CN107988241B - ptna基因片段在生产丁醇中的应用 - Google Patents
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Abstract
本发明公开了ptna基因片段在生产丁醇中的应用。具体为高产丁醇的过表达重组梭菌及其构建方法与应用。所述ptna基因的序列为SEQ ID NO.1。所述过表达重组梭菌的构建方法,包括以下步骤:(1)ptna基因过表达重组质粒构建;(2)过表达重组质粒的扩增(3)过表达重组质粒的甲基化;(4)ptna基因过表达重组菌株构建;(4)过表达重组菌株丁醇发酵性能检测。本发明还包括过表达重组梭菌在生产丁醇中的发酵应用。本发明将ptna基因在C.acetobutylicum ATCC 824中过表达能显著提高在ABE发酵中葡萄糖、果糖以及菊芋水解液的利用率及丁醇的产量。
Description
技术领域
本发明属于生物工程技术领域,涉及ptna基因片段在生产丁醇中的应用,具体为高产丁 醇的过表达重组梭菌及其构建方法与应用。
背景技术
近年来传统化石能源的日渐枯竭与能源需求的持续增长,社会经济的迅猛发展与温室效 应的逐年加剧,使得开发绿色可再生能源迫在眉睫,已成为全球环境与科研范畴内的研究热 点,也是人类社会未来可持续发展的重要议题。生物质资源则被认为是一种理想的可再生资 源,绿色环保、可再生的两大优点使之受到越来越多的关注,但目前生物质资源还存在利用 率低等缺点。
新型绿色环保、可持续发展的能源,已经成为全球共同的需求。生物液体燃料主要包括 生物乙醇,生物丁醇、生物柴油等,生物丁醇相对于其他生物燃料具有更多的优势。首先, 丁醇的能量密度及热值与汽油接近,可与汽油以任意比混溶,且无需对现有动力设备进行技 术改造,国际市场潜力巨大。其次,丁醇也是众多化学基产品的关键化工合成原料,对于解 决市场供需具有重要的经济意义。尽管生物丁醇展示了良好的发展态势,但仍然存在诸多技 术问题,总体来说,主要是发酵原料的成本较高、发酵过程中底物的利用不完全和发酵产物 中丁醇浓度较低等问题,因此开发一种廉价高效的生物工程策略,以提高丁醇发酵效率产值 及经济竞争能力具有极其重要的战略意义。
为有效解决原料成本问题,探索基于可再生物料的丁醇发酵工艺已经成为全球范围内的 研究热点之一。对于实际物料发酵,存在诸多问题,如原料利用率低,发酵周期长,丁醇产 量及产率低。实际上,在丁醇发酵生产中,丁醇的产量和产率是评价发酵性能的重要参数。
发明内容
本发明所要解决的技术问题是,克服现有生物丁醇发酵技术的不足,首先公开ptna基因 片段在生产丁醇中的应用,所述ptna基因片段(locus_tag="CA_P0066"),其具有如SEQ ID NO.1所示的核苷酸序列。所述ptna基因编码的蛋白质ptna mannose-specificphosphotransferase system component IIAB的氨基酸序列为SEQ ID NO.2。其中,该ptnamannose-specific phosphotransferase system component IIAB蛋白质全长325个氨基酸。
所述应用为构建过表达ptna基因片段相关的生物材料。具体涉及上述ptna基因片段相 关的生物材料为下述一种:
(1)含有上文所述的ptna基因的表达盒;
(2)含有上文所述的ptna基因的重组载体或含有(2)所述表达盒的重组载体;
(3)含有(2)所述重组载体的重组菌。
对于上述技术方案中,所述的(1)~(3)中任一项生物材料中,还含有核苷酸序列为 SEQ ID NO.3的硫解酶的启动子或其他能使ptna基因在梭菌内过表达的强启动子。
本发明还公开了通过在梭菌内过表达ptna基因来提高过表达重组菌株对葡萄糖、果糖和 菊芋水解液的利用率以及提高丁醇的产量。过表达重组梭菌含有核苷酸序列为SEQID NO.1 的ptna基因,且所述ptna基因在梭菌内过表达。
在优选的技术方案中,上文所述梭菌选自生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。
对于本发明上文所述的应用,还提供一种能提高丁醇发酵葡萄糖、果糖和菊芋水解液的 利用率及丁醇产量的梭菌的构建方法。具体包括以下步骤:
(1)过表达重组质粒的构建:将核苷酸序列为SEQ ID NO.3的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-thl,以丙酮丁醇梭菌C.acetobutylicum ATCC 824(购于American Type Culture Collection)基因组作为模板,利用PCR 扩增核苷酸序列为SEQ ID NO.1的ptna基因,将其与载体质粒pIMP1-thl进行连接从而构建 pIMP1-thl-ptna质粒;
