CN103911334A - 一种高抗逆性拜氏梭菌及其应用 - Google Patents
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Abstract
本发明公开了一种高抗逆性拜氏梭菌及其应用。该高抗逆性拜氏梭菌株基因Cbei_3304不能正常表达，缺失正常的Cbei_3304蛋白功能，对毒素物质阿魏酸、香草酸等具有高抗逆性，对未脱毒木质纤维酸解糖液中的毒素物质也具有高度抗逆性，能非常好地发酵生产丁醇；本发明提供了一种简单、高效的提高拜氏梭菌抗逆性的方法，只需使拜氏梭菌缺失正常的Cbei_3304蛋白功能即可获得对未脱毒木质纤维酸解糖液中的毒素物质具有高度抗逆性，且可高效生成丁醇的拜氏梭菌，有利于推广对木质纤维资源的高效利用。
Description
技术领域
本发明涉及一种高抗逆性拜氏梭菌及其应用，属于基因工程和发酵工程技术领域。
背景技术
随着化石能源的日益枯竭，利用可再生的木质纤维质原料（如玉米芯、甘蔗渣等）通过微生物发酵法生产生物能源和生物基化学品，成为研究的重点和热点之一。木质纤维原料必须经过预处理，得到微生物可发酵的糖，才能被微生物很好的利用。然而，木质纤维原料经预处理后，会产生有机酸、糠醛、酚类等抑制物，这些抑制物的除去成本较高、并对微生物生长有一定的抑制作用。因此，提高微生物的抗逆性尤其重要。
丁醇具有能量密度大、可与汽油任意比混合、可直接用于内燃机等优点，丁醇作为新型可再生的液体能源，受到广泛的重视。利用木质纤维原料发酵制备燃料丁醇，成为研究的热点之一。Thaddeus Ezeji等（Bioresource Technol.
2008, 99: 5915-5922）利用拜氏梭菌突变株BA101，以XAD-4 resin脱毒的玉米芯酸解和酶解糖液为底物发酵，总溶剂产量为9.30 g/L；但突变株BA101不能利用未脱毒的酸解糖液发酵产丁醇。中国专利ZL 201110020102.6报道：通过粒子束诱变育种得到的拜氏梭菌（Clostridium beijerinckii）IB4对酚类化合物具有较高的抗逆性，其能以未脱毒的玉米芯酸解糖液作为碳源，在2L发酵罐中总溶剂产量和丁醇产量分别达到了10.3g/L和7.1 g/L。然而，郭亭等（J and Microbiol
Biotechnol., 2012, 39(3), 401-407）研究发现，当玉米芯和甘蔗渣酸解糖液中的酚类化合物的浓度提升到1.5 g/L以上时，拜氏梭菌（Clostridium beijerinckii）IB4基本不生长。Tomas 等（Appl Environ Microbiol,
2003, 69(8): 4951−4965）在丙酮丁醇梭菌中过表达编码热激蛋白的groESL 基因，使丁醇对菌体细胞的抑制作用降低了85%，并最终使产物浓度提高了33%。Thaddeus Ezeji等（Biotechnol Bioeng 2007,
97(6):1460-1469）研究发现有机酸、糠醛等不影响丁醇的发酵，而酚类抑制物对丁醇发酵有明显的抑制效应。然而，酚类化合物对微生物生长与发酵的抑制机制比较复杂，其抑制机理还不清晰。
基于此，木质纤维预处理后的毒素抑制物（如阿魏酸、香兰酥等）严重抑制拜氏梭菌的生长和发酵性能；提高拜氏梭菌对酚类化合物等毒素的抗逆性，是木质纤维原料制备燃料丁醇产业化必须解决的关键问题之一。
发明内容
为了解决上述问题，本发明提供一种容易获得的高抗逆性拜氏梭菌，其对酚类化合物等毒素具有高抗逆性。
本发明的目的在于提供一种高抗逆性拜氏梭菌。
本发明的另一目的在于提供一种高抗逆性拜氏梭菌的构建方法。
本发明的再一目的在于提供一种高抗逆性拜氏梭菌的应用。
本发明所采取的技术方案是：
一种高抗逆性拜氏梭菌，其缺失正常的Cbei_3304蛋白功能。
进一步的，上述高抗逆性拜氏梭菌，其基因Cbei_3304不能正常表达。
进一步的，上述高抗逆性拜氏梭菌，其基因Cbei_3304被***失活。
上述一种高抗逆性拜氏梭菌在发酵制备丁醇中的应用。
进一步的，上述应用具体为：高抗逆性拜氏梭菌以不脱毒的木质纤维酸解糖液为原料直接发酵制备丁醇。
本发明的有益效果是：
本发明将拜氏梭菌中编码膜蛋白的Cbei_3304基因进行***失活后，使得此基因不能正常表达，获得Cbei_3304基因***失活的重组菌株。本发明提供了一种简单、高效的提高拜氏梭菌抗逆性的方法，借助此方法得到的重组菌株对毒素物质（阿魏酸、香草酸等）具有较高抗逆性，当以未脱毒的甘蔗渣酸解糖液为碳源时，在2L发酵罐中总溶剂产量和丁醇产量分别达到了7.0 g/L和5.1 g/L，而同等条件培养的出发菌株基本不生长。利用本发明方法构建的重组菌株，其抗逆性强、丁醇产量高，是一种适用于甘蔗渣等木质纤维原料发酵生产丁醇的优良菌种。
附图说明
图1为本发明***失活载体pWJ的质粒图谱；
图2为本发明使用二型内含子***失活的机理图；
图3为转化子菌落PCR电泳图。
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，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例
1
一、拜氏梭菌
Cbei_3304
基因***失活突变株的构建
构建拜氏梭菌Cbei_3304基因***失活突变株的原理如图2所示，具体构建过程包括以下步骤：
（
1
）
Cbei_3304
***失活载体的构建
1）设计内含子
根据NCBI数据库收录的拜氏梭菌的Cbei_3304基因序列（如SEQ ID No：1所示），借助软件设计合适的***基因位点（http://www.clostron.com），选择***在第101-102个碱基之间，并生成内含子序列，合成内含子序列S-101（其序列如SEQ ID NO：2所示），并设计以下引物。
克隆引物：
pWJ-OSC-101-S：5’- ggagtgtcgaggatcctcgagataattatccttacacttcgcc
-3’，其序列如SEQ ID NO：3所示；
