CN105505849B - 联产丁醇与2,3-丁二醇的基因工程菌及其构建方法和应用 - Google Patents
联产丁醇与2,3-丁二醇的基因工程菌及其构建方法和应用 Download PDFInfo
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- CN105505849B CN105505849B CN201610046293.6A CN201610046293A CN105505849B CN 105505849 B CN105505849 B CN 105505849B CN 201610046293 A CN201610046293 A CN 201610046293A CN 105505849 B CN105505849 B CN 105505849B
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- butanediol
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- butanol
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01005—Acetoin dehydrogenase (1.1.1.5)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01005—Acetolactate decarboxylase (4.1.1.5)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02T—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO TRANSPORTATION
- Y02T50/00—Aeronautics or air transport
- Y02T50/60—Efficient propulsion technologies, e.g. for aircraft
- Y02T50/678—Aviation using fuels of non-fossil origin
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- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了联产丁醇与2,3‑丁二醇的基因工程菌,它是导入了乙偶姻还原酶基因的产丁醇梭菌或者导入了乙酰乳酸脱羧酶基因和乙偶姻还原酶基因的产丁醇梭菌,所述的乙酰乳酸脱羧酶基因,其核苷酸序列为SEQ ID NO:1~7所示核苷酸序列中的任一种,所述的乙偶姻还原酶基因,其核苷酸序列为SEQ ID NO:8~11所示核苷酸序列中的任一种。本发明通过在丙酮丁醇梭菌中强化乙酰乳酸脱羧反应,将3‑羟基丁酮产量由初始1.8g/L左右大幅提高到6.4g/L左右,进而在乙偶姻还原酶的作用下将乙偶姻转化为高达8.05g/L的2,3‑丁二醇;而丙酮由4.5g/L左右降低至2.2g/L,显著提高了丙酮丁醇梭菌发酵的经济效益。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种联产丁醇与2,3-丁二醇的基因工程菌及其构建方法和应用。
背景技术
2,3-丁二醇是一种无色、无嗅的液体,具有较强的吸湿性。常温下为液体,黏度30.6mPa·s,沸点为178~182℃,冰点为-60℃,与水互溶,溶于乙醚、乙醇,在聚合体合成中用作单体。2,3-丁二醇是一种重要的生物基四碳平台化合物,广泛用于食品、化工、航空航天燃料等领域。
2,3-丁二醇生产方法主要有化学合成法和微生物发酵法两大类,化学合成法是利用化石原料生产2,3-丁二醇成本高,且条件苛刻、过程繁琐,容易对环境造成污染,而化石原料日益枯竭,因此化学合成法工业化生产较困难;生物转化法是利用可再生资源生产2,3-丁二醇,生物法既克服了化学法生产的原料问题,又符合绿色化工的要求,因此受到了人们越来越多的关注。目前2,3-丁二醇高产菌株主要是Klebsiella、Enterobacter和Serratia属的菌株,然而这些菌株具有潜在致病性,不符合工业化安全生产的要求。而目前报道的安全菌株如B.subtilis合成2,3-丁二醇的效率太低。