(2)过表达重组质粒的扩增:将重组质粒热激转化转入E.coli DH5α进行扩增,提取质 粒pIMP1-thl-ptna及测序验证核苷酸序列位点有无突变或缺失。
(3)过表达重组质粒的甲基化:将核苷酸测序正确的重组质粒热激转化转入E.coli DH10B(pAN1)中进行甲基化,得到甲基化质粒pIMP1-thl-ptna;
(4)通过电转化法,将步骤(2)所得甲基化质粒pIMP1-thl-ptna转化至丙酮丁醇梭菌 C.acetobutylicum ATCC 824中,通过涂布于含有红霉素(50μg/mL)抗性的TGY琼脂培养基 上,培养、筛选获得含有甲基化质粒pIMP1-thl-ptna的过表达丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMP1-thl-ptna)。
具体操作步骤如下,ptna基因过表达重组菌株的构建过程:在厌氧操作箱中,取50mL 梭菌活化培养基(TGY)培养至OD6200.4~0.6的丙酮丁醇梭菌C.acetobutylicum ATCC824细 胞培养液,4℃、4500rpm离心10min,去除上清液,加入30mL预冷的ETM电转缓冲液,吹打均匀后静置10分钟,4℃、4500rpm离心10min,除上清液后加入1.5mL的ET电转缓 冲液中,吹打均匀然后取190μL加入0.4cm的电转杯中,放置冰上用于后续电转化,加入 10μL步骤(3)所得甲基化质粒pIMP1-thl-ptna,置于冰上2~3min,将质粒加入电转杯中与 细胞液混合均匀,采用1.8kV脉冲电压和25μF的电容进行电转,随后将电转液加入800mL 梭菌活化培养基TGY中,37℃培养4h,4000rpm离心5min,去除800mL上清液,将余下 液体吹打均匀后涂布于含有红霉素(50μg/mL)抗性的TGY琼脂平板培养基上,培养22~30 h后,获得含有甲基化质粒pIMP1-thl-ptna的丙酮丁醇梭菌,命名为丙酮丁醇梭菌C. acetobutylicum ATCC824(pIMP1-thl-ptna)。
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发酵应用:
步骤(4)获得的丙酮丁醇梭菌接种于含有红霉素(50μg/mL)抗性的发酵培养基及菊芋 水解液培养基中进行厌氧发酵,发酵培养基初始pH调至5.5,发酵温度37.5℃,搅拌转速为 150rpm,发酵时间72~168h。
对于上述技术方案中所述的过表达重组梭菌的构建方法,所述电转化法中使用的电转缓 冲液:ETM溶液(270mM蔗糖,0.6mM Na2HPO4,4.4mM NaH2PO4及10mM MgCl2)和 ET溶液(270mM蔗糖,0.6mM Na2HPO4及4.4mM NaH2PO4)。
本发明中使用的活化培养基、种子培养基及发酵培养基为现有技术中丙酮丁醇梭菌所适 用的常规培养基;菊芋水解液培养基为实际物料培养基,除菊芋水解液外,其他成分均与常 规发酵培养基相同。本发明所使用的培养基配方如下:
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):葡萄糖或果糖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。
菊芋水解液培养基(g/L):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;加 菊芋水解液(含葡萄糖约12g/L,果糖约48g/L)定容至1L。
菊芋水解液制备:将菊芋块茎切片晒干,碾碎菊芋块茎成细小颗粒;称量500g至烧杯 中,加入纯净水定容至4L;使用浓度为1.5mol/L的硫酸调节pH至2.0;105℃酸解1h; 用纱布滤除残渣,得到菊芋水解液,置于冰箱待用;菊芋水解液使用前用3mol/L的氢氧化 钾调节pH至6.0。
活化培养基和种子培养基配好后分装到点滴瓶中,需要通入氮气15分钟,压严瓶盖, 121℃灭菌15分钟。
本发明另一方面涉及上述高效生产丁醇的重组梭菌的发酵应用,即所述过表达重组梭菌 在提高丁醇发酵中葡萄糖、果糖和菊芋水解液的利用率,以及提高丁醇产量方面的发酵应用。 通过本发明后面所述的具体发酵实验证明,本发明将ptna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中葡萄糖、果糖和菊芋水解液 的利用率及丁醇的产量,由于丁醇对菌体具有毒害作用,导致丁醇产量十分有限,传统技术 手段使菌体产丁醇浓度每提高1g/L都显得十分困难。而过表达重组菌株相比野生型菌株,丁 醇的产量和产率在三种碳源的发酵过程中都有所增加。ptna基因过表达重组菌株C. acetobutylicum ATCC 824(pIMP1-thl-ptna)在葡萄糖为碳源的发酵过程中,丁醇产量为13.41 g/L,与野生型菌株C.acetobutylicum ATCC 824相比,增加了14.81%;丁醇产率从0.16g/L/h 增加到0.24g/L/h。