CN112760256B - 一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌及其制备方法 - Google Patents
一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌及其制备方法 Download PDFInfo
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Abstract
本发明公开了一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌及其制备方法，所述菌株由食甲基丁酸杆菌Butyribacterium methylotrophicum（编号ATCC 33266）经过实验室适应性进化筛选得到食甲基丁酸杆菌突变菌株Butyribacterium methylotrophicum MT750，保藏编号为CCTCC NO：M 2020095，再将产丁醇基因导入该突变株获得。与出发菌株相比，经过实验室适应性进化筛选的食甲基丁酸杆菌可以耐受浓度700mM以上的甲醇，将含有产丁醇基因的穿梭质粒pJIR‑Pptb‑adhE2经甲基化后转化进入食甲基丁酸杆菌菌体内，使其可以利用甲醇生产丁醇，具有巨大的应用潜力。
Description
技术领域
本发明属于基因工程领域，具体涉及一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌及其制备方法。
背景技术
实验室适应性进化(ALE)已被用作一种有效的方法，提高菌体耐受甲醇的能力和提高丁酸的产量。限制修饰系统是广泛存在于细菌，主要由限制内切酶和甲基化酶组成的二元系统，限制酶功能是降解外源DNA，从而阻止其复制和整合到宿主细胞中，修饰酶作用是对自身某个碱基进行甲基化，保护自身DNA不被降解。
非模式菌株食甲基丁酸杆菌，简称Bm，是一种厌氧芽孢梭菌属，属于单碳厌氧型，可以同时利用多种C1原料进行发酵，如CO2、CO和甲醇等。另外，还能代谢多碳物质，包括葡萄糖、乳糖和丙酮酸，对这些物质代谢的主要产物是醋酸、丁酸或二者皆有，以及相应的醇类。Bm可用于生产石油和化学物质，开发食甲基丁酸杆菌作为甲醇利用的模式宿主，对促进甲醇生物转化具有重要意义。然而当前研究中该菌株遗传操作工具的缺乏，限制了其发展和应用。
提高食甲基丁酸杆菌对于甲醇的耐受性有利于提高菌体对于甲醇的利用和丁酸的合成，提高其在实际应用中的价值。目前对于食甲基丁酸杆菌遗传操作工具很少，需要建立一套甲基化工具，便于进行后续基因操作。
发明内容
本发明的第一目的在于提供一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌，其特征在于，由食甲基丁酸杆菌经过实验室适应性进化筛选得到食甲基丁酸杆菌突变菌株，再将产丁醇基因导入突变株获得所述耐高浓度甲醇、高产丁醇食甲基丁酸杆菌Butyribacterium methylotrophicum MT751，其中所述食甲基丁酸杆菌突变菌株分类命名为Butyribacterium methylotrophicum MT750，其保藏编号为 CCTCC NO：M 2020095。
所述食甲基丁酸杆菌为Butyribacterium methylotrophicum，来源于美国典型菌种保藏中心，编号为ATCC 33266。
本发明的第二目的在于提供上述耐高浓度甲醇、高产丁醇食甲基丁酸杆菌的制备方法，包括如下步骤：
(1)以食甲基丁酸杆菌作为出发菌株，采用实验室适应性进化逐步提高培养基中甲醇浓度筛选具有高甲醇耐受性的食甲基丁酸杆菌突变菌株；
(2)制备E.coli top10感受态；
(3)将含有甲基化酶基因的质粒转化进入E.coli top10；
(4)将步骤(3)获得的E.coli top10制备成感受态；
(5)构建含有产丁醇基因的穿梭质粒，并将其转化进步骤(4)得到的E.coli top10感受态细胞；
(6)从步骤(5)制备的E.coli top10中提取质粒；
(7)制备食甲基丁酸杆菌突变菌株感受态；
(8)将步骤(6)提取的质粒转化进入步骤(7)所述的食甲基丁酸杆菌突变菌株感受态中，进行电转，在含有甲砜霉素的平板上筛选单克隆菌株。
具体的，步骤(8)甲基化酶基因的质粒导入的作用是为了把含有丁醇基因的质粒甲基化。
具体的，所述实验室适应性进化周期为10个月，培养基为磷酸缓冲溶液培养基简称PBM，配方如下：KH2PO4，4g；K2HPO4,6g；NH4Cl，1g；MgCl2·7H20， 0.1g；CaCl2·2H20，0.1g；酵母粉，3g；微量元素溶液(DSM培养基141)，20ml；维生素溶液(DSM培养基141)，20ml，最终体积定容至1L，灭菌，冷却至室温后添加过膜灭菌的NaHCO3溶液，将pH调至7.3。碳源和还原剂以无菌厌氧环境在接种前添加，对于传代和转化，还需要添加葡萄糖(0.5％，27.7mM)，Na2S 0.2mM，DL-二硫苏糖醇0.5mM，L-半胱氨酸-HCl·H2O 0.2mM，以100-2000倍的稀释度接种细胞。其中甲醇浓度从500mM逐步提高至700mM，每次提高50 mM。
具体的，步骤(3)所述的含有甲基化酶基因的质粒为pMCljS。
具体的，步骤(5)所述的穿梭质粒为pJIR750。
具体的，步骤(8)所述电转条件为：电压1.6kv，电阻200Ω，电容25μF。
具体的，步骤(9)所述的产丁醇基因为adhE2，序列如SEQ ID NO:1所示。