丙酮丁醇梭菌(Clostridium acetobutylicum),是目前生产丁醇的主要菌种。其发酵产物除丁醇外,还有丙酮和乙醇,因此在生产中又被称为ABE(Acetone-Butanol-Ethanol)发酵。通常,60g/L的葡萄糖经丙酮丁醇梭菌发酵可产生12g/L的丁醇,6g/L的丙酮以及2g/L的乙醇,丁醇:丙酮:乙醇的比值为6:3:1(w/w)。在丙酮丁醇梭菌中导入乙酰乳酸脱羧反应酶和乙偶姻还原酶后,除ABE产物外,丙酮丁醇梭菌还会产生不少量的2,3-丁二醇。更重要的是,2,3-丁二醇生产过程中会有还原力(NADH或NADPH)的产生,在2,3-丁二醇的专性生产菌株中,这些还原力需被O2氧化的方式实现NAD+的再生,从而造成还原力的浪费。而丙酮丁醇梭菌中丁醇合成过程恰恰是一个缺乏NAD(P)H的过程,以致丁醇发酵中有大量的氧化态副产物丙酮的生成。因此,在丙酮丁醇梭菌中构建2,3-丁二醇合成途径,能够利用2,3-丁二醇合成过程中产生的还原力补偿丁醇合成过程所需的还原力,从而在NAD(P)H代谢平衡的基础上以丙酮为代价生产2,3-丁二醇。
提高丙酮丁醇梭菌的2,3-丁二醇产量有以下几方面的意义:
(1)提高传统ABE发酵的经济效益。目前丁醇价格约1.1万元/吨,丙酮0.8万元/吨,乙醇0.55万元/吨。按照3吨糖产1吨溶剂来算,ABE发酵的产品价值只是略高于原料(约3200元/吨糖),使得生物法生产ABE经济效益很差。然而2,3-丁二醇价格较高,市场价格约10万元/吨。因此提高ABE发酵中的2,3-丁二醇产量将显著提高ABE发酵的经济效益。
(2)有利于开发以2,3-丁二醇为代谢前体的其他重要化学品。ABE发酵中,丁醇是一个疏水性很强的化合物,对细胞有很大的毒性,限制了丁醇的发酵效率。相比丁醇,2,3-丁二醇或者2-丁醇对细胞的毒性较小。2,3-丁二醇经脱水反应可以生产1,3-丁二烯,1,3-丁二烯可以进一步用于合成橡胶、ABS树脂及SBS弹性体等,酯化产品是合成聚酯和聚氨酯的前体。由于2,3-丁二醇冰点低达-60℃,因此可以用作抗冻剂。2,3-丁二醇还可以通过脱氢反应生成双乙酰,双乙酰具有令人愉快的香味,是食品工业中一种具有高附加值的调味剂,同时双乙酰可以抑制食物中一些微生物的生长。另外,2,3-丁二醇通过脱氢反应还可以生产甲基乙基酮,它是一种有效的燃料添加剂,燃烧值比乙醇还要高,同时甲基乙基酮还可以用作树脂和涂料的溶剂。而2,3-丁二醇本身的燃烧热值达到27198J/g,可以和其它液体燃料如甲醇(22081J/g)和乙醇(29055J/g)相媲美,将等摩尔的乙醇和2,3-丁二醇混合后,燃烧热值可以达到27660J/g,因此,乙醇的添加并不影响2,3丁二醇在燃料工业的应用。
为了提高丙酮丁醇梭菌2,3--丁二醇的产量,研究通常先强化产生3-羟基丁酮的乙酰乳酸的合成反应,而非强化乙酰乳酸脱羧反应。因为通常认为,乙酰乳酸合成反应对3-羟基丁酮的生成贡献更大,而即使在缺乏ALDC(乙酰乳酸脱羧酶)的情况下乙酰乳酸仍可以自发脱羧生成3-羟基丁酮。然而,无论是在丙酮丁醇梭菌中表达其自身的ALS(乙酰乳酸合成酶)还是枯草芽孢杆菌的ALS,3-羟基丁酮产量均没有提高(Wardwe ll,S.A.Metabolismof acetoin in C.acetobutylicum ATCC824.Rice University.1999)。这表明丙酮丁醇梭菌中通过表达ALS来强化乙酰乳酸的合成反应对3-羟基丁酮产量的影响甚小。
发明内容
本发明要解决的技术问题是,提供一种联产丁醇与2,3-丁二醇的基因工程菌。
本发明还要解决的技术问题是,提供上述联产丁醇与2,3-丁二醇的基因工程菌的构建方法。
本发明最后要解决的技术问题是,提供上述联产丁醇与2,3-丁二醇的基因工程菌的应用。
为解决上述技术问题,本发明采用如下技术方案:
联产丁醇与2,3-丁二醇的基因工程菌,它是导入了乙偶姻还原酶基因的产丁醇梭菌。
联产丁醇与2,3-丁二醇的基因工程菌,它是导入了乙酰乳酸脱羧酶基因和乙偶姻还原酶基因的产丁醇梭菌。
其中,所述的乙酰乳酸脱羧酶基因,其核苷酸序列为SEQ ID NO:1~7所示核苷酸序列中的任一种,其核苷酸序列优选SEQ ID NO:1、SEQ ID NO:2、SEQ ID N O:7,最优选SEQID NO:2。
其中,所述的乙偶姻还原酶基因,其核苷酸序列为SEQ ID NO:8~11所示核苷酸序列中的任一种,其核苷酸序列优选SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:11,最优选SEQID NO:9、SEQ ID NO:11。