pWJ-OSC-101-A：5’-ggttctcctacagattgtacaaatgtggtgataacagataag-3’，其序列如SEQ ID NO：4所示。
验证引物：
Cbei-3304-T-S：5’-taaattacctacagcaaaactgtg-3’， 序列如SEQ ID NO：5所示；
Cbei-3304-T-A：5’-ggaattaagaaccttgaatctatc-3’， 序列如SEQ ID NO：6所示。
引物pWJ-OSC-101-S引入XhoⅠ酶切位点(下划线部分)，引物pWJ-OSC-101-A引入BsrGⅠ酶切位点(下划线部分)。
2）Cbei_3304-pWJ-101重组载体构建
用XhoⅠ和BsrGⅠ双酶切载体pWJ，pWJ质粒图谱如图1所示，其序列如SEQ ID NO：7所示。酶切产物经纯化试剂盒（Takara）纯化后，与内含子序列S-101通过一步克隆（ClonExpress）连接。将一步克隆连接的重组质粒转化到大肠杆菌E.coli DH5a，涂布到含有50μg/ml 氨苄霉素抗性LB平板，37℃培养12～16h，挑取转化子，接到液体含有50μg/ml 氨苄霉素LB培养基中，37℃、200rpm培养12h，提取重组质粒（AXYGEN），测序验证，获得Cbei_3304-pWJ-101重组载体。
3）Cbei_3304-pWJ-101重组载体的甲基化
制备E.coli Top 10/pAN2的化学感受态，将测序成功的Cbei_3304-pWJ-101重组载体转化到大肠杆菌E.coli Top 10，由于pAN2质粒具有四环素抗性，故涂布到含有50μg/ml氨苄霉素和10μg/ml四环素双抗性LB平板，37℃培养12～16h，挑取转化子，接到液体含有50μg/ml 氨苄霉素和10µg/ml四环素LB培养基中，37℃、200rpm培养12h，提取甲基化的Cbei_3304-pWJ-101重组载体（pAN2质粒含有一个枯草芽孢杆菌噬菌体基因，能编码甲基转移酶，能实现外源质粒在大肠杆菌中的甲基化），即Cbei_3304***失活载体。
（
2
）
Cbei_3304
***失活载体转化拜氏梭菌
(Clostridium
beijerinckiiNCIMB 8052)
1）将Clostridium beijerinckii NCIMB 8052接种至CGM培养基（酵母粉 3 g/L，蛋白胨 5 g/L，可溶性淀粉 10 g/L，乙酸铵 2 g/L，NaCl 2 g/L，MgSO4 ·7H2O 3 g/L，KH2PO4
1 g/L，K2HPO4
1 g/L，FeSO4·7H2O 0.1 g/L）37℃过夜培养，次日以5%比例接种到CGM培养基，37℃培养6-8h，以10%接种到2×YTG培养基（酵母粉16 g/L，蛋白胨 10 g/L，葡萄糖 5 g/L，氯化钠 5 g/L）37℃培养3h，OD600nm=1；
2）取50ml拜氏梭菌菌液，5000rpm，4℃离心10 min，弃上清。在以ETM缓冲液（270mM蔗糖，0.6mM Na2HPO4，4.4mM Na2HPO4，10mM MgCl2）重悬；同上离心，去上清，再次以ETM缓冲液重悬，同上离心，彻底取上清；
3）以1ml ET 缓冲液（270mM蔗糖，0.6mM Na2HPO4，4.4mM NaH2PO4重悬，取200µl，加入1μg Cbei_3304***失活载体，加入0.2cm预冷的电转杯，轻轻混匀；
4）使用MicroPulserTM电转仪电转，条件为电压1.8kV，电阻200Ω，电容2.5µF，电击后立刻加入1mL 2×YTG 培养基，转移到无菌离心管中复苏2～3h；
5）取200µl上述菌液，涂布到含有10 µg/ml红霉素的 CGM 固体培养基，培养2～3天。
（
3
）
Cbei_3304
***失活突变株的筛选
挑取上述步骤5）中培养2～3天的转化子，使用引物Cbei-3304-T-S和Cbei-3304-T-A对转化子进行菌落PCR验证，筛选出内含子***基因组的突变株（***后，PCR扩增出基因条带电泳图上比野生型大约1Kbp），如图3所示，将正确***的突变株传代三次，同时涂布在含有红霉素抗性和没有红霉素抗性的CGM 固体培养基上，筛选出敲除质粒丢失的突变株（在红霉素抗性平板上不能生长的突变株）。
二、
Cbei_3304
***失活突变株的传代稳定性
将上述构建的Cbei_3304基因***失活的拜氏梭菌突变株在固体培养基上连续转接7次，发现所得菌株与出发菌株在形态特征和培养特征上相同，生长状态良好。在以葡萄糖为碳源的发酵培养基中，检测重组菌的传代稳定性，***失活突变菌传代发酵试验结果如表1所示。
表1不同传代次数的Cbei_3304***失活突变菌的发酵情况
从实验结果可知，经过7次连续传代，两株突变株的总溶剂产量和丁醇产量较稳定，具有较好的传代稳定性，可作为进一步研究和开发的生产菌株。
三、
Cbei_3304
***失活的拜氏梭菌对模式酚类化合物阿魏酸的高抗逆性
培养基配方（%为质量百分比）：
平板培养基：酵母粉0.3%，蛋白胨0.5%，可溶性淀粉1%，乙酸铵0.2%，氯化钠0.2%，七水合硫酸镁0.3%，磷酸二氢钾0.1%，磷酸氢二钾0.1%，七水合硫酸亚铁0.01%，琼脂1.5%，其余为水，pH 6。
种子培养基：酵母粉0.3%，蛋白胨0.5%，可溶性淀粉1%，乙酸铵0.2%，氯化钠0.2%，七水合硫酸镁0.3%，磷酸二氢钾0.1%，磷酸氢二钾0.1%，七水合硫酸亚铁0.01%，其余为水，pH 6。
发酵培养基：葡萄糖3%，乙酸铵0.22%，磷酸二氢钾0.05%，磷酸氢二钾0.05%，氯化钠0.001%，七水合硫酸镁0.02%，七水合硫酸亚铁0.001%，一水合硫酸锰0.001%，玉米浆0.1%，阿魏酸 0.06%，其余为水，pH 6.6。
将上述Cbei_3304***失活的拜氏梭菌突变株接种至平板培养基厌氧培养，培养温度35℃，培养时间12 h。将平板培养的突变株接种到种子培养基中，培养温度35℃，50 mL 肖特厌氧瓶装液量30 mL，充氮气3min，培养温度35℃，培养时间12 h；将种子接种到发酵培养基中，接种量10%(v/v)，发酵温度35℃，100 mL 肖特厌氧瓶装液量50 mL，充氮气3min，发酵培养72 h后，检测丁醇产量达到了6.1 g/L，而同等条件培养的出发菌株NCIMB 8052基本不生长。
