CN105154348B - 一种提高酿酒酵母对纤维素水解液抑制物耐受性的方法 - Google Patents
一种提高酿酒酵母对纤维素水解液抑制物耐受性的方法 Download PDFInfo
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Abstract
本发明公开了一种可以高效地获得对纤维素水解液抑制物耐性显著提升的酿酒酵母工程菌株的方法。通过过表达ABC(ATP‑binding cassette)转运蛋白基因ADP1提高酿酒酵母对纤维素水解液中多种抑制物(甲酸、乙酸和糠醛)的抗性。本发明的方法所构建的ADP1过表达菌株相比于空载对照菌株，在含有1.0g/L甲酸抑制物的条件下，发酵时间缩短12h；在含有1.8g/L糠醛抑制物的条件下，乙醇产量提高18.5％；在含有3.0g/L乙酸抑制物的条件下，残糖含量降低55.7％。本发明为解决纤维素乙醇生产中的关键问题，提高微生物对纤维素水解液抑制物抗性，提供了重要方法。
Description
技术领域
本发明属于微生物技术领域，涉及一种过表达ABC转运蛋白基因ADP1提高酿酒酵母对纤维素水解液抑制物耐受性的方法。
背景技术
石油、天然气、煤炭等化石型能源的不可再生性严重威胁我国社会的可持续发展，以可再生的生物质能源替代不可再生的化石能源，摆脱对石油的依赖，实现工业原材料的根本转变，是我国转变经济增长模式、保障社会经济可持续发展的重大战略需求[Bai FW,Anderson WA,Moo-Young M.Ethanol fermentation technologies from sugar andstarch feedstocks. Biotechnol Adv,2008,26:89-105]。燃料乙醇是目前开发最快的生物质能源，它自身含氧量高，既可以作为燃料直接使用，也可以与汽油或柴油以一定比例混合使用。燃料乙醇因其具有较高的辛烷值[Chaudhari AB,Dandi ND,Vadnere NC,etal.Bioethanol:a critical appraisal.Oxford: The Haworth Press,2004:793-824.]，无毒，是公认的最有可能替代化石能源的环境友好型生物质能源之一。纤维素燃料乙醇由于其原料来源广泛，价格低廉，且不与人争粮，不与粮争地等特点受到国内外学者的广泛关注。
纤维素原料主要由纤维素、半纤维素以及木质素组成，各组分之间通过共价键和氢键紧密结合，结构复杂[Haiyan Yang,Qian Chen,Kun Wanga,Run-CangSun.Correlation between hemicelluloses-removal-induced hydrophilicityvariation and the bioconversion efficiency of lignocelluloses:BioresourceTechnology,2013(147):539–544.]。酵母菌不能直接利用纤维素、半纤维素等组分，因此需要对纤维素原料进行预处理和水解将其转化成酵母可直接利用的单糖。然而，当前高效的纤维素预处理方法(如蒸汽爆破法，酸法，碱法等)在处理过程中都会产生多种抑制物，影响酵母菌的后续发酵过程[Palmqvist,E.and Hahn-Hagerdal,B. Fermentation oflignocellulosic hydrolysates.II:inhibitors and mechanisms of inhibition.Bioresource Technology,2000,74(1):25-33.]。纤维素水解液中的抑制物主要有三类：弱酸(如甲酸、乙酸等)，呋喃醛类(如糠醛，5-羟甲基糠醛等)，酚类化合物(如4-水杨酸，苯酚等) [Palmqvist,E.and Hahn-Hagerdal,B..Fermentation of lignocellulosichydrolysates.II:inhibitors and mechanisms of inhibition.BioresourceTechnology,2000,74(1):25-33.]