具体的，含有产丁醇基因的穿梭质粒为pJIR-Pptb-adhE2。
本发明的第三目的在于提供所述耐高浓度甲醇食甲基丁酸杆菌在产丁醇上的应用。
本发明的有益技术效果是：与一般食甲基丁酸杆菌相比，经过适应性进化筛选的食甲基丁酸杆菌可以耐受浓度700mM以上的甲醇，将含有产丁醇基因的穿梭质粒pJIR-Pptb-adhE2转化进入食甲基丁酸杆菌菌体内，使其可以利用甲醇生产丁醇。
本发明实验室适应性进化获得的生物材料，其分类命名Butyribacteriummethylotrophicum MT750，已保藏于中国典型培养物保藏中心(简称CCTCC)，其保藏编号为CCTCC NO：M 2020095，保藏日期为：2020年4月29日，保藏地址为：中国.武汉.武汉大学。
附图说明
图1为在高浓度甲醇(>600mM)条件下，食甲基丁酸杆菌出发菌株和耐高浓度甲醇食甲基丁酸杆菌突变菌株分别的发酵特征。
图2为pJIR-Pptb-adhE2的限制性消化产物。
图3为ack基因扩增产物。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行完整的描述，显然，所描述的实施例仅是本发明一部分实施例。基于本发明的实施例，本领域的普通技术人员再没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
实施例1实验室适应性进化筛选耐高浓度甲醇食甲基丁酸杆菌
准备磷酸缓冲溶液培养基PBM：首先称取KH2PO4，4g；K2HPO4，6g；NH4Cl， 1g；MgCl2·7H2O，0.1g；CaCl2·2H20，0.1g；酵母粉，0.3g；微量元素溶液(DSM 培养基141)，20ml；维生素溶液(DSM培养基141)，20ml；定容至1L，灭菌，冷却至室温后添加过膜灭菌的NaHCO3溶液，将pH调至7.3。碳源和还原剂以无菌厌氧环境在接种前添加，对于传代和转化，还需要添加葡萄糖(0.5％， 27.7mM)，Na2S 0.2mM，DL-二硫苏糖醇0.5mM，L-半胱氨酸-HCl·H2O 0.2mM，以100-2000倍的稀释度接种细胞。
在适应性进化筛选过程中，定期向培养体系中补加甲醇，从而逐步提高菌体生长环境中甲醇浓度。甲醇浓度从500mM，550mM，600mM，650mM，700mM 逐步提高，10个月适应性驯化，筛选获得一株耐700mM浓度甲醇的突变菌株，命名为食甲基丁酸杆菌Butyribacterium methylotrophicum MT750。
Butyribacterium methylotrophicum MT750与出发菌株相比，优势在于：
经过17天的培养之后，如图1所示，出发菌株和突变菌株生产的丁酸/乙酸分别是84mM/30mM和103mM/26mM。经过8天的培养，进化菌株生产的丁酸超过了乙酸，然而原始菌株是在培养10天之后，生产的丁酸才超过了乙酸。另外，进化菌株比原始菌株消耗了更多的甲醇。
实施例2转化实验
感受态制备：将过夜的PBM-葡萄糖培养物稀释100倍至50ml新鲜的PBM- 葡萄糖中。当OD600至0.5-0.8，培养基在厌氧环境25℃中离心15分钟，倾倒上层清液。细胞重悬于25ml 270mM蔗糖中，在20℃条件下再次离心，最后再次重悬于1ml蔗糖中，并且分装为100μl用于转化。质粒准备：将含有仲氏梭菌甲基化酶基因的质粒pMCljS转化进入E.colitop10，用壮观霉素来筛选克隆。制备 E.colitop10(pMCljS)感受态。将质粒pJIR750转化进E.colitop10(pMCljS)，使用壮观霉素和氯霉素筛选克隆。从E.colitop10(pMCljS，pJIR750)中提取质粒为了进一步的转化做准备。转化：将100μl食甲基丁酸杆菌感受态和100-200ng 被甲基化的质粒，将混合物放进电转杯中冰浴10分钟，用以下程序进行电转：1.6kv，200Ω电容25μF，立即将转化好的细胞加入1ml预热的PBM-葡萄糖，复苏6-8h。第一轮用液态PBM-葡萄糖培养24h，在12.5mg/L甲砜霉素存在下筛选转化子，第二轮用含有25mg/L甲砜霉素的液态PBM-甲醇的培养基培养两天，将第二轮培养的液态PBM-甲醇培养基涂布在含有25mg/L甲砜霉素的平板上获得单克隆。含甲基化酶基因的质粒pAN1和穿梭质粒pSOS94也进行了测试。
转化结果如表1所示，由于限制修饰系统的存在，转化进食甲基丁酸杆菌的 DNA质粒必须要求被甲基化。穿梭质粒pSOS94没有被成功转化甚至没有被甲基化，穿梭质粒pJIR750甲基化后可以成功转化。
表1转化实验结果
| 质粒 | 阳性重组子 |
| pSOS94 | - |
| pSOS+pAN1 | - |
| pSOS+pMCljS | - |
| pJIR750 | - |
| pJIR750+pMCljS | + |
实施例3代谢工程改造食甲基丁酸杆菌生产丁醇
食甲基丁酸杆菌正常情况下，只会产酸(乙酸和丁酸)。为了使食甲基丁酸杆菌生产和积累丁醇，一个含有醇脱氢酶的质粒(pJIR-Pptb-adhE2)被构建出来。
将质粒pJIR-Pptb-adhE2转化进入食甲基丁酸杆菌中，通过沉淀细胞DNA，染色体形式和质粒为模板的PCR以及质粒纯化和限制性内切酶消化来验证转化。
因为质粒pJIR在食甲基丁酸杆菌中的拷贝数很低，所以从食甲基丁酸杆菌中提取的质粒不能在跑胶中验证。因此从食甲基丁酸杆菌中提取的 pJIR-Pptb-adhE2首先被转化进E.coli Top10。条带1和条带2是从E.coli Top10 中提取的pJIR-Pptb-adhE2的限制性消化产物，如图2所示。