四、
Cbei_3304
***失活的拜氏梭菌对模式酚类化合物香草酸的高抗逆性
培养基配方（%为质量百分比）：
平板培养基：同上述“三”。
种子培养基：同上述“三”。
发酵培养基：葡萄糖3%，乙酸铵0.22%，磷酸二氢钾0.05%，磷酸氢二钾0.05%，氯化钠0.001%，七水合硫酸镁0.02%，七水合硫酸亚铁0.001%，一水合硫酸锰0.001%，玉米浆0.1%，香草酸 0.05%，其余为水，pH 6.6。
将上述Cbei_3304***失活的拜氏梭菌突变株接种至平板培养基厌氧培养，培养温度35℃，培养时间12 h。将平板培养的突变株接种到种子培养基中，培养温度35℃，50 mL 肖特厌氧瓶装液量30 mL，充氮气3min，培养温度35℃，培养时间12 h；将种子接种到发酵培养基中，接种量10%(v/v)，发酵温度35℃，100 mL 肖特厌氧瓶装液量50 mL，充氮气3min，发酵培养72 h后，检测丁醇产量达到了4.6 g/L，而同等条件培养的出发菌株NCIMB 8052只能微弱的生长，但基本不产丁醇。
五、
Cbei_3304
***失活的拜氏梭菌对甘蔗渣酸解糖液的高抗逆性
本部分内容说明Cbei_3304***失活的拜氏梭菌利用甘蔗渣酸解糖液（可溶性总酚含量为2.4 g/L），在1L发酵罐中发酵生产丁醇的工艺。
培养基配方（%为质量百分比）：
平板培养基：同上述“三”。
种子培养基：同上述“三”。
发酵培养基：乙酸铵0.22%，磷酸二氢钾0.05%，磷酸氢二钾0.05%，氯化钠0.001%，七水合硫酸镁0.02%，七水合硫酸亚铁0.001%，一水合硫酸锰0.001%，玉米浆0.1%，用未脱毒的甘蔗渣酸解糖液（总还原糖为4 %）配置，其余为水，pH 6.6，培养基中可溶性总酚含量为2.4 g/L。
将上述Cbei_3304***失活的拜氏梭菌突变株接种至平板培养基厌氧培养，培养温度35℃，培养时间12 h。将平板培养的突变株接种到种子培养基中，250 mL 肖特厌氧瓶装液量150 mL，充氮气3min，培养温度35℃，培养时间12 h；将种子接种到装有1 L发酵培养基的2L发酵罐中，接种量10%（v/v），发酵温度35℃，连续通入氮气，流速为0.3 L/min，发酵培养90 h后，检测总溶剂产量和丁醇产量分别达到了3.1 g/L和2.2 g/L，而同等条件培养的出发菌株NCIMB 8052基本不生长。
六、
Cbei_3304
***失活的拜氏梭菌对甘蔗渣酸解糖液的高抗逆性
本部分内容说明Cbei_3304***失活的拜氏梭菌利用甘蔗渣酸解糖液（可溶性总酚含量为2.0 g/L），在2 L发酵罐中发酵生产丁醇的工艺。
培养基配方（%为质量百分比）：
平板培养基：同上述“三”。
种子培养基：同上述“三”。
发酵培养基：乙酸铵0.22%，磷酸二氢钾0.05%，磷酸氢二钾0.05%，氯化钠0.001%，七水合硫酸镁0.02%，七水合硫酸亚铁0.001%，一水合硫酸锰0.001%，玉米浆0.1%，用未脱毒的甘蔗渣酸解糖液（总还原糖为3.6%）配置，其余为水，pH 6.6，培养基中可溶性总酚含量为2.0 g/L。
将上述Cbei_3304***失活的拜氏梭菌突变株接种至平板培养基厌氧培养，培养温度35℃，培养时间12 h。将平板培养的突变株接种到种子培养基中，250 mL 肖特厌氧瓶装液量150 mL，充氮气3min，培养温度35℃，培养时间12 h；将种子接种到装有1 L发酵培养基的2L发酵罐中，接种量10%（v/v），发酵温度35℃，连续通入氮气，流速为0.3 L/min，发酵培养90 h后，检测总溶剂产量和丁醇产量分别达到了7.0 g/L和5.1g/L，而同等条件培养的出发菌株NCIMB 8052基本不生长。
<110> 广州甘蔗糖业研究所
<120> 一种高抗逆性拜氏梭菌及其应用
<130>
<160> 7
<170> PatentIn version
3.5
<210> 1
<211> 723
<212> DNA
<213> 拜氏梭菌
<400> 1
atgacaaagg ttaataaatt acctacagca aaactgtgga atccgaaatc atttattata 60
ttttccgtat ttttctcatt tctgccagct ggaatcatgt gtgctttgaa ttatggacga 120
tctggaagtc aaaaaaagaa gtggatattt cttttagcaa gcatcttagt gtttatagca 180
cttatagcac ttctgcctat attatcaatt aatacttcta tcatattttt cagtattaat 240
atagctttag gcataattct tatgtttaca cagttaaagc tgtataatga acatattcaa 300
aatggcgggc aaagtgcttc gtatttattt cctataatca tagggctttt gattttttca 360
ttgtcagctg cttcaattct atactcaatt tacgtgccaa aaaatgctct tgattatggc 420
gaaaatcact tattttatac taataagata acagaatccc aagcgaaaaa acttggagat 480
tatttaaatt cagaaggata cttcactcct agttcaaaag tggacgttaa gattgataaa 540
caagatacac tctatatttt atctctcgtt gtagaaggtg actataaaag tgatacaagc 600
tatgtagaac ctatgaaagc aatatcaaaa gagatttcaa aaaatgtttt tgaaaataat 660
aaagttagaa tagatttatg caatgataga ttcaaggttc ttaattccat caatgttgat 720
taa
723