。弱酸类抑制物主要通过引起胞内环境酸化和阴离子积累，从而降低生物量，降低乙醇产率等[Russell JB.Another explanation for thetoxicity of fementation acids at low PH:anion accumulation versus uncoupling.J ppl Bacreriol,1992,73:363-370.]。呋喃类化合物对细胞代谢过程中重要的酶(如己糖激酶等) 有抑制作用，有研究表明糠醛可以使细胞内活性氧的含量增加，对细胞产生毒害作用。[Allen, S.A.,Clark,W.,McCaffery,J.M.,et al.,Furfural induces reactiveoxygen species accumulation and cellular damage in Saccharomycescerevisiae.Biotechnology for Biofuels,2010:1754-6834.]，酚类化合物对酵母的毒性最大，主要通过对细胞膜的损伤(影响细胞膜的完整性和选择透过性等) 来影响细胞代谢。因此，获得对抑制物耐受性提高的工业酵母对于纤维素乙醇的发展和推广至关重要。
ABC(ATP-binding cassette)转运蛋白，是一种广泛存在于各种生物中的核酸结合蛋白 [Jamie Snider,Asad Hanif,HMapping the functional yeast ABC transporterinteractome,Nat Chem Biol.2013；9(9)]，其中酿酒酵母大约含有30种ABC转运蛋白，根据其核酸结合区域的不同分属于ABCB，ABCC，ABCD，和ABCG四个亚族，然而关于这些转运蛋白的功能研究还不是很清楚[Jungwirth,H.,Kuchler,K.Yeast transporter-a tale ofsex,stress,drugs and aging.FEBS Lett.2006,580,1131-1138]。ADP1是ABC转运蛋白的一种，通常被认为是一种“多药物/溶剂”排出泵基因，推测其可能与膜转运蛋白通过质子动力排出胞内有毒物质的功能相关 [Rojas,A.,Duque,E.etal.Three efflux pumps arerequired to provide efficient tolerance to toluene in Pseudomoas putida DOT-T1E.Bacteriol,2001；183:3967-3973]。但是，ADP1基因在纤维素水解液抑制物耐受性方面的研究还很少，没有关于该基因提高酿酒酵母抑制物耐受性的相关报道。
发明内容
本发明的目的在于利用基因工程的方法，构建过表达ADP1基因的重组酿酒酵母，使其能够在含有纤维素水解液抑制物的条件下直接发酵底物生产乙醇，解决纤维素水解液中抑制物对酵母发酵产生负面影响的问题。
所述的过表达的目的基因是ABC(ATP-binding cassette)转运蛋白基因ADP1，来源于供体菌S.cerevisiae 959基因组；所述ABC转运蛋白ADP1基因序列的GenBank登录号为NM001178724.1。
本发明的具体技术方案是：通过过表达转运蛋白基因ADP1的方法为在酿酒酵母中导入含有ADP1基因的多拷贝质粒载体构建阳性重组菌株，实现目的基因的过表达，从而提高其对纤维素水解液抑制物的耐受性。
本发明实施例中所用多拷贝质粒载体为pRS424，此处所述的多拷贝质粒载体还有多种，如pRS系列，如pRS424、pRS 324、pRS414、pYES2.0系列等。只要能实现目标蛋白的过表达的附加型载体或整合表达载体都在本发明保护内。
本发明的实施例中还在多拷贝质粒载体中导入了强启动子基因，所述的强启动子为3- 磷酸甘油酸激酶基因(3-phosphoglycerate kinase)启动子PGK；其基因序列的GenBank登录号为BK006937.1。对于本研究领域的研究人员来说,显而易见,启动子可换用其他在酿酒酵母中具有较强活性的启动子如ADH(乙醇脱氢酶),GAL(半乳糖苷酶),TPS1(海藻糖合成酶基因)。
