性能。
对于BP-01963菌株的自克隆得到的TAL基因、ADH基因和PDC基因的导入,例如可以是通过下述方法进行的。
对于希望导入的基因及其终止子部(下文称作基因+终止子部)进行PCR扩增。对导入中想要使用的启动子部进行PCR扩增。它们均由本发明中使用的菌株即季也蒙毕赤酵母(Meyerozyma guilliermondii)的染色体进行PCR扩增。
将PCR扩增后的DNA片段按照启动子、基因+终止子部的顺序使用融合(In-Fusion)法克隆到大肠杆菌用的市售载体中。将克隆后的载体转化到大肠杆菌中,进行载体的扩增。利用限制酶由扩增后的载体切出启动子以及基因+终止子部,或者由扩增后的载体进行PCR扩增,从而得到同源重组用的DNA片段。
将所得到的DNA片段同源重组到菌株中,得到所期望的菌株。同源重组中使用电穿孔法(electroporation method)。通过该方法导入基因时,能够在染色体上导入多个拷贝,因而能够增强所导入的酶的活性。
作为同源重组用DNA片段,例如可以使用木糖还原酶的启动子、转醛醇酶+终止子。这是由于,可以认为通过使用在木糖同化时发挥功能的木糖还原酶的启动子,转醛醇酶能够有效地发挥作用。
木糖还原酶的启动子具体地说使用下述序列编号1和序列编号2的引物进行扩增,转醛醇酶基因和终止子部分使用下述序列编号3和4的引物进行扩增。
序列编号1:AAGGCTTGGGAACTTTCTTT
序列编号2:AGCAATTGATGATTAATTTT
序列编号3:ATGACCAATTCTCTTGAACA
序列编号4:AAATTGTGCCGTGTCAAACT
另外,也可以使用GAPDH的启动子、醇脱氢酶+终止子。GAPDH是存在于糖酵解系统中的强力的启动子,因而通过将其用作作为糖酵解系统的酶的醇脱氢酶的启动子,可有效地发挥出作用。醇脱氢酶具有将乙醛转换为乙醇的作用,同时由于在NADH依赖性的情况下生成NAD+,因而具有将NAD+依赖的木糖醇脱氢酶的作用进行强化的作用。
GAPDH的启动子具体地使用下述序列编号5和序列编号6的引物进行扩增,醇脱氢酶基因和终止子部分使用下述序列编号7和8的引物进行扩增。
序列编号5:GTTGTAGCGGAGGCTCAATT
序列编号6:TGTATAATTTAAATGTGGGT
序列编号7:ATGTCAATTCCAGAATCCAT
序列编号8:CACCTTGGCTGGAAGTGCTG
PDC基因通过将PDC基因的启动子转换为GAPDH的启动子从而得以强化。通过向用序列编号9和序列编号10所表示的序列之间导入用序列编号5和序列编号6的引物扩增的GAPDH的启动子所得到的DNA片段通过同源重组进行置换。序列编号9的序列表示PDC基因的启动子的末端,序列编号10的序列表示PDC基因的起始端。
序列编号9:AGATTGCTGCAAAAATCATC
序列编号10:ATGACAGAAATTACTTTGGG
此外,虽然通过该方法得到的菌株导入了基因,但由于是自克隆,因此依据卡塔赫纳法的规定,上述菌株属于非重组菌的范畴。
接着,利用源自稀硫酸处理的玉米秸秆的酶糖化液,对BP-01963菌株、BP-01962菌株、N菌株的发酵收率进行比较。
所述源自稀硫酸处理的玉米秸秆的酶糖化液是使用下述方式得到的酶糖化液。首先,将美国爱荷华州产的玉米秸秆浸渍到2倍量的3.7质量%硫酸中,并在170℃温度下浸渍10分钟后,恢复至常温,由此进行前处理。接着,向已施以所述前处理的玉米秸秆中添加NaOH水溶液从而调节至pH4后,添加糖化酶(Meiji Seika Pharma株式会社制,商品名:支顶孢属纤维素酶),在50℃温度下保持72小时进行酶糖化,得到含有酶糖化液的浆液。接着,将所述浆液通过离心分离来进行固液分离,将所回收的液体成分用NaOH水溶液调节至pH6,作为所述源自稀硫酸处理的玉米秸秆的酶糖化液。所述源自稀硫酸处理的玉米秸秆的酶糖化液例如含有3~15质量%的葡萄糖和1~10质量%的木糖。
接着,以15质量%的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基,向该培养基中添加BP-01963菌株的培养液,以使得培养基的OD600达到2.0,于30℃温度下进行100小时培养。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖45g/L、木糖38g/L,且为pH6。并且,进行所述培养后采集所述培养基,通过GC-FID(GL Sciences Inc.株式会社制,商品名:GC390B)测定乙醇的浓度,通过下式(1)算出发酵收率。结果示于图1。