C.acetobutylicum ATCC 824(pIMP1-thl-ptna)在果糖为碳源的发酵过程中, 丁醇产量为10.75g/L,与野生型菌株C.acetobutylicum ATCC824相比,增加了117.61%;丁 醇产率从0.03g/L/h增加到0.07g/L/h。C.acetobutylicumATCC 824(pIMP1-thl-ptna)在菊芋 水解液为碳源的发酵过程中,丁醇产量为7.65g/L,与野生型菌株C.acetobutylicum ATCC 824 相比,增加了42.72%;丁醇产率从0.06g/L/h增加到0.11g/L/h。
附图说明
图1为重组质粒pIMP1-thl的结构示意图;
图2为重组表达质粒pIMP1-thl-ptna的结构示意图;
图3为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824 (pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)在 70g/L葡萄糖中的残糖和丁醇的发酵动力学曲线;
图4为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824 (pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)在 70g/L果糖中的残糖和丁醇的发酵动力学曲线;
图5为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824 (pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)在菊 芋水解液中的残糖和丁醇的发酵动力学曲线。
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,所用活化培养基、种子培养基及发酵培养基为现有技术 中丙酮丁醇梭菌所适用的常规培养基,菊芋水解液培养基为实际物料培养基,除菊芋水解液 外,其他成分均为常规实验试剂。
实施例1
本实施例包括以下步骤:
(1)ptna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(购于American TypeCulture Collection)基因组DNA,利用引物:thl-F:GACACCTGCAGTTTTTAACAAAATATATTGA(划线部分为Pst I酶切位点)和thl-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核苷酸序列(具体序列见SEQ IDNO.3),将PCR扩增得到的硫解酶启动子DNA片段用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 of cloned homologous fermentative genes inClostridium acetobutylicum ATCC 824.Nature Biotechnology,1992,10(2):190-5.]载体进行连接,从而构建载体质粒pIMP1-thl;图 1为重组质粒pIMP1-thl的结构示意图;利用引物:ptna-F:5’- GCGTCGACATGGTAGGAATTATTCTTGC(划线部分为Sal I酶切位点);ptna-R:5’- GGGGTACCTTATTTTTGTTTATTTAGTT(划线部分为Kpn I酶切位点);PCR扩增978bp 的ptna基因(具体序列见SEQ ID NO.1),PCR产物经Sal I和Kpn I进行酶切,与使用Sal I 和Kpn I进行酶切后的pIMP1-thl质粒载体使用T4连接酶连接,从而构建过表达重组质粒 pIMP1-thl-ptna;图2为过表达重组质粒pIMP1-thl-ptna的结构示意图;
(2)过表达重组质粒的扩增:将重组质粒热激转化转入E.coli DH5α进行扩增,提取质 粒pIMP1-thl-ptna及测序验证核苷酸序列位点有无突变或缺失。
(3)过表达重组质粒pIMP1-thl-ptna的甲基化:将过表达重组质粒热激转化转入E.coli DH10B(pAN1)[Mermelstein,L.D.&Papoutsakis,E.T.In vivo methylation inEscherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase toprotect plasmids from restriction upon transformation of Clostridiumacetobutylicum ATCC 824.Applied and Environmental Microbiology, 1993,59(4),1077-1081.]中进行甲基化,得到甲基化过表达重组质粒pIMP1-thl-ptna;
