CN106399215B - 一种高效生产丁醇的重组梭菌、构建方法与应用 - Google Patents
一种高效生产丁醇的重组梭菌、构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种能提高丁醇发酵葡萄糖麦芽糖利用效率及生产强度的梭菌及其构建方法与应用,属于生物化工技术领域。所述梭菌含有序列为SEQ ID NO.1的glcG基因,glcG基因编码的蛋白质GlcG的氨基酸序列为SEQ ID NO.2。所述梭菌的构建方法包括以下步骤:(1)glcG基因过表达重组质粒构建;(2)glcG基因过表达重组菌株构建;(3)重组菌株丁醇发酵性能检测。本发明还包括所述能提高丁醇发酵葡萄糖麦芽糖利用效率及生产强度的梭菌在生产丁醇中的应用。本发明将glcG基因在C.acetobutylicum ATCC 824中过表达能显著提高葡萄糖麦芽糖利用效率及丁醇生产强度。
Description
技术领域
本发明涉及一种高效生产丁醇的重组梭菌及其构建方法与应用,尤其是涉及一种可用于生产丁醇的梭菌及其构建方法及应用。
背景技术
近年来,随着全球石油资源日渐枯竭、世界石油价格不断上涨,全球气候呈现的不断恶化趋势,能源问题成为当前世界经济及工业可持续发展亟待解决的问题,开发新型可再生、绿色环保能源,已成为世界各国能源开发战略之一。当今,生物质能源已成为仅次于石油、天然气和煤炭的世界第四大能源,因其具有可再生和绿色环保的优点,对社会和经济发展具有重要意义,具有广阔的应用前景。
生物丁醇,不仅是生物基化学品,可作为优良的有机溶剂及重要的化工原料,更是一种极具潜力的第二代新型生物燃料,其与生物乙醇相比,具有高于乙醇的热值及良好的燃料经济性。事实上,生物丁醇具有极为广阔的消费市场,在全球化工及能源领域需求量保持持续增长。一般来说,其化工合成法以石油为原料,投资大,技术设备要求高,因此生物丁醇的相关生产研究成为当前可再生绿色能源开发利用的热点之一。
传统丙酮丁醇发酵以淀粉基、糖基物料进行,当前已发展成为基于廉价可再生物料的新型生物丁醇发酵工艺。两者同样存在一系列制约因素,主要体现在丁醇产量及产率较低,发酵迟滞或周期过长,原料利用效率较低等问题。而在众多种类原料及其水解液成分中,葡萄糖均占有较高比例因而为丁醇发酵菌株提供高效碳源。事实上,丙酮丁醇发酵工业生产中,丁醇产量和产率(生产强度)是评价发酵性能的重要参数,提高丁醇产量有利于后续分离和提纯成本的降低,而产率对资本投入具有很大影响,GBL公司研究发现提高一倍产率,可降低20%固定投入,已有应用流式细胞监测和固定化连续发酵技术提高丁醇发酵产率的研究报道。
进一步提高生产菌株的丁醇产量及生产强度,有效办法之一是提高菌株对底物的代谢利用效率,缩短发酵周期及迟滞期,进而满足工业化指标。
2001年,丙酮丁醇梭菌基因组的测序工作完成,为研究工作者针对生物丁醇的分子水平调控提供了可能性。目前,基于转录组学、代谢组学及蛋白组学的相关研究进展有着广泛报道。
虽然目前对丙酮丁醇梭菌生理代谢及调控机制的认识已经取得很多进展,但通过菌株改造来提高丁醇发酵底物利用效率及生产强度的研究进展却不大,主要原因是缺少有效的靶蛋白进行代谢工程的定向改造。
发明内容
本发明所要解决的技术问题是,克服现有技术的不足,提供一种能提高丁醇发酵葡萄糖麦芽糖利用效率及生产强度的重组梭菌。
具体通过在梭菌内过表达glcG基因来提高重组菌对葡萄糖麦芽糖利用效率及丁醇发酵生产强度。所述重组梭菌含有核苷酸序列为SEQ ID NO.1的glcG基因,且所述glcG基因在梭菌内过表达,所述glcG基因编码的蛋白质GlcG的氨基酸序列为SEQ ID NO.2。其中,该GlcG蛋白质全长665个氨基酸,其理论分子量为70.54kDa。
在优选的技术方案中,上文所述的重组梭菌还含有核苷酸序列为SEQ ID NO.3的硫解酶的启动子。
在优选的技术方案中,上文所述梭菌选自产丁醇的丙酮丁醇梭菌(Clostridiumacetobutylicum),拜氏梭菌(Clostridium beijerinckii),糖乙酸多丁醇梭菌(Clostridium saccharoperbutylacetonicum)及糖丁酸梭菌(Clostridiumsaccharobutylicum);可以为野生型菌株,也可为经过诱变或遗传改造后的菌株。