条带3和条带4是用从食甲基丁酸杆菌中提取的pJIR-Pptb-adhE2作为模板的PCR产物。条带5和条带6是用食甲基丁酸杆菌的染色体作为模板的PCR产物。条带3，4，5和6使用引物S-adh(tggatacctaggattagctgttgt)，A-adh (gtggcataatagaccatgacgat)。
更进一步，我们设计了寡聚物S-TM(cggaatatacggataccacagccact)，A-TM(gcagtgagcgcaacgcaattaatgt)来验证抗性基因是否插入染色体(条带2)和S-ACK(cttgttattaattgcggtagct)，A-ACK(gattaaccaactaattcaacggt)来扩增天然ack基因 (条带3)来验证染色体提取的正确性。结果显示没有抗性基因被扩增(条带2) 然而天然的ack基因可以被正确的扩增(条带3)，如图3。结果显示PCR过程是正确的，质粒存在染色体整合。
结论：(1)虽然pJIR-Pptb-adhE2的拷贝数是低的，但是它还是成功转化进入食甲基丁酸杆菌。(2)adhE2基因(序列SEQ ID NO:1)只存在于质粒上而没有发现整合到染色体上。
实施例5重组食甲基丁酸杆菌(pJIR-Pptb-adhE2)的发酵
将重组食甲基丁酸杆菌Butyribacterium methylotrophicum MT751进行发酵，以食甲基丁酸杆菌耐受菌株Butyribacterium methylotrophicum MT750和食甲基丁酸杆菌(pJIR750)作为对照，所用培养基为实施例1所述培养基PBM，甲醇为碳源，发酵时间为12天。
表2食甲基丁酸杆菌发酵结果
结果显示，相比较于在对照菌株检测到的丁醇浓度，在食甲基丁酸杆菌 MT751中发挥作用的pJIR-Pptb-adhE2可以使菌体最终积累11mM丁醇。
序列表
<110> 南京工业大学
<120> 一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌及其制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2577
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 1
atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60
aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120
gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180
cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240
aaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300
gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360
attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420
cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480
ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540
ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600
tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660
gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720
ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780
aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840
aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900
ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960
gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020
atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080
ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140
aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200
ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260
acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320
agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380
aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440