发酵收率=生成乙醇浓度/(葡萄糖浓度+木糖浓度)/0.5114···(1)
(葡萄糖浓度和木糖浓度为培养开始前的初始浓度)
接着,以20质量%的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基,向该培养基中添加BP-01962菌株的培养液,以使得培养基的OD600达到0.5,于30℃温度下进行100小时培养。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖64g/L、木糖48g/L,且为pH6。并且,在所述培养后采集所述培养基,通过GC-FID(GL Sciences Inc.公司制,商品名:GC390B)测定乙醇的浓度,通过式(1)算出发酵收率。结果示于图1。
接着,以26质量%的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基,分别向该培养基中添加季也蒙毕赤酵母(Meyerozyma guillier mondii)N菌株的培养液,以使得培养基的OD600达到0.5,于30℃温度下进行100小时培养。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖64g/L、木糖48g/L,且为pH6。并且,在所述培养之后采集所述培养基,通过GC-FID(GL Sciences Inc.公司制,商品名:GC390B)测定乙醇的浓度,通过式(1)算出发酵收率。结果示于图1。
由图1明显可知,BP-01963菌株与N菌株相比,对于低浓度的源自前述稀硫酸玉米秸秆的酶糖化液,具备比BP-01962菌株和N菌株更优异的乙醇发酵性能。
接着,以26质量%的源自氨处理的稻秆的酶糖化液作为培养基,向该培养基中添加BP-01963菌株的培养液,以使得培养基的OD600达到2.0,于30℃进行120小时培养。所述源自氨处理的稻秆的酶糖化液含有葡萄糖73.8g/L、木糖28.3g/L,且为pH6。并且,每隔规定时间采集所述培养基,分别通过HPLC(东曹公司制,商品名:LC-8020)测定木糖的浓度、通过GC-FID(GL Sciences Inc.公司制,商品名:GC390B)测定乙醇的浓度。结果示于图2。
由图2可知,从培养开始起120小时后,葡萄糖和木糖的全部量被消化,乙醇浓度随着培养时间越长而变得越高。此外,从培养开始起48小时后,葡萄糖浓度几乎变为零,但其后木糖浓度也下降,乙醇浓度继续增加,因此明显可知,BP-01963菌株在将葡萄糖的全部量消化后,以木糖作为底物进行乙醇发酵。
附图标记说明
无标记。
Claims (1)
1.一种高效乙醇发酵菌,其是由五碳糖和六碳糖有效地产生乙醇的发酵菌,该发酵菌的特征在于,
其是在以保藏号NITE BP-01962被保藏于专利微生物保藏中心的菌中导入了自克隆得到的转醛醇酶基因、醇脱氢酶基因以及丙酮酸脱羧酶基因而得到的菌,
其以保藏号NITE BP-01963被保藏于专利微生物保藏中心。
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CN107429219A (zh) | 2017-12-01 |
BR112017010660A2 (pt) | 2017-12-26 |
EP3228698B1 (en) | 2019-08-14 |
EP3228698A1 (en) | 2017-10-11 |
JP6145583B2 (ja) | 2017-06-14 |
US20170369906A1 (en) | 2017-12-28 |
JPWO2016088275A1 (ja) | 2017-05-25 |
US10059965B2 (en) | 2018-08-28 |
WO2016088275A1 (ja) | 2016-06-09 |
EP3228698A4 (en) | 2018-05-23 |
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