本发明的另一个目的是,提供一种能提高丁醇发酵葡萄糖麦芽糖利用效率及生产强度的梭菌的构建方法。具体包括以下步骤:
(1)glcG基因过表达重组质粒的构建:将核苷酸序列为SEQ ID NO.1的glcG基因与pIMP1-Pthl质粒进行连接从而构建pIMP1-Pthl-glcG质粒;将重组质粒转入E.coli DH10B(pAN1)中进行甲基化,得到甲基化质粒pIMP1-Pthl-glcG;
其中,步骤(1)中所述pIMP1-Pthl质粒的构建方法如下:
以丙酮丁醇梭菌C.acetobutylicum ATCC 824基因组作为模板,利用PCR扩增153bp的硫解酶的启动子序列,将硫解酶的启动子序列经Pst I和Sal I酶切后与pIMP1质粒连接,得到载体质粒pIMP1-Pthl;
(2)glcG基因过表达重组菌株的构建:
通过电转化法,将步骤(1)所得甲基化质粒pIMP1-Pthl-glcG转化至丙酮丁醇梭菌C.acetobutylicum ATCC 824中,通过涂布于含有红霉素抗性的TGY琼脂培养基上,培养、筛选获得含有glcG基因过表达质粒pIMP1-Pthl-glcG的重组梭菌。
更为具体的,上文所述的glcG基因过表达重组菌株的构建过程如下:厌氧条件下,取50-100mL梭菌活化培养基(TGY)培养的对数中期(OD6201.0~1.5左右)的丙酮丁醇梭菌C.acetobutylicum ATCC 824细胞液,4℃、3000rpm离心10min,去除上清液,加入50mL预冷的电转缓冲液(270mmol/L蔗糖,5mmol/LNaH2PO4,pH7.4),洗涤两次,并重悬至1.5mL的电转缓冲液中,然后取80~100μL转入0.4cm的电转杯中,放置冰浴中用于电转化,加入10~20μL步骤(1)所得甲基化质粒pIMP1-Pthl-glcG,置于冰浴中2~3min,采用2000V脉冲电压和25μF的电容进行电转化,随后将电转液加入到梭菌活化培养基TGY中,37℃培养4~6h,2000~3000rpm离心10min收集菌体细胞,将收集的细胞涂布于含有红霉素抗性的TGY琼脂平板培养基上,培养36~40h后,获得含有glcG基因过表达质粒pIMP1-Pthl-glcG的丙酮丁醇梭菌,命名为丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMT-glcG)。
本发明进一步的目的是,提供一种利用上文所述的梭菌在生产丙酮丁醇中的应用:
步骤(2)获得的丙酮丁醇梭菌接种于含有红霉素抗性的发酵培养基中进行厌氧发酵,发酵温度37~38℃,搅拌转速为150rpm,发酵培养基初始pH调至5~6,发酵48~96h
本发明中使用的活化培养基、种子培养基及发酵培养基应理解为现有技术中任何可实现丙酮丁醇梭菌所适用的常规培养基,本发明实施例中所使用的配方如下:
活化培养基(g/L):葡萄糖20,胰蛋白胨30,酵母粉10。
种子培养基(g/L):葡萄糖70,乙酸铵3.22,酵母粉2.0,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
发酵培养基(g/L):葡萄糖或麦芽糖或混合糖70,乙酸铵3.22,酵母粉2,MgSO4·7H2O 0.2,KH2PO4 0.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
通过本发明后文所述的具体发酵实验证明,本发明将glcG基因在丙酮丁醇梭菌C.acetobutylicum ATCC 824中过表达能显著提高菌株的丁醇发酵葡萄糖麦芽糖利用效率及生产强度。
附图说明
图1为重组表达质粒pIMP1-Pthl-glcG的结构示意图;
图2为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L葡萄糖中的发酵动力学曲线;
图3为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L麦芽糖中的发酵动力学曲线;
图4为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L混合糖(葡萄糖:木糖=2:1)中的发酵动力学曲线。
具体实施方式
以下结合具体实施例对本发明作进一步详细说明。
