CN106636168A - 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 - Google Patents
一种调控丙酮丁醇梭菌胞外聚合物合成的方法 Download PDFInfo
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Abstract
本发明公开了一种调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，该方法是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列。本发明提供了一种简单、高效的调控EPS合成的方法，借助此方法得到的重组菌株对EPS具有较高的调控作用，当以葡萄糖为碳源，在100ml厌氧瓶中利用载玻片生物膜培养法培养丙丁菌生物膜时，生物膜厚度为5.81μm，而同等条件培养的出发菌株生物膜厚度达到24.6μm。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法。
背景技术
生物被膜又称生物膜(biofilm)是在生物或非生物介质表面由微生物及其胞外基质(Extracellular polymeric substance,EPS)形成的致密、复杂的生物聚集体。EPS作为生物膜的主要组成物质，对保持生物膜的结构和功能的完整性、代谢物质传质通道顺畅具有重要作用。EPS是细胞在固体介质诱导下自主合成的富含大分子多糖的胞外结构，具有良好的持水性、溶胀性、透过性，能够稳定细胞表面的水化层、及时泵出细胞的反应物、为细胞创造有益的微环境，可认为是生物相容性最佳的固定介质。同时，EPS为细胞隔离外界毒性物质提供了一道天然屏障，因而提高了EPS中微生物细胞的抗逆能力。因此，细胞在EPS中能够大量增殖形成密集的菌群进而展示集群效应。集群效应可使群体成员实现生理协同作用，借助个体间的互相刺激而使个体的行为、生理、形态发生变化，使多个生物个体在行为上相互协作，从而提高整个种群的代谢及适存能力。
在临床医学领域，超过80％的慢性感染疾病都与体内细菌形成EPS有关，而细菌形成EPS后对抗生素等杀菌剂和宿主免疫系统的敏感性显著降低，因此，大部分有关EPS机理的研究都集中在如何清除人体器官及外源性生物植入材料表面上的EPS。然而，作为EPS的另一种存在形式——固定化发酵过程中细胞形成的EPS却至今鲜有研究，尤其是固定化细胞形成EPS后表现出的较高底物耐受性和较快的发酵效率的机制机理到现在还不是很清楚。
发明内容
本发明所要解决的技术问题是提供一种调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法。
本发明还要解决的技术问题是提供利用调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法构建得到的丙酮丁醇基因工程菌。
本发明最后要解决的技术问题是提供利用上述基因工程菌在EPS催化体系中的应用。
为解决上述技术问题，本发明采用的技术方案如下：
一种调控丙酮丁醇梭菌胞外聚合物合成的方法，该方法是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列。在Spo0A基因中插入内含子序列，最终使Spo0A基因不能正常表达，从而引起丙酮丁醇梭菌胞外聚合物(EPS)的合成。
其中，所述的丙酮丁醇梭菌保藏编号为CGMCC No.5234，该菌株的具体信息在申请号为201210075094.X的中国专利中详细公开。
其中，所述的Spo0A基因为CA_C2071，其核苷酸序列如SEQ ID No.：1所示。Spo0A基因编码一种孢子生成蛋白，在发育早期通过多组分磷酸化途径而被激活，是双组份应答性调节蛋白大家族的成员之一，调控并起始孢子形成作用所需基因的转录。
其中，利用二型内含子基因敲除技术将SEQ ID No.：2所示的内含子序列插入Spo0A基因的第242bp和第243bp之间
其中，所述二型内含子基因敲除技术的操作方法如下：
(1)分析SEQ ID No：1所示的序列，得到内含子的核苷酸序列和基因的插入位点；
(2)内含子序列构建到二型内含子基因敲除质粒中，得到重组质粒；
(3)将步骤(2)得到的重组质粒转化丙酮丁醇梭菌，筛选得到Spo0A基因失活的菌株
其中，所述的所述二型内含子基因敲除质粒为PMTL007C-E2质粒，该质粒的核苷酸序列如SEQ ID No：5所示。