本发明使用的受体菌株为模式菌株酿酒酵母菌株S.cerevisiae 959，显而易见,其他的模式菌株酵母菌株如S.cerevisiae280或工业用酵母菌株也可实现。
本发明的实施例中最终构建的阳性重组菌株为S.cerevisiae 959/ADP。其具体是通过分子生物学方法将PGK基因和ADP1基因插入过表达载体pRS424中，再将构建好的过表达载体用热激法导入E.coli DH5α中，其筛选标记为Amp抗性基因。从大肠杆菌中提取质粒，经过酶切验证后，以醋酸锂转化法将其导入菌株S.cerevisiae 959中，实现目的基因的过表达。其中，大肠杆菌培养、感受态制备及转化等基因工程操作均为常规操作方法，可以参照《分子克隆实验指南》(J.Sambrook,et al.第二版，1996)。
本发明为模式酿酒酵母菌株中携带目的基因的多拷贝游离质粒的过表达实验。启动子 PGK和目的基因ADP1之间通过NotI酶切位点连接，PGK-ADP1组合片段通过SacI和XhoI酶切位点与过表达载体连接，表达载体通过色氨酸营养缺陷型标记进行筛选。
本发明所述的纤维素水解液抑制物是甲酸、乙酸或糠醛。即本发明实施例的结果证明获得的阳性重组菌株S.cerevisiae 959/ADP具有对甲酸、乙酸和糠醛的抗性。
本发明的有益成果是，通过本方法构建的过表达菌株与其空载对照菌株相比具有更高的纤维素水解液抑制物耐受性，在分别含有甲酸(1.0g/L)，乙酸(3.0g/L)，糠醛(1.8g/L) 的YNB培养基中的发酵性能与其对照菌株相比都有明显提高。
附图说明
图1为PGK启动子的基因序列及其酶切验证电泳图谱。
图2为目的基因ADP1的基因序列及其酶切验证电泳图谱。
图3为含有1.0g/L甲酸的条件下过表达菌株和对照菌株发酵性能对比图。
图4为含有1.8g/L糠醛的条件下过表达菌株和对照菌株发酵性能对比图。
图5为含有3.0g/L乙酸的条件下过表达菌株和对照菌株发酵性能对比图。
具体实施方式
本发明实施例1～3所涉及的生物材料和培养基等试验材料，如无特殊说明，均可以由商业途径购买或者常规方法制备获得，其中：实验室模式菌株S.cerevisiae 959，pRS424载体，E.coli DH5α为本实验室保存也可以由商业途径购买；LB选择培养基(氯化钠10g/L，酵母浸粉5g/L，蛋白胨10g/L，Amp 100mg/L)、YNB培养基(Arg 20mg/L,Asp100mg/L,Glu 100mg/L,Iso 30mg/L,Lys 30mg/L,Val 150mg/L,Met 20mg/L,Phe 50mg/L,Ser375mg/L, Tyr 30mg/L,Thr 200mg/L,Ade 40mg/L,His 20mg/L,Leu 60mg/L,Ura 20mg/L,YNB 6.7g/L，葡萄糖20g/L)、YNB/Trp+固体培养基(琼脂粉30g/L,其它成分同YNB培养基)、种子培养基(葡萄糖30g/L,其它成分同YNB培养基)均经过无菌处理。
实施例1PGK-ADP1-PRS424过表达载体的构建
根据启动子PGK(GenBank：BK006937.1)序列，设计引物SEQ ID NO.1～2；根据目的基因ADP1(GenBank：NM001178724.1)序列，设计引物SEQ ID NO.3～4。
| 引物 | 序列 | SEQ ID NO |
| p-pgk forward primer | TCCGAGCTCGGAACTGTAATTGCTTTTAGTTG(Sac I) | 1 |
| p-pgk reverse primer | TTGCGGCCGCAATGTTTTATATTTGTTGTAAA(Not I) | 2 |
| p-adp forward primer | TTGCGGCCGCAAATGGGAAGTCATCGACGTTA(Not I) | 3 |
| p-adp reverse primer | CCGCTCGAGGGCCTACTTTTGTTCCACAACTA(Xho I) | 4 |