以下实施例中所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,无特殊说明,均可从商业途径获取,所用培养基应理解为现有技术中任何可实现产丁醇梭菌所适用的常规培养基。
实施例1
本实施例包括以下步骤:
(1)pIMP1-Pthl质粒的构建
采用Sangon Biotech(上海生工)Ezup柱式细菌基因组DNA抽提试剂盒(货号:B518255)提取丙酮丁醇梭状芽孢杆菌C.acetobutylicum ATCC 824(购自美国标准生物品收藏中心)基因组DNA,利用引物:Pthl-F:GACACCTGCAGTTTTTAACAAAATATATTGA(划线部分为Pst I酶切位点)和Pthl-R:GACACGTCGACTTCTTTCATTCTAACTAACCTC(划线部分为Sal I酶切位点)从基因组DNA中扩增硫解酶的启动子序列(具体序列见SEQ ID NO.3),将PCR扩增的硫解酶启动子DNA用Pst I和Sal I进行双酶切,与使用Pst I和Sal I双酶切后的pIMP1质粒[Mermelstein L.D.,Welker N.E.,Bennett G.N.,Papoutsakis E.T.Expression ofcloned homologous fermentative genes in Clostridium acetobutylicum ATCC824.Nature Biotechnology,1992,10(2):190-5.]载体进行连接,从而构建载体质粒pIMP1-Pthl;
(2)glcG基因过表达重组质粒的构建
采用Sangon Biotech(上海生工)Ezup柱式细菌基因组DNA抽提试剂盒(货号:B518255)提取丙酮丁醇梭状芽孢杆菌C.acetobutylicum ATCC 824(购自美国标准生物品收藏中心)基因组DNA,利用引物:glcG-F:5’-GACACGTCGACATGGGTAATAAGATATTTGC(划线部分为Sal I酶切位点);glcG-R:5’-GACACGGTACCTTATTTTACGGTAACATCCG(划线部分为Kpn I酶切位点);利用PCR扩增1998bp的glcG基因(具体序列见SEQ ID NO.1),PCR产物经Sal I和Kpn I进行双酶切,与使用Sal I和Kpn I双酶切后的pIMP1-Pthl质粒载体进行连接,从而构建重组质粒pIMP1-Pthl-glcG;图1为重组表达质粒pIMP1-Pthl-glcG的结构示意图;将重组质粒转入E.coli DH10B(pAN1)[Mermelstein,L.D.&Papoutsakis,E.T.In vivomethylation in Escherichia coli by the Bacillus subtilis phage phi 3T Imethyltransferase to protect plasmids from restriction upon transformation ofClostridium acetobutylicum ATCC 824.Applied and Environmental Microbiology,1993,59(4),1077-1081.]中进行甲基化,得到甲基化重组质粒pIMP1-Pthl-glcG;
(3)glcG基因过表达重组菌株的构建
厌氧条件下,取50-100mL梭菌活化培养基(TGY)培养的对数中期(OD620 1.0左右)的丙酮丁醇梭菌C.acetobutylicum ATCC 824细胞液,4℃、3000rpm离心10min,去除上清液,加入50mL预冷的电转缓冲液(270mmol/L蔗糖,5mmol/LNaH2PO4,pH7.4),洗涤两次,并重悬至1.5mL的电转缓冲液中,然后取100μL转入0.4cm的电转杯中,放置冰浴中用于电转化,加入10μL步骤(1)所得甲基化质粒pIMP1-Pthl-glcG,置于冰浴中2min,采用2000V脉冲电压和25μF的电容进行电转化,随后将电转液加入到梭菌活化培养基TGY中,37℃培养4h,2000~3000rpm离心10min收集菌体细胞,将收集的细胞涂布于含有红霉素抗性的TGY琼脂培养基上,培养36h后,获得含有glcG基因过表达质粒pIMP1-Pthl-glcG的丙酮丁醇梭菌,命名为丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMT-glcG);