其中,所述产丁醇梭菌为丙酮丁醇梭菌(C.acetobutylicum)、拜氏梭菌(C.beijerinck ii)、糖丁醇梭菌(C.saccharobutylicum)、糖丁醇丙酮梭菌(C.saccharoperbutylacetonicum)、丁酸梭菌(C.butyricum)、产芽孢梭菌(C.sporogenes)、巴氏梭菌(C.pasteurianum)、酪丁酸梭菌(C.tyrobutyricum)、产气荚膜梭菌(C.perfringens)或肉毒梭菌(C.botulinum)。
其中,所述丙酮丁醇梭菌为丙酮丁醇梭菌ATCC 824或丙酮丁醇梭菌CGMCC 5234。以下实施例中丙酮丁醇梭菌B3
上述联产丁醇与2,3-丁二醇的基因工程菌的构建方法,从梭菌属、芽孢杆菌属、沙雷氏菌属、肠杆菌属或克雷伯氏菌属细菌的细胞中克隆乙偶姻还原酶基因,并将其克隆到表达载体中,构建表达乙偶姻还原酶的重组质粒,将重组质粒导入产丁醇的梭菌中,即得到联产丁醇与2,3-丁二醇的基因工程菌。
上述联产丁醇与2,3-丁二醇的基因工程菌的构建方法,从梭菌属、芽孢杆菌属、沙雷氏菌属、肠杆菌属或克雷伯氏菌属细菌的细胞中克隆乙酰乳酸脱羧酶基因和乙偶姻还原酶基因,并将两种基因克隆到表达载体中,构建融合表达乙酰乳酸脱羧酶和乙偶姻还原酶的重组质粒,并将重组质粒导入产丁醇的梭菌中,即得到联产丁醇与2,3-丁二醇的基因工程菌。
优选将乙酰乳酸脱羧酶BSU36000和乙偶姻还原酶Cbei_1464融合表达,最优选将乙酰乳酸脱羧酶BSU36000和乙偶姻还原酶BSU06240之间插入如下RBS序列:AGGAGGTTAGTTAGA。
其中,所述的表达载体为pIMPI-ptb。
上述联产丁醇与2,3-丁二醇的基因工程菌在生产丁醇和2,3-丁二醇中的应用在本发明的保护范围之内。
本发明通过在产丁醇梭菌中引入乙偶姻还原酶,将代谢过程中产生的3-羟基丁酮(乙偶姻)进一步转化为2,3-丁二醇。同时,通过强化乙酰乳酸脱羧反应来提高3-羟基丁酮(乙偶姻)产量,使反映过程中生成丙酮进一步代谢生成更多的3-羟基丁酮(乙偶姻),进一步在乙偶姻还原酶的作用下将乙偶姻转化为2,3-丁二醇,而丁醇产量不受影响,提高了梭菌发酵的产品得率,也提高了梭菌发酵的产品价值。
有益效果:本发明通过在丙酮丁醇梭菌中强化乙酰乳酸脱羧反应,将3-羟基丁酮产量由初始1.8g/L左右大幅提高到6.4g/L左右,进而在乙偶姻还原酶的作用下将乙偶姻转化为高达8.05g/L的2,3-丁二醇;而丙酮由4.5g/L左右降低至2.2g/L。显著提高了丙酮丁醇梭菌发酵的经济效益。
附图说明
图1是丙酮丁醇梭菌代谢网络示意图。
图2重组丙酮丁醇梭菌B3(pIMP1-BSU36000-RBS-BSU06240)发酵液气相色谱图谱,按出峰先后顺序依次为:3.062min,丙酮;3.472min,乙醇;4.811min,丁醇;6.077min,3-羟基丁酮;7.074min,乙酸;7.937min,2,3-丁二醇;8.539min,丁酸。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
下述实施例中如无特殊说明,所用方法均为常规方法,如《分子克隆:实验室手册》(New York:Cold Spring Habor Laboratary Press,1989)中所述的方法。所用试剂均可从商业途径获得。
下述实施例以丙酮丁醇梭菌为例阐明本发明的联产梭菌丁醇与2,3-丁二醇的方法。
酵母粉购自英国OXIOD公司,目录号1023098;蛋白胨购自英国OXIOD公司,目录号594566。
LB培养基配方为:10g/L蛋白胨,5g/L酵母粉,10g/L NaCl,固体培养基另加15g/L琼脂粉。用于大肠杆菌的常规培养。
2xYTG培养基配方为:16g/l蛋白胨,10g/l酵母粉,4g/l NaCl,5g/l葡萄糖。用于培养制备电转化感受态的丙酮丁醇梭杆菌。
P2平板培养基:葡萄糖10g/L,酵母膏5g/L,蛋白胨3g/L,七水合硫酸镁3g/L,乙酸铵2g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,琼脂粉15g/L。
实施例1:构建含有乙酰乳酸脱羧酶基因CAC2967、BSU36000、BL02479、A3UG_05860、KPN2242—13255、EL23_18310和CK00_RS0123920表达质粒的丙酮丁醇梭菌。
表1乙酰乳酸脱羧酶基因名称及其来源
乙酰乳酸脱羧酶基因名称 | 来源 | 序列号 |