(4)ptna基因过表达重组菌株的构建:在厌氧操作箱中,取50mL梭菌活化培养基(TGY)培养至OD6200.4~0.6的丙酮丁醇梭菌C.acetobutylicum ATCC 824细胞培养液,4℃、4500rpm离心10min,去除上清液,加入30mL预冷的ETM电转缓冲液,吹打均匀后静置 10分钟,4℃、4500rpm离心10min,除上清液后加入1.5mL的ET电转缓冲液中,吹打均 匀然后取190μL加入0.4cm的电转杯中,放置冰上用于后续电转化,加入10μL步骤(3) 所得甲基化质粒pIMP1-thl-ptna,置于冰上2~3min,将质粒加入电转杯中与细胞液混合均匀, 采用1.8kV脉冲电压和25μF的电容进行电转,随后将电转液加入800mL梭菌活化培养基 TGY中,37℃培养4h,4000rpm离心5min,去除800mL上清液,将余下液体吹打均匀后 涂布于含有红霉素抗性的TGY琼脂平板培养基上,培养22~30h后,获得含有甲基化质粒 pIMP1-thl-ptna的丙酮丁醇梭菌,命名为丙酮丁醇梭菌C.acetobutylicum ATCC 824 (pIMP1-thl-ptna)。
实施例2
重组菌株发酵生产丁醇,本实施例包括以下步骤:
首先活化菌种,将实施例1中所得重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC824 (pIMP1-thl-ptna)和空载质粒菌株C.acetobutylicum ATCC 824(pIMP1-thl)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50μg/mL红霉素抗性)。在 厌氧环境中,37.5℃静置培养20h,将活化的菌种按10%(v/v)接种量接种于种子培养基中(含 50μg/mL红霉素抗性),在摇床中培养,培养温度为37.5℃,转速为150rpm,培养24~30h; 使用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,在3L发酵罐 (含50μg/mL红霉素抗性)中发酵液量为1.1L,发酵温度37.5℃,转速为150rpm,接种前 发酵罐通入15min N2以除去发酵培养基中的溶氧,接种后通过添加稀硫酸或氢氧化钾溶液将 发酵液初始pH调至5.5,发酵72~168h,期间定时取样检测溶剂(丙酮、乙醇和丁醇)及残 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10min,取上清液,上清液中溶剂含量采用气相色谱法测定,色谱分离条件:毛细管色谱柱AgilentHP-INNOWAX(30cm×0.25mm×0.50um),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0.2uL,分流比50:1, 内标物为异丁醇。
葡萄糖含量测定:发酵样品10000×g离心10min,取上清液,上清液果糖浓度稀释至小 于2g/L,采用DNS法测定,通过计算得出发酵液中葡萄糖浓度。
图3为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna) 在70g/L葡萄糖中的残糖和丁醇的发酵动力学曲线;结果表明空载菌株C.acetobutylicum ATCC 824(pIMP1-thl)生产丁醇12.05g/L,野生型菌株生产丁醇11.68g/L。ptna基因过表 达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)对葡萄糖的利用率、丁醇产量增加, 丁醇产量达到13.41g/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ptna基因在丙酮丁醇梭菌C.acetobutylicumATCC 824 中过表达能显著提高菌株对葡萄糖的利用率以及丁醇的产量。
实施例3
过表达重组菌株发酵生产丁醇,本实施例包括以下步骤:
首先活化菌种,将实施例1中所得过表达重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC 824(pIMP1-thl-ptna)和对照空载质粒菌株C.acetobutylicumATCC 824(pIMP1-thl)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50μg/mL红霉素抗 性)。在厌氧环境中37.5℃静置培养20h,将活化的菌种按10%(v/v)接种量接种于种子培养 基中(含50μg/mL红霉素抗性),在摇床中培养,培养温度为37.5℃,转速为150rpm,培 养24~30h;使用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, 在3L发酵罐中发酵液(含50μg/mL红霉素抗性)量为1.1L,发酵温度37.5℃,转速为150rpm, 接种前发酵罐通入15min N2以除去发酵培养基中的溶氧,接种后通过添加稀硫酸或氢氧化钾 溶液将发酵液初始pH调至5.5,发酵72~168h,期间定时取样检测溶剂(丙酮、乙醇和丁醇) 及残糖含量。