实施例2
重组菌株发酵生产丁醇,本实施例包括以下步骤:
将实施例1中所得重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMT-glcG)和对照空载质粒菌株C.acetobutylicum ATCC 824(pIMP1)及其出发野生型菌株C.acetobutylicum ATCC 824分别接种至活化培养基中(含10μg/mL红霉素抗性),置于厌氧环境中静置培养,培养温度为37.5℃,活化培养20h用于种子培养;将活化的菌种按10%(v/v)接种量接种于种子培养基中(含10μg/mL红霉素抗性),置于厌氧环境中摇瓶培养,培养温度为37.5℃,转速为150rpm,培养24~30h用于厌氧发酵培养;采用Biotec-3BG-4发酵罐(上海保兴生物设备工程有限公司)进行厌氧发酵,3L发酵罐培养时发酵培养基(含10μg/mL红霉素抗性)装液量为1.1L,发酵温度控制在37~38℃,搅拌转速为150rpm,通过添加稀硫酸或氢氧化钾溶液将接种后发酵培养基初始pH调至5.5,接种前发酵罐通入N2以除去发酵培养基中的溶氧,发酵48~96h,期间定时取样检测溶剂(丙酮、乙醇和丁醇)及葡萄糖含量。
本实施例中所涉及培养基分别按照如下方法制备:
活化培养基(g/L):葡萄糖20,胰蛋白胨30,酵母粉10。
种子培养基(g/L):葡萄糖70,乙酸铵3.22,酵母粉2.0,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
发酵培养基(g/L):葡萄糖70,乙酸铵3.22,酵母粉2,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
溶剂(丙酮、乙醇和丁醇)含量测定:发酵样品10000×g离心5min,取上清液,上清液组分浓度采用气相色谱法测定,色谱分离条件:色谱柱:毛细管色谱柱Agilent HP-INNOWAX(30m×0.25mm×0.50μm);柱温:100℃;进样口温度:250℃;FID检测器温度:300℃;H2流速:40mL/min;空气流速:400mL/min;载气N2流速:30mL/min;进样量:0.2μL;分流比:50:1;采用内标法进行定量分析,内标物使用异丁醇。
葡萄糖含量测定:发酵样品10000×g离心5min,取上清液,上清液葡萄糖浓度稀释至小于1g/L,使用SBA-40C生物传感分析仪(山东省科学院生物研究所)测定,取25μL上清稀释液直接进样,通过计算得出发酵液中葡萄糖浓度。
图2为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L葡萄糖中的发酵动力学曲线;结果表明glcG基因过表达重组菌株C.acetobutylicumATCC 824(pIMT-glcG)利用葡萄糖速率及合成丁醇速率加快,发酵28h左右已消耗近55g/L葡萄糖,丁醇产量达到12.6g/L;同一时刻,野生型C.acetobutylicum ATCC 824消耗葡萄糖近39g/L,生产丁醇9.1g/L,空载质粒菌株C.acetobutylicum ATCC 824(pIMP1)消耗葡萄糖近38g/L,生产丁醇8.0g/L。
发酵结果如下表1所示:
表1重组菌株、对照菌株及野生菌株葡萄糖发酵性能比较
本实施例实验结果表明,本发明将glcG基因在丙酮丁醇梭菌C.acetobutylicumATCC 824中过表达能显著提高菌株葡萄糖利用效率及丁醇发酵生产强度。
实施例3
重组菌株发酵生产丁醇,本实施例包括以下步骤:
将实施例1中所得重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMT-glcG)和对照空载质粒菌株C.acetobutylicum ATCC 824(pIMP1)及其出发野生型菌株C.acetobutylicum ATCC 824分别接种至活化培养基中(含10μg/mL红霉素抗性),置于厌氧环境中静置培养,培养温度为37.5℃,活化培养20h用于种子培养;将活化的菌种按10%(v/v)接种量接种于种子培养基中(含10μg/mL红霉素抗性),置于厌氧环境中摇瓶培养,培养温度为37.5℃,转速为150rpm,培养24~30h用于厌氧发酵培养;采用Biotec-3BG-4发酵罐(上海保兴生物设备工程有限公司)进行厌氧发酵,3L发酵罐培养时发酵培养基(含10μg/mL红霉素抗性)装液量为1.1L,发酵温度控制在37~38℃,搅拌转速为150rpm,通过添加稀硫酸或氢氧化钾溶液将接种后发酵培养基初始pH调至5.5,接种前发酵罐通入N2以除去发酵培养基中的溶氧,发酵48~96h,期间定时取样检测溶剂(丙酮、乙醇和丁醇)及麦芽糖含量。
本实施例中所涉及培养基分别按照如下方法制备:
活化培养基(g/L):葡萄糖20,胰蛋白胨30,酵母粉10。
种子培养基(g/L):葡萄糖70,乙酸铵3.22,酵母粉2.0,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
发酵培养基(g/L):麦芽糖70,乙酸铵3.22,酵母粉2,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
溶剂(丙酮、乙醇和丁醇)含量测定:发酵样品10000×g离心5min,取上清液,上清液组分浓度采用气相色谱法测定,色谱分离条件:色谱柱:毛细管色谱柱Agilent HP-INNOWAX(30m×0.25mm×0.50μm);柱温:100℃;进样口温度:250℃;FID检测器温度:300℃;H2流速:40mL/min;空气流速:400mL/min;载气N2流速:30mL/min;进样量:0.2μL;分流比:50:1;采用内标法进行定量分析,内标物使用异丁醇。
麦芽糖含量测定:发酵样品10000×g离心5min,取上清液,上清液麦芽糖浓度稀释至小于2g/L,采用DNS法测定测定,通过计算得出发酵液中麦芽糖浓度。
图3为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L麦芽糖中的发酵动力学曲线;结果表明glcG基因过表达重组菌株C.acetobutylicumATCC 824(pIMT-glcG)利用麦芽糖速率及合成丁醇速率加快,发酵20h左右已消耗近58g/L麦芽糖,丁醇产量达到8.0g/L;同一时刻,野生型C.acetobutylicum ATCC 824消耗麦芽糖近47g/L,生产丁醇6.3g/L,空载质粒菌株C.acetobutylicum ATCC 824(pIMP1)消耗麦芽糖近45g/L,生产丁醇6.0g/L。
发酵结果如下表2所示:
表2重组菌株、对照菌株及野生菌株麦芽糖发酵性能比较
本实施例实验结果表明,本发明将glcG基因在丙酮丁醇梭菌C.acetobutylicumATCC 824中过表达能显著提高菌株麦芽糖利用效率及丁醇发酵生产强度。
实施例4
重组菌株发酵生产丁醇,本实施例包括以下步骤:
将实施例1中所得重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMT-glcG)和对照空载质粒菌株C.acetobutylicum ATCC 824(pIMP1)及其出发野生型菌株C.acetobutylicum ATCC 824分别接种至活化培养基中(含10μg/mL红霉素抗性),置于厌氧环境中静置培养,培养温度为37.5℃,活化培养20h用于种子培养;将活化的菌种按10%(v/v)接种量接种于种子培养基中(含10μg/mL红霉素抗性),置于厌氧环境中摇瓶培养,培养温度为37.5℃,转速为150rpm,培养24~30h用于厌氧发酵培养;采用Biotec-3BG-4发酵罐(上海保兴生物设备工程有限公司)进行厌氧发酵,3L发酵罐培养时发酵培养基(含10μg/mL红霉素抗性)装液量为1.1L,发酵温度控制在37~38℃,搅拌转速为150rpm,通过添加稀硫酸或氢氧化钾溶液将接种后发酵培养基初始pH调至5.5,接种前发酵罐通入N2以除去发酵培养基中的溶氧,发酵48~96h,期间定时取样检测溶剂(丙酮、乙醇和丁醇)及麦芽糖含量。
本实施例中所涉及培养基分别按照如下方法制备:
活化培养基(g/L):葡萄糖20,胰蛋白胨30,酵母粉10。