CAC2967 | C.acetobutylicum ATCC 824 | SEQ ID NO:1 |
BSU36000 | B.Subtilis 168 | SEQ ID NO:2 |
BL02479 | B.licheniformis ATCC14580 | SEQ ID NO:3 |
A3UG_05860 | E.cloacae SDM | SEQ ID NO:4 |
KPN2242—13255 | K.pneumoniae KCTC 2242 | SEQ ID NO:5 |
EL23_18310 | P.polymyxa DSM 365 | SEQ ID NO:6 |
CK00_RS0123920 | S.marcescens ATCC 14041 | SEQ ID NO:7 |
采用细菌基因组试剂盒提取对数生长中后期的丙酮丁醇梭菌C.acetobutylicumAT CC 824基因组DNA,用以下引物进行PCR扩增其乙酰乳酸脱羧酶基因CAC2967:
CAC2967-s(SEQ ID NO:12):1:CATATGATTGAAGAAGTGATCCCTAATCAT(划线部分为NdeI识别位点);
CAC2967-as(SEQ ID NO:13):GAAATAAGTAAAGTTGAGAAATAACATATG(划线部分为NdeI识别位点)。
采用TAKARA公司的高效保真酶Primerstart进行PCR扩增,扩增程序为:95℃3min;98℃10s、55℃15s、72℃1min,30个循环;72℃10min。PCR产物经测序后确定其序列如SEQ IDNO:1所示。
将所得PCR产物纯化回收后,与经Nde I酶切后的pIMPI-ptb载体(Mermelstein,L.D.,Welker,N.E.,Bennett,G.N.,Papoutsakis,E.T.,1992.Expression of clonedhomolo gous fermentative genes in Clostridium acetobutylicum ATCC824.Biotechnology 10,190–195)在T4DNA连接酶作用下连接,构建得到载体pIMP1-CAC2967。
采用细菌基因组试剂盒提取对数生长中后期的枯草芽孢杆菌B.Subtilis 168基因组DNA,用以下引物进行PCR扩增其乙酰乳酸脱羧酶基因BSU36000:
BSU36000-s(SEQ ID NO:14):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACAT(划线部分为NdeI识别位点);
BSU36000-as(SEQ ID NO:15):GTACTGAGAGTGCACCATATGTTATTCAGGGCTTCCTTCAG(划线部分为NdeI识别位点)。
采用TAKARA公司的高效保真酶Primerstart分别进行PCR扩增,扩增程序为:94℃5min;94℃30s、52℃30s、72℃2min,30个循环;72℃10min。将所得PCR产物纯化回收后,与经Nde I酶切后的pIMPI-ptb载体经一步克隆连接后,构建得到载体pIMP1-BSU36000。经测序后确定其序列如SEQ ID No:2所示。
其中pIMP1-BL02479、pIMP1-A3UG_05860、pIMP1-KPN2242_13255、pIMP1-EL23_18310和pIMP1-CKOO_RS0123920表达质粒由金唯智生物技术有限公司亚克隆获得。相应的基因序列见SEQ ID No:3至SEQ ID No:7
将质粒pIMP1-CAC2967、pIMP1-BSU36000、pIMP1-BL02479、pIMP1-A3UG_05860、pIMP1-KPN2242_13255、pIMP1-EL23_18310和pIMP1-CKOO_RS0123920转化到E.coli TOP10(pAN2)中,E.coli Top10购自北京天根生化科技有限公司(目录号CB104)。pAN2为甲基化质粒(Heap,J.T.,Pennington,O.J.,Cartman,S.T.,Carter,G.P.,Minton,N.P.,2007.TheClosTron:a universal gene knock-out system for the genus Clostridium.JMicrobiol Methods 70,452-464)。并将转化后细胞涂布于含有氨苄青霉素和四环素素的LB平板上,挑取单菌落富集培养后提取质粒,提取到的pIMP1-CAC2967、pIMP1-BSU36000、pIMP1-BL02479、pIMP1-A3UG_05860、pIMP1-KPN2242_13255、pIMP1-EL23_18310和pIMP1-CKOO_RS0123920质粒即是甲基化后的质粒。