tttatagtaa cagataaaga tctttttaaa cttggatatg ttaataaaat aacaaaggta 1500
ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560
gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620
attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680
gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740
ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800
tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860
acttcttatg aattgacccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920
cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980
gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040
tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100
atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160
cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220
gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280
ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340
aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400
ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460
ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520
aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaa 2577
Claims (8)
1.一株耐高浓度甲醇、高产丁醇食甲基丁酸杆菌，其特征在于，由食甲基丁酸杆菌经过实验室适应性进化筛选得到食甲基丁酸杆菌突变菌株，再将产丁醇基因导入突变株获得所述耐高浓度甲醇、高产丁醇食甲基丁酸杆菌Butyribacterium methylotrophicum MT751，其中所述食甲基丁酸杆菌突变菌株分类命名为Butyribacterium methylotrophicumMT750，其保藏编号为CCTCC NO：M 2020095；所述食甲基丁酸杆菌为Butyribacteriummethylotrophicum，编号为ATCC 33266；所述的产丁醇基因为adhE2，序列如SEQ ID NO:1所示。
2.权利要求1所述耐高浓度甲醇、高产丁醇食甲基丁酸杆菌的制备方法，其特征在于，包括如下步骤：
(1)以食甲基丁酸杆菌作为出发菌株，采用实验室适应性进化逐步提高培养基中甲醇浓度筛选具有高甲醇耐受性的食甲基丁酸杆菌突变菌株；所述食甲基丁酸杆菌为Butyribacterium methylotrophicum，编号为ATCC 33266；所述食甲基丁酸杆菌突变菌株分类命名为Butyribacterium methylotrophicum MT750，其保藏编号为CCTCC NO：M2020095；
(2)制备E.coli top10感受态；
(3)将含有甲基化酶基因的质粒转化进入E.coli top10；
(4)将步骤(3)获得的E.coli top10制备成感受态；
(5)构建含有产丁醇基因的穿梭质粒，并将其转化进步骤(4)得到的E.coli top10感受态细胞；所述的产丁醇基因为adhE2，序列如SEQ ID NO:1所示；
(6)从步骤(5)制备的E.coli top10中提取质粒；
(7)制备食甲基丁酸杆菌突变菌株感受态；
(8)将步骤(6)提取的质粒转化进入步骤(7)所述的食甲基丁酸杆菌突变菌株感受态中，进行电转，在含有甲砜霉素的平板上筛选单克隆菌株。
3.根据权利要求2所述的制备方法，其特征在于，所述实验室适应性进化周期为10个月，培养基为磷酸缓冲溶液培养基简称PBM，其中甲醇浓度从500mM逐步提高至700mM，每次提高50mM。
4.根据权利要求2所述的制备方法，其特征在于，步骤(3)所述的含有甲基化酶基因的质粒为pMCljS。
5.根据权利要求2所述的制备方法，其特征在于，步骤(5)所述的穿梭质粒为pJIR750。
6.根据权利要求2所述的制备方法，其特征在于，步骤(8)所述电转条件为：电压1.6kv，电阻200Ω，电容25μF。
7.根据权利要求2所述的制备方法，其特征在于，含有产丁醇基因的穿梭质粒为pJIR-Pptb-adhE2。
8.权利要求1所述耐高浓度甲醇、高产丁醇食甲基丁酸杆菌在产丁醇上的应用。
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