种子培养基(g/L):葡萄糖70,乙酸铵3.22,酵母粉2.0,MgSO4·7H2O 0.2,KH2PO40.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
发酵培养基(g/L):混合糖(葡萄糖:木糖=2:1)70,乙酸铵3.22,酵母粉2,MgSO4·7H2O 0.2,KH2PO4 0.5,K2HPO4 0.5,FeSO4·7H2O 0.01,MnSO4·7H2O 0.01,生物素0.01,对氨基苯甲酸0.01。
溶剂(丙酮、乙醇和丁醇)含量测定:发酵样品10000×g离心5min,取上清液,上清液组分浓度采用气相色谱法测定,色谱分离条件:色谱柱:毛细管色谱柱Agilent HP-INNOWAX(30m×0.25mm×0.50μm);柱温:100℃;进样口温度:250℃;FID检测器温度:300℃;H2流速:40mL/min;空气流速:400mL/min;载气N2流速:30mL/min;进样量:0.2μL;分流比:50:1;采用内标法进行定量分析,内标物使用异丁醇。
葡萄糖及木糖含量测定:发酵样品10000×g离心5min,取上清液,葡萄糖及木糖浓度采用Waters 1525高效液相色谱测定。色谱分离条件:色谱柱:有机酸分析柱Aminex HPX-87H(300mm×7.8mm;Bio-Rad,Hercules);流动相:5mmol/L H2SO4;流速:0.5mL/min;进样量:20μL;柱温:50℃;PDA检测器检测波长:210nm。
图4为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1)、glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)在70g/L混合糖(葡萄糖:木糖=2:1)中的发酵动力学曲线;结果表明glcG基因过表达重组菌株C.acetobutylicum ATCC 824(pIMT-glcG)利用麦芽糖速率及合成丁醇速率加快,发酵24h左右已消耗全部葡萄糖,丁醇产量达到10.5g/L;同一时刻,野生型C.acetobutylicumATCC 824消耗葡萄糖近33g/L,生产丁醇7.6g/L,空载质粒菌株C.acetobutylicum ATCC824(pIMP1)消耗葡萄糖近33g/L,生产丁醇7.8g/L。
发酵结果如下表3所示:
表3重组菌株、对照菌株及野生菌混合糖发酵性能比较
本实施例实验结果表明,本发明将glcG基因在丙酮丁醇梭菌C.acetobutylicumATCC 824中过表达能显著提高菌株混合糖利用效率及丁醇发酵生产强度。
Claims (4)
1.一种高效生产丁醇的重组梭菌,其特征在于,所述重组梭菌含有glcG基因的过表达载体,且所述glcG基因在梭菌内过表达;所述梭菌为丙酮丁醇梭菌C.acetobutylicum ATCC824,所述glcG基因的核苷酸序列为SEQ ID NO.1;所述glcG基因编码的蛋白质的氨基酸序列为SEQ ID NO.2。
2.如权利要求1所述的高效生产丁醇的重组梭菌,其特征在于,所述重组梭菌的过表达载体还含有核苷酸序列为SEQ ID NO.3的硫解酶的启动子。
3.一种如权利要求1所述的重组梭菌的构建方法,其特征在于,包括以下步骤:
(1)glcG基因过表达重组质粒的构建:以丙酮丁醇梭菌C.acetobutylicum ATCC 824基因组作为模板,利用PCR扩增核苷酸序列为SEQ ID NO.1的glcG基因,再将其与pIMP1-Pthl质粒进行连接从而构建pIMP1-Pthl-glcG质粒;将pIMP1-Pthl-glcG重组质粒转入E.coliDH10B(pAN1)中进行甲基化,得到甲基化质粒pIMP1-Pthl-glcG;
(2)通过电转化法,将步骤(1)所得甲基化质粒pIMP1-Pthl-glcG转化至丙酮丁醇梭菌C.acetobutylicum ATCC 824中,通过涂布于含有红霉素抗性的TGY琼脂培养基上,培养、筛选获得含有glcG基因过表达质粒pIMP1-Pthl-glcG的重组梭菌。
4.一种如权利要求1所述的重组梭菌在生产丁醇中的应用,其特征在于,所述的重组梭菌能够缩短发酵周期,提高丁醇发酵葡萄糖麦芽糖利用效率及生产强度。
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