厌氧条件下,取2xYTG培养基培养的生长至对数中期的C.acetobutylicum B3(保藏于中国微生物菌种保藏管理委员会普通微生物中心,CGMCC No.5234;地址:北京市朝阳区北辰西路1号院3号,该菌株的信息在申请号为201210075094.X的中国专利中详细公开)培养液60ml,4℃、4000rpm离心10min弃去上清,加入足量预冷的电转缓冲液EPB(270mM蔗糖,5mM NaH2PO4,pH 7.4),洗涤两次,并用2.3ml EPB重悬。然后取570ul加入0.4cm电转杯放置在冰浴中冷却,并加入20ul甲基化的pIMP1-CAC2967质粒,冰浴放置2min。2.0kV电压,25uF电容进行电转化。随后将电转液加入到1ml 37℃的2xYTG培养基中复苏培养4h,离心收集细胞100ul并将细胞涂布于含有20ug/ml甲砜霉素的P2平板中。厌氧培养24-36h后,获得含有pIMP1-CAC2967质粒的重组丙酮丁醇梭菌,命名为C.acetobutylicum B3(pIMP1-CAC2967)。
依照上述电转方法分别获得pIMP1-BSU36000、pIMP1-BL02479、pIMP1-A3UG_05860、pIMP1-KPN2242_13255、pIMP1-EL23_18310和pIMP1-CKOO_RS0123920质粒的重组丙酮丁醇梭菌,命名为C.acetobutylicum B3(pIMP1-BSU36000)、C.acetobutylicum B3(pIMP1-BL02479)、C.acetobutylicum B3(pIMP1-A3UG_05860)、C.acetobutylicum B3(pIMP1-KPN2242_13255)、C.acetobutylicumB3(pIMP1-EL23_18310)、C.acetobutylicumB3(pIMP1-CKOO_RS0123920)。
丙酮丁醇梭杆菌(C.acetobutylicum)B3(pIMP1-CAC2967)、B3(pIMP1-BSU36000)、B3(pIMP1-BL02479)、B3(pIMP1-A3UG_05860)、B3(pIMP1-KPN2242_13255)、B3(pIMP1-EL23_18310)和B3(pIMP1-CKOO_RS0123920)在P2种子培养基(不加琼脂,其他组分同P2平板培养基)中37℃静止培养12h。分别以10%(v/v)的接种量接种至发酵培养基中。发酵培养基的配方如下:K2HPO4 0.5g/L;KH2PO4 0.5g/L;CH3COO NH4 2.2g/L;MgSO4·7H2O 0.2g/L;MnSO4·H2O 0.01g/L;NaCl 0.01g/L;FeSO4·7H2O 0.01g/L;对氨基苯酸1mg/L;硫胺1mg/L;生物素0.01mg/L。37℃厌氧发酵96h。
发酵液组分用气相色谱检测,气相色谱检测条件如下:火焰离子检测器(FID),Agilent HP-INNOWAX 19091N-236毛细管色谱柱(60m×0.25mm×0.25um),N2为载气,流速2mL/min,分流比10:1,H2流速30ml/min,空气流速300ml/min,进样口温度240℃,检测器250℃,柱温(程序升温):70℃保留0.5min,然后以20℃/min的速率升温到190℃,保留4min。气相色谱出峰先后顺序见图2。发酵结果见表1,其中丙酮丁醇梭杆菌B3(pIMP1-CAC2967)、B3(pIMP1-BSU36000)、B3(pIMP1-BL02479)、B3(pIMP1-A3UG_05860)、B3(pIMP1-KPN2242_13255)、B3(pIMP1-EL23_18310)、B3(pIMP1-CKOO_RS0123920)所对应的数据为任意挑选的10株重组菌的平均数据。
表2重组含乙酰乳酸脱羧酶的丙酮丁醇梭菌及对照菌株发酵结果
由表2可见,表达乙酰乳酸脱羧蛋白的重组菌株3-羟基丁酮产量明显提高,实现了底物由生成丙酮向生成3-羟基丁酮的转变。其中,丙酮丁醇梭杆菌B3(pIMP1-BSU36000)的3-羟基丁酮产量与野生菌B3相比,提高了241%,同时丁醇产量不受影响。与表达前相比,总溶剂(ABE和3-羟基丁酮)得率也提高了20%。