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):果糖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。
活化培养基和种子培养基配好后分装到点滴瓶中,需要通入氮气15分钟,盖盖压严, 121℃灭菌15分钟。
溶剂(丙酮、乙醇和丁醇)含量测定:发酵样品10000×g离心10min,取上清液,上清液中溶剂含量采用气相色谱法测定,色谱分离条件:毛细管色谱柱AgilentHP-INNOWAX(30cm×0.25mm×0.50um),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0.2uL,分流比50:1, 内标物为异丁醇。
果糖含量测定:发酵样品10000×g离心10min,取上清液,上清液果糖浓度稀释至小于 2g/L,采用DNS法测定,通过计算得出发酵液中果糖浓度。
图4为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna) 在70g/L果糖中的残糖和丁醇的发酵动力学曲线。结果表明野生型C.acetobutylicum ATCC 824消耗43.64g/L的果糖,产生4.94g/L丁醇,空载质粒菌株C.acetobutylicum ATCC 824 (pIMP1-thl)消耗果糖43.07g/L,产生丁醇4.39g/L,ptna基因过表达重组菌株C. acetobutylicum ATCC 824(pIMP1-thl-ptna)对果糖的利用率及丁醇产量增加,至发酵结束利 用了57.38g/L的混合糖,产生丁醇10.75g/L;相比野生型菌株,果糖利用率提高了23.95%, 丁醇产量提高了117.61%。
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果糖发酵性能比较
本实施例实验结果表明,本发明将ptna基因在丙酮丁醇梭菌C.acetobutylicumATCC 824 中过表达能显著提高菌株对果糖的利用率及丁醇的产量。
实施例4
重组菌株发酵生产丁醇,本实施例包括以下步骤:
首先活化菌种,将实施例1中所得重组菌株丙酮丁醇梭菌C.acetobutylicum ATCC824 (pIMP1-thl-ptna)和对照空载质粒菌株C.acetobutylicum ATCC 824(pIMP1-thl)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50μg/mL红霉素抗性)。 在厌氧环境中37.5℃静置培养20h,将活化的菌种按10%(v/v)接种量接种于种子培养基中(含 50μg/mL红霉素抗性),在摇床中培养,培养温度为37.5℃,转速为150rpm,培养24~30h; 使用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,在3L发酵罐 中发酵液(含50μg/mL红霉素抗性)量为1.1L,发酵温度37.5℃,转速为150rpm,接种前 发酵罐通入15min N2以除去发酵培养基中的溶氧,接种后通过添加稀硫酸或氢氧化钾溶液将 发酵液初始pH调至5.5,发酵72~168h,期间定时取样检测溶剂(丙酮、乙醇和丁醇)及残 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。
菊芋水解液发酵培养基(g/L):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;加 菊芋水解液(含葡萄糖约12g/L,果糖约48g/L)定容至1L。
菊芋水解液制备:将菊芋块茎切片晒干,碾碎菊芋块茎成细小颗粒;称量500g至烧杯 中,加入纯净水定容至4L;使用浓度为1.5mol/L的硫酸调节pH至2.0;105℃酸解1h; 用纱布滤除残渣,得到菊芋水解液,置于冰箱待用;菊芋水解液使用前用3mol/L的氢氧化 钾调节pH至6.0。
活化培养基和种子培养基配好后分装到点滴瓶中,需要通入氮气15分钟,盖盖压严, 121℃灭菌15分钟。
溶剂(丙酮、乙醇和丁醇)含量测定:发酵样品10000×g离心10min,取上清液,上清液中溶剂含量采用气相色谱法测定,色谱分离条件:毛细管色谱柱AgilentHP-INNOWAX(30cm×0.25mm×0.50um),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0.2uL,分流比50:1, 内标物为异丁醇。