<210> 2
<211> 344
<212> DNA
<213> 人工合成序列
<400> 2
ctcgagataa ttatccttac acttcgcccg cgtgcgccca gatagggtgt taagtcaagt 60
agtttaaggt actactctgt aagataacac agaaaacagc caacctaacc gaaaagcgaa 120
agctgatacg ggaacagagc acggttggaa agcgatgagt tacctaaaga caatcgggta 180
cgactgagtc gcaatgttaa tcagatataa ggtataagtt gtgtttactg aacgcaagtt 240
tctaatttcg gttaagtgtc gatagaggaa agtgtctgaa acctctagta caaagaaagg 300
taagttatgg cgggcgactt atctgttatc accacatttg taca 344
<210> 3
<211> 43
<212> DNA
<213> 人工引物
<400> 3
ggagtgtcga ggatcctcga gataattatc cttacacttc gcc 43
<210> 4
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<212> DNA
<213> 人工引物
<400> 4
ggttctccta cagattgtac aaatgtggtg ataacagata ag 42
<210> 5
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<212> DNA
<213> 人工引物
<400> 5
taaattacct acagcaaaac tgtg
24
<210> 6
<211> 24
<212> DNA
<213> 人工引物
<400> 6
ggaattaaga accttgaatc tatc
24
<210> 7
<211> 8601
<212> DNA
<213> pWJ质粒
<400> 7
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 60
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 120
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 180
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 240
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 300
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 360
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 420
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 480
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 540
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 600
taataatagt atatacttaa tgtgctgcag tataaaatat aaataatttt ctaaaaaact 660
taacttcatg tgaaaagttt gttaaaatat aaatgagcac gttaatcatt taacatagat 720
aattaaatag taaaagggag tgtcgaggat cctcgagata attatcctta aaggacgtag 780
atgtgcgccc agatagggtg ttaagtcaag tagtttaagg tactactctg taagataaca 840
cagaaaacag ccaacctaac cgaaaagcga aagctgatac gggaacagag cacggttgga 900
aagcgatgag ttacctaaag acaatcgggt acgactgagt cgcaatgtta atcagatata 960
aggtataagt tgtgtttact gaacgcaagt ttctaatttc ggtttccttc cgatagagga 1020
aagtgtctga aacctctagt acaaagaaag gtaagttaac atctacgact tatctgttat 1080
caccacattt gtacaatctg taggagaacc tatgggaacg aaacgaaagc gatgccgaga 1140
atctgaattt accaagactt aacactaact ggggataccc taaacaagaa tgcctaatag 1200
aaaggaggaa aaaggctata gcactagagc ttgaaaatct tgcaagggta cggagtactc 1260
gtagtagtct gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact 1320
aaaattaaaa attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag 1380
aaagaatcag taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt 1440
atcttttacg tccagatatt tattacgtgg cgacgcgttg ggaaatggca atgatagcga 1500
aacaacgtaa aactcttgtt gtatgctttc attgtcatcg tcacgtgatt cataaacaca 1560