实施例2:构建含有乙偶姻还原酶BSU06240、Cbei_1464、KPN_02061、CAETHG_0385表达质粒的丙酮丁醇梭菌。
表3乙偶姻还原酶基因名称及其来源
采用细菌基因组试剂盒提取对数生长中后期的枯草芽孢杆菌B.Subtilis 168和拜氏梭菌(Clostridium beijerinckii)NCIMB 8052。用以下引物进行PCR扩增其乙偶姻还原酶基因BSU06240和Cbei_1464:
BSU06240-s(SEQ ID NO:16):AAAAGGGAGTGTCGACATATGAAGGCAGCAAGATG(划线部分为NdeI识别位点);
BSU06240-as(SEQ ID NO:17):GTACTGAGAGTGCACCATATGTTAGTTAGGTACAAGGA(划线部分为NdeI识别位点)。将扩增产物按实施例1一步克隆的方法构建得到载体pIMP1-BSU06240。经测序后确定其序列如SEQ ID No:8所示。
Cbei_1464-s(SEQ ID NO:18):AAAAGGGAGTGTCGACATATGAAAGCAGCATTATGG(划线部分为NdeI识别位点);
Cbei_1464-as(SEQ ID NO:19):GTACTGAGAGTGCACCATATGTTAAGATTTAGATACAAGTTCTTTGTC(划线部分为NdeI识别位点)。将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-Cbei_1464。经测序后确定其序列如SEQ ID No:9所示。
将重组质粒按照实施例1电转的方法导入丙酮丁醇梭菌获得含有pIMP1-BSU06240和pIMP1-Cbei_1464、pIMP1-KPN_02061、pIMP1-CAUT质粒的重组丙酮丁醇梭菌,命名为丙酮丁醇梭杆菌B3(pIMP1-BSU06240)、B3(pIMP1-Cbei_1464)、丙酮丁醇梭菌B3(pIMP1-KPN_02061)和B3(pIMP1-CAETHG_0385),其中pIMP1-KPN_02061、pIMP1-CAETHG_0385表达载体由金唯智生物科技有限公司亚克隆获得,对应的基因序列见SEQ ID No:10和SEQ ID No:11将重组的丙酮丁醇梭菌进行发酵培养,发酵结果见表4。
表4重组含乙偶姻还原酶的丙酮丁醇梭菌及对照菌株发酵结果
由表4可见,表达乙偶姻还原酶的重组菌株3-羟基丁酮产量明显降低,实现了由3-羟基丁酮向2,3-丁二醇的转变,同时丁醇产量不受影响。其中,丙酮丁醇梭杆菌B3(pIMP1-KPN_0206)的2,3-丁二醇产量明显高于其他菌株。
实施例3:构建含有乙酰乳酸脱羧酶基因BSU36000分别与乙偶姻还原酶BSU06240、Cbei_1464、KPN_02061、CAETHG_0385相连接的表达质粒的丙酮丁醇梭菌。以上4种基因序列中乙酰乳酸脱羧酶基因BSU36000的终止密码子后紧邻乙偶姻还原酶BSU06240、Cbei_1464、KPN_02061、CAETHG_0385的起始密码子。
用以下引物进行PCR扩增能使乙酰乳酸脱羧酶基因BSU36000和乙偶姻还原酶基因BSU06240、Cbei_1464、KPN_02061和CAETHG_0385相连的基因片段:
BSU36000BSU06240-s(SEQ ID NO:20):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACATTC(划线部分为NdeI识别位点);
BSU36000BSU06240-as(SEQ ID NO:21):TGCCATCTTGCTGCCTTCATTTATTCAGGGCTTCCTTCAG;
BSU06240-s(SEQ ID NO:22):CTGAAGGAAGCCCTGAATAAATGAAGGCAGCAAGATG;
BSU06240-as(SEQ ID NO:23):GTACTGAGAGTGCACCATATGTTAGTTAGGTCTAACAAGGA(划线部分为NdeI识别位点)。
将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-BSU06240。
BSU36000Cbei_1464-s(SEQ ID NO:24):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACATTC(划线部分为NdeI识别位点);
