CN109593662B - 一种乙酸和木糖共利用的马克斯克鲁维酵母菌株及筛选方法 - Google Patents
一种乙酸和木糖共利用的马克斯克鲁维酵母菌株及筛选方法 Download PDFInfo
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Abstract
一种乙酸和木糖共利用的马克斯克鲁维酵母菌株菌株,生物保藏编号为:CGMCC No.16757。该菌株由马克斯克鲁维酵母经纤维素水解液长期定向驯化后获得,步骤如下:从YPD平板上挑取酵母单菌落,40℃过夜培养;将收集的细胞重新接种至含有一定浓度纤维素水解液的新鲜培养基中,使其在620nm的光密度值≈0.3;40℃,150rmp/min培养24‑48h,当其OD620≥2.0(5‑7代)时,收集细胞;重复上述步骤,直至在此浓度的抑制物胁迫条件下细胞生长速度明显提高;提高纤维素水解液的浓度,开始新的重复培养;连续驯化后,将收集的细胞涂布于含有纤维素水解液的YPD平板上,40℃培养24‑48h;挑取生长状态较好的单菌落于YPD液体培养基中,40℃,150rmp/min培养24‑48h;将优势抗性克隆保存在30%甘油中。
Description
技术领域
本发明属于工业微生物遗传育种领域,涉及一种乙酸和木糖共利用的马克斯克鲁维酵母菌株和筛选方法。
背景技术
木质纤维素是最为丰富的可再生生物质资源,利用其作为底物进行燃料乙醇生产,可满足我国对能源的需求并且有利于环境保护。以木质纤维素为原料生产燃料乙醇,经济和社会意义重大。纤维素水解液中的抑制物尤其是乙酸对微生物的毒害作用是纤维素乙醇发展面临的主要瓶颈问题之一(Palmqvist,E. &Hahn-Hagerdal,B.Fermentation oflignocellulosichydrolysates.I: inhibition and detoxification.Bioresour.Technol.2000,74,17–24)。从目前的文献报道来看,可通过定向进化和过表达某些基因来提高纤维素水解液发酵菌株的乙酸抗性(Piper P,Calderon CO,Hatzixanthis K,MollapourM.Weak acidadaptation:the stress response that confers yeasts with resistanceto organic acid food preservatives.Microbiology,
2001.147:2635–2646;Yingying Chen,Lisa Stabryl,Na Wei.Improved aceticacid resistance in Saccharomyces cerevisiae by overexpression of the WHI2gene identified through inverse metabolic engineering.Appl EnvironMicrobiol.2016,82(7):2156-2166.)。而如果发酵菌株具有乙酸的利用特性,则可以进一步提高纤维素乙醇生产效率。(Na Wei,Josh Quarterman,Soo Rin Kim,Jamie H.D.Cate&Yong-Su Jin.Enhanced biofuel production through coupledacetic acid and xyloseconsumption by engineeredyeast。Nature communications,2013,4:2580-2588)。