上述调控丙酮丁醇梭菌胞外聚合物合成的方法构建得到的丙酮丁醇基因工程菌。
上述丙酮丁醇基因工程菌在发酵生产丙酮丁醇中的应用。
有益效果：
(1)本发明将丙酮丁醇梭菌B3中Spo0A基因进行插入失活后，使得此基因不能正常表达，获得CA_C2071基因插入失活的重组菌株。
(2)本发明提供了一种简单、高效的调控EPS合成的方法，借助此方法得到的重组菌株对EPS具有较高的调控作用，当以葡萄糖为碳源，在100ml厌氧瓶中利用载玻片生物膜培养法培养丙丁菌生物膜时，生物膜厚度为5.81μm，而同等条件培养的出发菌株生物膜厚度达到24.6μm。
附图说明
图1为本发明二型内含子基因敲除质粒PMTL007C-E2的质粒图谱。
图2为本发明使用二型内含子插入失活的机理图。
图3为转化子菌落PCR电泳图，泳道1：出发菌基因组验证PCR产物(846bp)；泳道2-23为挑取的红霉素抗性单菌落的菌液作为模板验证PCR产物(2755bp)；泳道M：DNA MarkerDL2000。
图4为自制反应器中细胞固定化材料的填装方法图。
图5为出发菌与敲除菌EPS宏观形态图。(在不锈钢反应器中，固定化材料与不锈钢丝网交替缠绕呈圆柱状，我们定义固定化材料靠近罐壁的一面为正面，另一面为反面反面。)
图6为CLSM观察出发菌与敲除菌EPS形成过程图。
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。
实施例1：丙酮丁醇梭菌CA_C2071基因失活突变株的构建方法。
1、设计内含子：
根据NCBI数据库收录的丙酮丁醇梭菌的CA_C2071基因序列(如SEQ ID No：1所示)，借助软件设计合适的插入基因位点(http://www.clostron.com)，选择插入在第242-243个碱基之间，并生成内含子序列，合成内含子序列S-242其序列如SEQ ID NO：2所示。
2、CA_C2071插入失活载体的构建：
用XhoⅠ和BsrGⅠ双酶切载体PMTL007C-E2，其序列如SEQ ID NO：5所示。酶切产物经纯化试剂盒(Takara)化后，与内含子序列S-242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(本实验室)，涂布到含有25ug/ml氯霉素抗性LB平板，37℃培养12-16h，挑取转化子，接到液体含有25ug/ml氯霉素LB培养基中，37℃、200rpm培养12h，提取重组质粒(AXYGEN)，测序验证,获得CA_C2071插入失活载体PMTL007C-E2-2071。
3、载体PMTL007C-E2-2071甲基化：
由于已经证明丙酮丁醇梭菌(Clostridium acetobutlicum)ATCC 824中含有限制性内切酶Cac824I，可以切割未甲基化的外源DNA，因此在对丙酮丁醇梭菌进行转化前对重组失活载体PMTL007C-E2-2071进行甲基化修饰。具体操作步骤为制备E.coli Top 10/pAN2(本实验室)的化学感受态，将测序成功的失活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25ug/ml氯霉素和10ug/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25ug/ml氯霉素和10ug/ml四环素LB培养基中，37℃、200rpm培养12h，提取甲基化缺失载体(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)。
4、电转化甲基化的敲除质粒至丙酮丁醇梭菌：
厌氧条件下，取2xYTG培养基培养的生长至对数中期的C.acetobutylicumB3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，5mMNaH2PO4，pH7.4)，洗涤两次，并用2.3mlEPB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1ml37℃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。
5、筛选CA_C2071插入失活突变株：