agtgaatgtc gacagtgaat ttttacgaac gaacaataac agagccgtat actccgagag 1620
gggtacgtac ggttcccgaa gagggtggtg caaaccagtc acagtaatgt gaacaaggcg 1680
gtacctccct acttcaccat atcattttct gcagccccct agaaataatt ttgtttaact 1740
ttaagaagga gatatacata tatggctaga tcgtccattc cgacagcatc gccagtcact 1800
atggcgtgct gctagcgcta tatgcgttga tgcaatttct atgcactcgt agtagtctga 1860
gaagggtaac gccctttaca tggcaaaggg gtacagttat tgtgtactaa aattaaaaat 1920
tgattaggga ggaaaacctc aaaatgaaac caacaatggc aattttagaa agaatcagta 1980
aaaattcaca agaaaatata gacgaagttt ttacaagact ttatcgttat cttttacgtc 2040
cagatattta ttacgtggcg tatcaaaatt tatattccaa taaaggagct tccacaaaag 2100
gaatattaga tgatacagcg gatggcttta gtgaagaaaa aataaaaaag attattcaat 2160
ctttaaaaga cggaacttac tatcctcaac ctgtacgaag aatgtatatt gcaaaaaaga 2220
attctaaaaa gatgagacct ttaggaattc caactttcac agataaattg atccaagaag 2280
ctgtgagaat aattcttgaa tctatctatg aaccggtatt cgaagatgtg tctcacggtt 2340
ttagacctca acgaagctgt cacacagctt tgaaaacaat caaaagagag tttggcggcg 2400
caagatggtt tgtggaggga gatataaaag gctgcttcga taatatagac cacgttacac 2460
tcattggact catcaatctt aaaatcaaag atatgaaaat gagccaattg atttataaat 2520
ttctaaaagc aggttatctg gaaaactggc agtatcacaa aacttacagc ggaacacctc 2580
aaggtggaat tctatctcct cttttggcca acatctatct tcatgaattg gataagtttg 2640
ttttacaact caaaatgaag tttgaccgag aaagtccaga aagaataaca cctgaatatc 2700
gggagctcca caatgagata aaaagaattt ctcaccgtct caagaagttg gagggtgaag 2760
aaaaagctaa agttctttta gaatatcaag aaaaacgtaa aagattaccc acactcccct 2820
gtacctcaca gacaaataaa gtattgaaat acgtccggta tgcggacgac ttcattatct 2880
ctgttaaagg aagcaaagag gactgtcaat ggataaaaga acaattaaaa ctttttattc 2940
ataacaagct aaaaatggaa ttgagtgaag aaaaaacact catcacacat agcagtcaac 3000
ccgctcgttt tctgggatat gatatacgag taaggagatc tggaacgata aaacgatctg 3060
gtaaagtcaa aaagagaaca ctcaatggga gtgtagaact ccttattcct cttcaagaca 3120
aaattcgtca atttattttt gacaagaaaa tagctatcca aaagaaagat agctcatggt 3180
ttccagttca caggaaatat cttattcgtt caacagactt agaaatcatc acaatttata 3240
attctgaact ccgcgggatt tgtaattact acggtctagc aagtaatttt aaccagctca 3300
attattttgc ttatcttatg gaatacagct gtctaaaaac gatagcctcc aaacataagg 3360
gaacactttc aaaaaccatt tccatgttta aagatggaag tggttcgtgg gggatcccgt 3420
atgagataaa gcaaggtaag cagcgccgtt attttgcaaa ttttagtgaa tgtaaatccc 3480
cttatcaatt tacggatgag ataagtcaag ctcctgtatt gtatggctat gcccggaata 3540
ctcttgaaaa caggttaaaa gctaaatgtt gtgaattatg tgggacgtct gatgaaaata 3600
cttcctatga aattcaccat gtcaataagg tcaaaaatct taaaggcaaa gaaaaatggg 3660
aaatggcaat gatagcgaaa caacgtaaaa ctcttgttgt atgctttcat tgtcatcgtc 3720
acgtgattca taaacacaag tgaatgtcga gcacccgttc tcggagcact gtccgaccgc 3780