马克斯克鲁维酵母(Kluyveromycesmarxianus)作为乳酸克鲁维酵母(Kluyveromyceslactis)的姊妹酵母,是一种能够同时天然发酵代谢多种碳源 (己糖、戊糖等)的耐热微生物。由于克鲁维酵母耐热性好,生长迅速,底物谱广泛、可同时利用纤维素水解液中葡萄糖、木糖等优点,被公认为是有望在生物质能源工业化中起到重要作用的非传统酵母。但对于其在抑制物抗性方面的研究还是比较欠缺,限制了其在纤维素乙醇生产中的应用。相关文献请参阅: Karin D,Breunig H,Steensma Y,Kluyveromyceslactis:genetics, physiology,and application.Springer Berlin Heidelberg,2003,2: 171-205;Fonseca G G,Heinzle E,Wittmann C,et al.The yeast Kluyveromycesmarxianusand its biotechnological potential. ApplMicrobiolBiotechnol.2008,79(3):339–354;Goshima T,Tsuji M,Inoue H,Yano S,Hoshino T,Matsushika A.Bioethanolproduction from lignocellulosic biomass by a novel Kluyveromycesmarxianus strain.Biosci.Biotech.Bioch.2013,77:1505-1510;Lane MM,MorrisseyJP.Kluyveromycesmarxianus:a yeast emerging from its sister's shadow. FungalBiol.Rev.2010,24,17-26;侯胜博,冯华良,高教琪,李益民,袁文杰,白凤武。马克斯克鲁维酵母的木糖和***糖发酵。生物工程学报,2017, 33(6):923-935。
发明内容
本发明针对木质纤维素乙醇生产中的纤维素水解液抑制微生物发酵问题,通过长期定向进化的方法,获得了一株对纤维素水解液抑制物抗性提高且能够利用乙酸的菌株,并且乙酸和木糖可同时利用,乙酸的添加还可提高木糖醇的产率,为纤维素乙醇生产菌株的选育提供了新的菌株和筛选方法。
为实现上述目的,本发明提供的乙酸和木糖共利用的马克斯克鲁维酵母菌株,该菌株分类学命名为马克斯克鲁维酵母(Kluyveromycesmarxianus)DX-5,已保藏于中国微生物保藏管理委员会普通微生物保藏中心(CGMCC),保藏日期: 2018年11月21日;保藏号:CGMCC No.16757。
上述菌株的可以利用乙酸为唯一碳源,且浓度可达到13g/L。在含13g/L 乙酸,10g/L酵母粉,20g/L蛋白胨的培养基中,酵母菌可以消耗全部乙酸,OD 达到2.左右。在含4g/L乙酸,1g/L K2PO4,0.1g/L CaCl2,0.4g/L MgCl·H2O, 0.5g/L NaCl,5g/L(NH4)2SO4,0.1g/L KH2PO4的培养基中,酵母菌可以消耗全部乙酸,OD达到0.7左右。在含10g/L乙酸,1g/L K2PO4,0.1g/L CaCl2,0.4 g/L MgCl·H2O,0.5g/L NaCl,5g/L(NH4)2SO4,0.1g/L KH2PO4的培养基中,酵母菌可以消耗部分乙酸,OD达到0.7左右。
本发明提供的乙酸和木糖共利用的马克斯克鲁维酵母菌株筛选为定向进化筛选法,主要步骤如下:
(1)菌株定向驯化:从YPD平板上挑取酵母单菌落,40℃过夜培养,收集细胞;
(2)将收集的细胞重新接种至含有一定浓度纤维素水解液的新鲜培养基中,使其在620nm的光密度值(OD620)≈0.3;
(3)40℃,150rmp/min培养24-48h,当其OD620≥2.0(5-7代)时,停止培养,收集细胞;
(4)重复上述步骤,直至在此浓度的抑制物胁迫条件下细胞生长速度明显提高;
(5)提高纤维素水解液的浓度,开始新的重复培养;
(6)继续驯化一段时间后,将收集的细胞涂布于含有纤维素水解液的YPD 平板上,40℃培养24-48h;
(7)挑取生长状态较好的单菌落于YPD液体培养基中,40℃,150rmp/min 培养24-48h;
(8)菌种保存:将优势抗性克隆保存在30%甘油中。
本发明所提供菌株的可以在添加有纤维素水解液混合抑制物的培养基上生长良好,并且能够同时利用乙酸和木糖,并且乙酸的添加能提高木糖醇的得率。定向进化筛选获得,为近一步发展具有综合抗逆能力、能够有效发酵不脱毒木质纤维素水解液的混合糖生产菌株奠定了基础。本发明选育的能够发酵不脱毒水解液的抗逆性耐高温菌株,能够有效降低木质纤维素乙醇生产成本。