葡萄糖及果糖总含量测定:发酵样品10000×g离心10min,取上清液,葡萄糖及果糖浓 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AminexHPX-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5为野生型菌株C.acetobutylicum ATCC 824、空载质粒菌株C.acetobutylicumATCC 824(pIMP1-thl)、ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna) 在菊芋水解液(葡萄糖约12g/L,果糖约48g/L)中的残糖和丁醇的发酵动力学曲线;结果 表明ptna基因过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)对菊芋水解液中 糖的利用率以及丁醇产量均有较大提升。发酵终止,野生型C.acetobutylicum ATCC 824利用 糖33.27g/L,生产丁醇5.36g/L,丁醇产率为0.06;空载质粒菌株C.acetobutylicum ATCC 824 (pIMP1-thl)利用糖34.18g/L,生产丁醇5.35g/L;过表达重组菌株C.acetobutylicum ATCC 824(pIMP1-thl-ptna)利用了42.82g/L糖,产生7.65g/L的丁醇;相比野生型菌株,糖利用 率提高了28.70%,丁醇产量提高了42.72%,丁醇产率为0.11g/L/h。
发酵结果如下表3所示:
表3重组菌株、对照菌株及野生菌株菊芋水解液发酵性能比较
本实施例实验结果表明,本发明将ptna基因在丙酮丁醇梭菌C.acetobutylicumATCC 824 中过表达能显著提高菌株对菊芋水解液中糖的利用率及丁醇的产量。
序列表
<110> 大连理工大学
<120> ptna基因片段在生产丁醇中的应用
<130> 2011
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 978
<212> DNA
<213> ptna基因的核苷酸序列
<400> 1
atggtaggaa ttattcttgc tagtcacgga gaatttgcta agggtatctt gcaatcgggt 60
gcgatgattt ttggagacca agagaatgtg caagcggtta cattaatgcc tagtgaaggc 120
cctgatgatg ttaaagcaaa aatgaaagac gcaattgcat cctttgacaa ccaggatgag 180
gttttattct tagttgatct ttggggtggt acaccattca accaggctaa tagtctattt 240
gaagaacata aagataaatg ggcaatcgta gctggtatga atctaccaat ggtaattgaa 300
gcttatggtg cacgtctttc aatggaatct gcacatgaga ttgcagctag cattataagc 360
acagctaaag aaggagttaa agttaagcct gaagaattag aaccagaaga tgctggtaaa 420
gcttctcagg gttctgcaaa gcaatctaat acaggtgcac ctggatcatt cgaatatgtt 480
ttagctcgta ttgattctcg tttacttcat ggtcaagtag caactgcttg gacaaaagct 540
atgcaaccta caagaattat tgtagtatca gatgcagtag ctaaagacga gcttcgtaag 600
aaattgattc aacaagctgc tcctccagga gttaaagcac atgttgtacc aattaatcac 660
atgattaaac ttgcaaaaga cgatcaacac tttggaggac aacgtgcaat gcttcttttt 720
gagaatccag aagatgtact aagagtagta gaaggtggcg tacctcttaa aacaatcaat 780
gttggttcta tggctcactc tactggtaag gttcaaccaa ataaagtact tgctttcaat 840
caagaagata ttgatacctt caataagctt aaacaatctg ggttaacttt tgatgtccgt 900