tttggccgcc gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc 3840
atggcgacca cacccgtcct gtggatcgcc aagccgccga tggtagtgtg gggtctcccc 3900
atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 3960
gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 4020
ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 4080
taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 4140
ctacaaactc ttcctgtcgt catatctaca agcatatggt gcactctcag tacaatctgc 4200
tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 4260
cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 4320
atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 4380
cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 4440
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4500
tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4560
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4620
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4680
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 4740
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4800
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4860
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4920
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4980
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 5040
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 5100
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 5160
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 5220
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5280
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5340
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5400
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5460
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5520
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5580
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5640
ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5700
gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 5760
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5820
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5880
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5940
gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 6000
cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 6060
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 6120
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6180
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6240
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6300
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6360
gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 6420
cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 6480
ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 6540
attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt 6600
tggctaacac acacgccatt ccaaccaata gttttctcgg cataaagcca tgctctgacg 6660
cttaaatgca ctaatgcctt aaaaaaacat taaagtctaa cacactagac ttatttactt 6720
cgtaattaag tcgttaaacc gtgtgctcta cgaccaaaag tataaaacct ttaagaactt 6780
tcttttttct tgtaaaaaaa gaaactagat aaatctctca tatcttttat tcaataatcg 6840
catcagattg cagtataaat ttaacgatca ctcatcatgt tcatatttat cagagctcgt 6900
gctataatta tactaatttt ataaggagga aaaaataaag agggttataa tgaacgagaa 6960
aaatataaaa cacagtcaaa actttattac ttcaaaacat aatatagata aaataatgac 7020
aaatataaga ttaaatgaac atgataatat ctttgaaatc ggctcaggaa aagggcattt 7080
tacccttgaa ttagtacaga ggtgtaattt cgtaactgcc attgaaatag accataaatt 7140
atgcaaaact acagaaaata aacttgttga tcacgataat ttccaagttt taaacaagga 7200
tatattgcag tttaaatttc ctaaaaacca atcctataaa atatttggta atatacctta 7260
taacataagt acggatataa tacgcaaaat tgtttttgat agtatagctg atgagattta 7320
tttaatcgtg gaatacgggt ttgctaaaag attattaaat acaaaacgct cattggcatt 7380
atttttaatg gcagaagttg atatttctat attaagtatg gttccaagag aatattttca 7440
tcctaaacct aaagtgaata gctcacttat cagattaaat agaaaaaaat caagaatatc 7500
acacaaagat aaacagaagt ataattattt cgttatgaaa tgggttaaca aagaatacaa 7560
gaaaatattt acaaaaaatc aatttaacaa ttccttaaaa catgcaggaa ttgacgattt 7620
aaacaatatt agctttgaac aattcttatc tcttttcaat agctataaat tatttaataa 7680
gtaagttaag ggatgcataa actgcatccc ttaacttgtt tttcgtgtac ctattttttg 7740
tgaatcgata atttacagaa aagaaaatta tagaatttag tatgattaat tatactcatt 7800
tatgaatgtt taattgaata caaaaaaaaa tacttgttat gtattcaatt acgggttaaa 7860
atatagacaa gttgaaaaat ttaataaaaa aataagtcct cagctcttat atattaagct 7920
accaacttag tatataagcc aaaacttaaa tgtgctacca acacatcaag ccgttagaga 7980
actctatcta tagcaatatt tcaaatgtac cgacatacaa gagaaacatt aactatatat 8040
attcaattta tgagattatc ttaacagata taaatgtaaa ttgcaataag taagatttag 8100
aagtttatag cctttgtgta ttggaagcag tacgcaaagg cttttttatt tgataaaaat 8160
tagaagtata tttatttttt cataattaat ttatgaaaat gaaagggggt gagcaaagtg 8220
acagaggaaa gcagtatctt atcaaataac aaggtattag caatatcatt attgacttta 8280
gcagtaaaca ttatgacttt tatagtgctt gtagctaagt agtacgaaag ggggagcttt 8340
aaaaagctcc ttggaataca tagaattcat aaattaattt atgaaaagaa gggcgtatat 8400
gaaaacttgt aaaaattgca aagagtttat taaagatact gaaatatgca aaatacattc 8460
gttgatgatt catgataaaa cagtagcaac ctattgcagt aaatacaatg agtcaagatg 8520
tttacataaa gggaaagtcc aatgtattaa ttgttcaaag atgaaccgat atggatggtg 8580
tgccataaaa atgagatgtt t
8601
Claims (5)
1.一种高抗逆性拜氏梭菌，其特征在于：其缺失正常的Cbei_3304蛋白功能。