BSU36000Cbei_1464-as(SEQ ID NO:25):TACCATAATGCTGCTTTCATTTATTCAGGGCTTCCTTCAG;
Cbei_1464-s(SEQ ID NO:26):CTGAAGGAAGCCCTGAATAAATGAAAGCAGCATTATGG;
Cbei_1464-as(SEQ ID NO:27):GTACTGAGAGTGCACCATATGTTAAGATTTAGATACAAGTTCTTTGTC(划线部分为NdeI识别位点)。
将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-Cbei_1464。
BSU36000CAETHG_0385-s(SEQ ID NO:28):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACATTC(划线部分为NdeI识别位点);
BSU36000CAETHG_0385-as(SEQ ID NO:29):TTACAATAAGGATTTGTCAGTTATTCAGGGCTTCCTTCA;
CAETHG_0385-s(SEQ ID NO:30):CTGAAGGAAGCCCTGAATAAATGAAAGCTGTATTGTGGTA;
CAETHG_0385-as(SEQ ID NO:31):GTACTGAGAGTGCACCATATGTTACAATAAGGATTTGTCAGGAG(划线部分为NdeI识别位点)。将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-CAETHG_0385。
BSU36000KPN_02061-s(SEQ ID NO:32):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACATTC(划线部分为NdeI识别位点);
BSU36000KPN_02061-as(SEQ ID NO:33):TTAGTTAAACACCATCCCGCTTATTCAGGGCTTCCTTCA;
KPN_02061-s(SEQ ID NO:34):CTGAAGGAAGCCCTGAATAAATGAAAAAAGTCGCACTTG;
KPN_02061-as(SEQ ID NO:35):GTACTGAGAGTGCACCATATGTTAGTTAAACACCATCCCGC(划线部分为NdeI识别位点)。将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-KPN_02061。
将重组质粒按照实施例1电转的方法导入丙酮丁醇梭菌获得含有pIMP1-BSU36000-BSU06240、pIMP1-BSU36000-CBEI1464、pIMP1-BSU36000-KPN_02061和pIMP1-BSU36000-CAETHG_0385质粒的重组丙酮丁醇梭菌。将重组丙酮丁醇梭菌进行发酵培养,发酵结果见表5。
表5重组含BSU36000和乙偶姻还原酶基因的丙酮丁醇梭菌及对照菌株发酵结果
由表5可见,表达枯草乙酰乳酸脱羧蛋白和乙偶姻还原酶的重组菌株后,除了丙酮丁醇梭杆菌B3(pIMP1-BSU36000-Cbei_1464)发酵产物正常,其他菌株2,3-丁二醇和3-羟基丁酮产量均很低。
实施例4:构建含有乙酰乳酸脱羧酶基因BSU36000与RBS连接后又分别与乙偶姻还原酶BSU06240、Cbei_1464、CAETHG_0385、KPN_02061相连接的表达质粒的丙酮丁醇梭菌。RBS的序列如下:AGGAGGTTAGTTAGA。
用以下引物进行PCR扩增能使乙酰乳酸脱羧酶基因BSU36000和乙偶姻还原酶基因BSU06240、Cbei_1464、CAETHG_0385和KPN_02061相连接的基因片段:
BSU36000RBS-s(SEQ ID NO:36):AAAAGGGAGTGTCGACATATGAAACGAGAAAGCAACATTC(划线部分为NdeI识别位点);
BSU36000RBS-as(SEQ ID NO:37):TCTAACTAACCTCCT TTATTCAGGGCTTCCTTCAG;
RBSBSU06240-s(SEQ ID NO:38):AGGAGGTTAGTTAGA ATGAAGGCAGCAAGATG;