以S.cerevisiae 959基因组为模板扩增PGK和ADP1基因。PCR程序：95℃预变性5min； 94℃ 30s，55℃ 1min，72℃ 3min，30个循环；72℃延伸10min。PCR产物经胶回收纯化后，PGK片段经Sac I和Not I酶切后与经过同样处理的pRS424载体进行连接。连接产物转化E.coli DH5α感受态细胞，在LB选择培养基(Amp终浓度为100mg/L)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，得到空载质粒pRS424-PGK；ADP1片段经NotI和XhoI酶切后与经过同样处理的pRS424-PGK载体进行连接，重复上述步骤，得到过表达载体pRS424-PGK-ADP1。
实施例2S.cerevisiae 959的转化及转化子验证
采用醋酸锂转化法将空载质粒pRS424-PGK和过表达载体pRS424-PGK-ADP1分别导入 S.cerevisiae 959的感受态细胞，于YNB/Trp+固体平板上进行筛选。30℃培养2-3天，挑选转化子，提取酵母中质粒，将酵母质粒重新转化进E.coli DH5α感受态细胞进行验证。1％琼脂糖电泳显示转化入E.coli DH5α的质粒经Sac I和Not I酶切后获得872bp左右片段，与PGK 大小相符合；经Not I和Xho I酶切后获得3.15kb左右的片段，与ADP1大小相符合，证明获得阳性转化子，酶切图谱如图1和2所示。将空载菌株命名为S.cerevisiae 959-PGK，过表达菌株命名为S.cerevisiae 959-ADP。
实施例3转化子在含有抑制物的条件下发酵性能考察
取S.cerevisiae 959-PGK和S.cerevisiae 959-ADP各100μL，接至50mL的YNB培养基中，30℃培养24h后，按10％接种到种子培养基，然后将种子按初始OD620≈0.6接种分别含有一定浓度抑制物(甲酸1.0g/L，乙酸3.0g/L，糠醛1.8g/L)的YNB发酵培养基(50g/L 葡萄糖)中，100/250mL摇瓶，30℃培养，每12h取样。发酵液经适当稀释后，测定菌体密度(OD620)；发酵液5000rpm离心5min后，上清液用于测定发酵产物和残糖含量。转化子 S.cerevisiae959-ADP的生物量在各抑制物培养基中均略高于空载对照菌株，降糖速率和乙醇产率比对照菌株都有明显提高。
结果：由图3可以看出，在含有1.0g/L甲酸抑制物的条件下，过表达菌株从12h就开始消耗葡萄糖生产乙醇，在60h发酵结束，乙醇含量达到最大17.5g/L；空载菌株从24h开始消耗葡萄糖，在72h到达发酵终点，乙醇产量为17.2g/L，过表达菌株的延迟期比对照相比，缩短了12h；耗糖速率也明显高于对照菌株。
由图4的结果可以看出，在含有1.8g/L糠醛抑制物的条件下，过表达菌株菌株的降糖速率和乙醇产率都高于对照菌株。发酵结束时，过表达菌株的残糖含量为1.5g/L，空载菌株的发酵残糖含量为7.1g/L；过表达菌株的乙醇产量为15.8g/L，空载菌株为13.3g/L，比空载菌株提高了18.5％。
图5的结果反应了两株菌在含有3.0g/L乙酸的条件下的发酵情况，从图中也可以明显看出过表达菌株无论从降糖速率，残糖含量(13.8g/L和21.5g/L)，乙醇产量(9.7g/L和8.5g/L) 都优于对照空载菌株。
综上，过表达ABC转运蛋白基因ADP1的方法可以显著提高酿酒酵母对纤维素水解液抑制物(甲酸，乙酸，糠醛)的耐受性。
Claims (1)
1.一种提高酿酒酵母对纤维素水解液抑制物耐受性的方法，其特征在于：过表达转运蛋白基因ADP1，所述ADP1基因的GenBank登录号为NM001178724.1，所述的过表达转运蛋白基因ADP1的方法为，在酿酒酵母中导入含有ADP1基因的多拷贝质粒载体构建阳性重组菌株；所述的多拷贝质粒载体为pRS424、pRS324、pRS414或pYES2.0；所述的多拷贝质粒载体中还导入了强启动子基因ADH、GAL、TPS1或PGK；所述的酿酒酵母为S. cerevisiae 959或S. cerevisiae280；所述的纤维素水解液抑制物是甲酸、乙酸或糠醛。
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