2.根据权利要求1所述的一种高抗逆性拜氏梭菌，其特征在于：其基因Cbei_3304不能正常表达。
3.根据权利要求2所述的一种高抗逆性拜氏梭菌，其特征在于：其基因Cbei_3304被***失活。
4.权利要求1～3中任一所述一种高抗逆性拜氏梭菌在发酵制备丁醇中的应用。
5.根据权利要求4所述的应用，其特征在于：权利要求1所述的高抗逆性拜氏梭菌以不脱毒的木质纤维酸解糖液为原料直接发酵制备丁醇。
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| PCT/CN2014/085468 WO2015158094A1 (zh) | 2014-04-16 | 2014-08-29 | 一种高抗逆性拜氏梭菌及其应用 |
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Cited By (4)
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| CN104894048A (zh) * | 2015-06-12 | 2015-09-09 | 南京工业大学 | 一种提高拜氏梭菌阿魏酸抗逆性的方法 |
| WO2015158094A1 (zh) * | 2014-04-16 | 2015-10-22 | 广州甘蔗糖业研究所 | 一种高抗逆性拜氏梭菌及其应用 |
| CN105567603A (zh) * | 2016-02-03 | 2016-05-11 | 广州甘蔗糖业研究所 | 一种提高拜氏梭菌对4-羟基肉桂酸抗逆性的方法 |
| CN106047754A (zh) * | 2016-06-07 | 2016-10-26 | 广州甘蔗糖业研究所 | 一种提高拜氏梭菌产电的方法及其应用 |
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| CN102174433A (zh) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | 一株高抗逆性贝氏梭菌及其应用 |
| CN103667147A (zh) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | 一株高阿魏酸抗逆性拜氏梭菌及其应用 |
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| US8685729B2 (en) * | 2007-02-26 | 2014-04-01 | The Regents Of The University Of California | Heterologous expression of extremophile heat shock proteins and chaperones in microorganisms to increase tolerance to toxic compounds |
| WO2009143455A2 (en) * | 2008-05-22 | 2009-11-26 | Microbia, Inc. | Engineering resistance to aliphatic alcohols |
| GB201205795D0 (en) * | 2012-03-30 | 2012-05-16 | Univ Nottingham | Vector |
| CN103911334B (zh) * | 2014-04-16 | 2016-04-20 | 广州甘蔗糖业研究所 | 一种高抗逆性拜氏梭菌及其应用 |
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2014
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| CN102174433A (zh) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | 一株高抗逆性贝氏梭菌及其应用 |
| CN103667147A (zh) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | 一株高阿魏酸抗逆性拜氏梭菌及其应用 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015158094A1 (zh) * | 2014-04-16 | 2015-10-22 | 广州甘蔗糖业研究所 | 一种高抗逆性拜氏梭菌及其应用 |
| CN104894048A (zh) * | 2015-06-12 | 2015-09-09 | 南京工业大学 | 一种提高拜氏梭菌阿魏酸抗逆性的方法 |
| CN104894048B (zh) * | 2015-06-12 | 2018-03-09 | 南京工业大学 | 一种提高拜氏梭菌阿魏酸抗逆性的方法 |
| CN105567603A (zh) * | 2016-02-03 | 2016-05-11 | 广州甘蔗糖业研究所 | 一种提高拜氏梭菌对4-羟基肉桂酸抗逆性的方法 |
| CN105567603B (zh) * | 2016-02-03 | 2019-08-09 | 广东省生物工程研究所（广州甘蔗糖业研究所） | 一种提高拜氏梭菌对4-羟基肉桂酸抗逆性的方法 |
| CN106047754A (zh) * | 2016-06-07 | 2016-10-26 | 广州甘蔗糖业研究所 | 一种提高拜氏梭菌产电的方法及其应用 |
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| WO2015158094A1 (zh) | 2015-10-22 |
| CN103911334B (zh) | 2016-04-20 |
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