附图说明
图1是出发菌株和Kluyveromycesmarxianus DX-5在不同浓度混合抑制物情况下的生长情况。
a:g/L:甲酸1.0,乙酸1.5,糠醛1.0,5-HMF 0.6;
b:g/L:甲酸1.0,乙酸2.0,糠醛1.0,5-HMF 0.6;
c:g/L:甲酸1.0,乙酸1.5,糠醛1.0,5-HMF 0.8;
d:g/L:甲酸0.8,乙酸1.0,糠醛0.8,5-HMF 0.6。
图2是Kluyveromycesmarxianus DX-5在不同浓度乙酸为唯一碳源时(有机氮源)的生长情况。
a:4g/L乙酸;氮源:10g/L酵母粉,20g/L蛋白胨
b:7g/L乙酸;氮源:10g/L酵母粉,20g/L蛋白胨
c:10g/L乙酸;氮源:10g/L酵母粉,20g/L蛋白胨
d:13g/L乙酸;氮源:10g/L酵母粉,20g/L蛋白胨
图3是Kluyveromycesmarxianus DX-5在不同浓度乙酸为唯一碳源时(无机氮源)的生长情况。
a:氮源:1g/L K2PO4,0.1g/L CaCl2,0.4g/L MgCl·H2O,0.5g/L NaCl,5
g/L(NH4)2SO4,0.1g/L KH2PO4,4g/L乙酸;
b:氮源相同,7g/L乙酸
c:氮源相同,10g/L乙酸
d:氮源相同,13g/L乙酸
图4是Kluyveromycesmarxianus DX-5在含有乙酸和木糖时的生长情况。
a:20g/L木糖和10g/L乙酸;b:20g/L木糖
具体实施方式
以下通过实施例和附图详细叙述本发明的具体实施例。
实施例1:水解液抗性酵母Kluyveromycesmarxianus DX-5的筛选
秸秆汽爆预处理液是使用粉粹后秸秆,料水比1:10,1%硫酸,130℃,2h 获得。水解液中含木糖30g/L,乙酸3g/L,甲酸0.9g/L。在10%提取水解液中添加葡萄糖2%、酵母提取物0.2%、蛋白胨1%做成起始定向进化的培养基。接种Kluyveromycesmarxianus出发菌株,620nm的光密度值(OD620)≈0.3;40℃,150rmp/min培养48h,当其OD620≥2.0时,收集细胞;按OD620≈0.3 在15%提取水解液中添加葡萄糖2%、酵母提取物0.2%、蛋白胨1%继续定向驯化。重复该步骤,提高驯化培养基中的水解液含量为100%。但OD620≥2.0 时,收集细胞,涂布含有1.5%琼脂的100%水解液培养基平板上,挑取生长状况好的单菌落进行培养和菌种保存。出发菌株Kluyveromycesmarxianus1727和 Kluyveromycesmarxianus DX-5在含有不同浓度抑制物的平板上生长情况,见图 1所示。
实施例2:Kluyveromycesmarxianus DX-5在不同浓度乙酸中的生长状况
Kluyveromycesmarxianus DX-5在YPD培养基,40℃,培养24h后,按 OD620≈0.3接种至不同浓度和不同种类的培养基中。Kluyveromycesmarxianus DX-5可以以不同浓度的乙酸为唯一碳源进行生长,与无机氮源相比,添加有机氮源会促进乙酸的利用。随乙酸浓度的升高,酵母细胞生长速率降低,乙酸耗尽时间延长。在有机氮源中,13g/L的乙酸可以在130h全部消耗完,OD620达到2左右。具体结果见附图2,图3。
实施例3:KluyveromycesmarxianusDX-5共利用木糖和乙酸状况
Kluyveromycesmarxianus DX-5在YPD培养基,40℃,培养24h后,按 OD620≈0.3接种至含10g/L的乙酸和20g/L木糖培养基中。
Kluyveromycesmarxianus DX-5可以同时利用乙酸和木糖。与不添加乙酸的木糖生长情况相比,添加高浓度乙酸会降低木糖的利用速率,但可以提高木糖醇产量。具体结果见附图4。
Claims (1)
1.一种乙酸和木糖共利用的马克斯克鲁维酵母(Kluyveromyces marxianus)菌株 DX-5,生物保藏编号为:CGMCC No.16757。
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