RBSBSU06240-as(SEQ ID NO:39):GTACTGAGAGTGCACCATATGTTAGTTAGGTCTAACAAGGA(划线部分为NdeI识别位点)。将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-RBS-BSU06240。
RBSCbei_1464-s(SEQ ID NO:40):AGGAGGTTAGTTAGA ATGAAAGCAGCATTATGGTATG;
RBSCbei_1464-as(SEQ ID NO:41):GTACTGAGAGTGCAC CATATGTTAAGATTTAGATACAAGTTCTTTGTC(划线部分为NdeI识别位点)。
将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-RBS-1464。
RBSKPN_02061-s(SEQ ID NO:42):AGGAGGTTAGTTAGAATGAAAAAAGTCGCACTTG;
RBSKPN_02061-as(SEQ ID NO:43):GTACTGAGAGTGCACCATATGTTAGTTAAACACCATCCCGC(划线部分为NdeI识别位点)。
将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-RBS-KPN_02061。
RBSCAETHG_0385-s(SEQ ID NO:44):AGGAGGTTAGTTAGAATGAAAGCTGTATTGTGGTATG;
RBSCAETHG_0385-as(SEQ ID NO:45):GTACTGAGAGTGCACCATATGTTACAATAAGGATTTGTCAGGAG(划线部分为NdeI识别位点)。将扩增产物按实施例1一部克隆的方法构建得到载体pIMP1-BSU36000-RBS-CAETHG_0385。
将扩增产物按照实施例1一步克隆的方法导入丙酮丁醇梭菌获得含有pIMP1-BSU36000-RBS-BSU06240、pIMP1-BSU36000-RBS-Cbei_1464、pIMP1-BSU36000-RBS-CAETHG_0385和pIMP1-BSU36000-RBS-KPN_02061质粒的重组丙酮丁醇梭菌,将重组后的丙酮丁醇梭菌进行发酵培养,发酵结果见表6。
表6重组丙酮丁醇梭菌及对照菌株发酵结果
由表6可见,重组菌株丙酮丁醇梭杆菌B3(pIMP1-BSU36000-RBS-BSU06240)将3-羟基丁酮转化为2,3-丁二醇效率较高,2,3-丁二醇产量可达8.05g/L,同事丁醇产量不受影响。
虽然以上实施例仅以C.acetobutylicum ATCC 824和C.acetobutylicum B3(CGMCC 5234)为例进行了说明,但本领域的技术人员通过本发明的公开,很容易看出本发明的方法同样适用于其他产梭菌,这些菌株包括但不限于:C.acetobutylicum,C.beijerinckii,C.saccharobutylicum,C.saccharoperbutylacetonicum,C.butyricum,C.sporogenes,C.thermosaccharotyticum,C.pasteurianum,C.tyrobutyricum,C.perfringens,C.botulinum。因此这些内容同样应当属于本发明的范围。
Claims (4)
1.联产丁醇与2,3-丁二醇的基因工程菌,其特征在于,它是导入了乙酰乳酸脱羧酶基因和乙偶姻还原酶基因的产丁醇梭菌;
所述的乙酰乳酸脱羧酶基因,其核苷酸序列如SEQ ID NO:2所示;
所述的乙偶姻还原酶基因,其核苷酸序列如SEQ ID NO:9所示;
所述乙酰乳酸脱羧酶基因与乙偶姻还原酶基因之间插入RBS序列,所述RBS序列如AGGAGGTTAGTTAGA所示;
所述产丁醇梭菌为丙酮丁醇梭菌CGMCC 5234。
2.权利要求1所述的联产丁醇与2,3-丁二醇的基因工程菌的构建方法,其特征在于,克隆乙酰乳酸脱羧酶基因和乙偶姻还原酶基因,并将两种基因克隆到表达载体中,构建融合表达乙酰乳酸脱羧酶和乙偶姻还原酶的重组质粒,并将重组质粒导入产丁醇的梭菌中,即得到联产丁醇与2,3-丁二醇的基因工程菌。
3.根据权利要求2所述的联产丁醇与2,3-丁二醇的基因工程菌的构建方法,其特征在于,所述的表达载体为pIMPI-ptb。
4.权利要求1所述联产丁醇与2,3-丁二醇的基因工程菌在生产丁醇和2,3-丁二醇中的应用。
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