aaggttccaa atgattcaaa aggaaatatg gacgaaataa ttaaaaaggc ccaagacgaa 960
ctaaataaac aaaaataa 978
<210> 2
<211> 325
<212> Protein
<213> ptna基因编码的蛋白质ptna mannose-specific phosphotransferasesystem component IIAB的氨基酸序列
<400> 2
MVGIILASHG EFAKGILQSG AMIFGDQENV QAVTLMPSEG PDDVKAKMKD AIASFDNQDE 60
VLFLVDLWGG TPFNQANSLF EEHKDKWAIV AGMNLPMVIE AYGARLSMES AHEIAASIIS 120
TAKEGVKVKP EELEPEDAGK ASQGSAKQSN TGAPGSFEYV LARIDSRLLH GQVATAWTKA 180
MQPTRIIVVS DAVAKDELRK KLIQQAAPPG VKAHVVPINH MIKLAKDDQH FGGQRAMLLF 240
ENPEDVLRVV EGGVPLKTIN VGSMAHSTGK VQPNKVLAFN QEDIDTFNKL KQSGLTFDVR 300
KVPNDSKGNM DEIIKKAQDE LNKQK 325
<210> 3
<211> 153
<212> DNA
<213> 硫解酶启动子(thl)基因的核苷酸序列
<400> 3
tttttaacaa aatatattga taaaaataat aatagtgggt ataattaagt tgttagagaa 60
aacgtataaa ttagggataa actatggaac ttatgaaata gattgaaatg gtttatctgt 120
taccccgtat caaaatttag gaggttagtt aga 153
Claims (6)
1.ptna基因片段在生产丁醇中的应用,所述ptna基因片段如SEQ ID NO.1所示的核苷酸序列;所述ptna基因片段编码如SEQ ID NO.2所示的氨基酸序列;
所述应用为构建过表达ptna基因片段相关的生物材料;
所述ptna基因片段在梭菌内过表达;
所述梭菌为丙酮丁醇梭菌(Clostridium acetobutylicum)。
2.根据权利要求1所述应用,其特征在于,所述ptna基因片段相关的生物材料为下述一种:
(1)含有权利要求1所述的ptna基因片段的表达盒;
(2)含有权利要求1所述的ptna基因片段的重组载体或含有(1)所述表达盒的重组载体。
3.根据权利要求2所述应用,其特征在于,所述的(1)~(2)中任一项的生物材料中,还包含核苷酸序列为SEQ ID NO.3的硫解酶的启动子。
4.根据权利要求1所述应用,其特征在于,包括以下步骤:
(1)过表达重组质粒的构建:将核苷酸序列为SEQ ID NO.3的硫解酶启动子thl序列经Pst I和Sal I酶切后与pIMP1质粒连接,得到载体质粒pIMP1-thl,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ptna基因片段,将其与载体质粒pIMP1-thl进行连接从而构建pIMP1-thl-ptna质粒;
(2)过表达重组质粒的扩增:将pIMP1-thl-ptna质粒热激转化转入E.coli DH5α进行扩增,提取质粒pIMP1-thl-ptna及测序验证核苷酸序列位点有无突变或缺失;
(3)过表达重组质粒的甲基化:将核苷酸测序正确的重组质粒热激转化转入E.coliDH10B(pAN1)中进行甲基化,得到甲基化质粒pIMP1-thl-ptna;
(4)通过电转化法,将步骤(3)所得甲基化质粒pIMP1-thl-ptna转化至丙酮丁醇梭菌C.acetobutylicum ATCC 824中,通过涂布于含有50μg/mL红霉素抗性的TGY琼脂培养基上,培养、筛选获得含有甲基化质粒pIMP1-thl-ptna的过表达丙酮丁醇梭菌C.acetobutylicumATCC 824(pIMP1-thl-ptna)。
5.根据权利要求4所述应用,其特征在于,所述电转化法中使用的电转缓冲液为ETM溶液和ET溶液;其中ETM溶液的配方为:270mM蔗糖,0.6mM Na2HPO4,4.4mM NaH2PO4及10mMMgCl2;ET溶液的配方为:270mM蔗糖,0.6mM Na2HPO4及4.4mM NaH2PO4。
6.根据权利要求4所述应用,其特征在于,所述过表达丙酮丁醇梭菌C.acetobutylicumATCC 824(pIMP1-thl-ptna)能提高丁醇发酵中葡萄糖、果糖和菊芋水解液的利用率,以及提高丁醇的产量。
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