挑取转化子，使用引物CA_C2071-T-F(其序列如SEQ ID NO：3所示)和CA_C2071-T-R(其序列如SEQ ID NO：4所示)，对转化子进行菌落PCR验证，筛选出内含子插入基因组的突变株(插入后，PCR扩增出基因条带电泳图上比野生型大约1Kbp)。将正确插入的突变株传代三次，同时涂布在含有红霉素抗性和红霉素、甲砜霉素抗性的P2固体培养基上，筛选出敲除质粒丢失的突变株(在同时含红霉素和甲砜霉素抗性平板上不能生长的突变株)。
实施例2：敲除菌与出发菌分别在2L发酵罐中固定化发酵合成胞外聚合物(EPS)的工艺。
平板培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，琼脂15g/L，其余为水。
种子培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，其余为水。
发酵培养基：葡萄糖60g/L，乙酸铵2.2g/L，磷酸二氢钾0.5g/L，磷酸氢二钾0.5g/L，氯化钠0.01g/L，七水合硫酸镁0.2g/L，七水合硫酸亚铁0.01g/L，一水合硫酸锰0.01g/L，其余为水。
将实施例1中构建的CA_C2071基因插入失活的丙酮丁醇梭菌重组菌和出发菌株CGMCC No.5234接种至平板培养基厌氧培养，培养温度37℃，培养时间12h。将平板培养的重组菌接种到种子培养基(不加琼脂，其他组分同P2平板培养基)中37℃静止培养12h。
固定化发酵：吸附固定化细胞发酵放大工艺在自制的不锈钢反应器中进行。反应器柱状，有效容积约2L，内径10cm，高30cm，外壁有夹套，可通过硅胶管与外置恒温水浴锅相连，进而实现发酵保温。反应器上下两端和侧面在适当位置装有用于取样、液体循环、探头检测的管道和阀门。材料填装时，固定化材料CTF与不锈钢丝网(孔径约2mm，厚度不足1mm)交替缠绕，松紧适度，如图4所示，然后置于2L不锈钢反应器中。发酵液静止反应12h，维持发酵温度37℃，通过蠕动泵对反应器中的发酵液进行循环，循环流速约20-35mL/min，每12h换一次液，发酵培养120h。
由图5可知，出发菌EPS宏观形态为白色粘液状团聚体，其中含有大量孔穴和通道；敲除菌肉眼基本观察不到EPS。
实施例3：利用激光共聚焦显微镜观察敲除菌与出发菌生物膜的结构与厚度。
利用载玻片生物膜培养法培养丙丁菌生物膜，用异硫氰酸荧光素(FITC)标记的刀豆蛋白A(FITC-ConA)和碘化吡啶(PI)双重免疫荧光技术染色，用激光共聚焦显微镜(CLSM)观察生物膜结构和其中胞外多糖的分布,并对生物膜进行逐层水平扫描,根据三维坐标系Z轴距离计算生物膜厚度。
具体操作如下：
(1)生物膜标本的制备
在100mL肖特瓶内放入25mm×75mm载玻片，装入50mL发酵液厌氧静置培养。在培养6、24和72h后取出载玻片，用0.1M，pH＝7.4PBS缓冲液反复冲洗,除掉表面浮游菌,无菌滤纸吸干多余液体,用2.5％戊二醛4℃固定玻片1.5h后，悬浮于0.1M，pH＝7.4的PBS缓冲溶液中，待用。
(2)生物膜标本的染色
多糖染色：FITC-ConA与生物膜中的多糖结合发出绿色荧光。将FITC-ConA配成1mg/mL溶液，呈黄色，样品4℃避光染色15-30min后，用PBS缓冲液清洗去除残留染色剂。FITC-ConA激发/发射波长：488/505nm。
细胞染色：PI与菌体内的DNA结合发出红色荧光，但无膜通透性，不能透过活细胞膜，只能染死细胞。样品4℃避光染色15-30min后，用PBS缓冲液清洗去除残留染色剂。PI激发/发射波长为：536/617nm。
(3)CLSM观察生物膜及厚度测量
生物膜观察:采用LSM 510型激光共聚焦扫描显微镜,37℃恒温载物台,物镜倒置。扫描模式:HeNe/Ar激光,Ex/Em为490/525nm和536/617nm。
生物膜厚度确定:建立三维坐标体系，调节聚焦平面位置，对生物膜标本由外(生物膜游离面)向内(生物膜与玻片相贴面)逐层水平扫描,扫描30层，并进行3D图像重建，则第一张与最后一张能观察到生物膜的扫描图片间的Z轴距离为生物膜厚度。
由图6可知，在初始黏附期，出发菌株生物膜中多糖含量较多，而细菌含量相比较少，多糖形成多处较大的积聚区域，但相互之间的联系并不紧密，细菌形成较小团块，形如细菌镶嵌于多糖基质中；而敲除菌中细菌大部分成散落分布，大部分区域细菌和多糖关系并不密切。在对数生长期，出发菌株生物膜中细菌大量繁殖，细菌和多糖的关联开始紧密，已开始形成多糖和细菌相互包裹的网络状结构；而敲除菌中菌落间的细菌分布厚薄不均，形成沟壑间隙。在成熟稳定期，出发菌株多糖染色明显多于细菌染色，细菌镶嵌在由大量多糖形成的基质中，结构紧密，形成菌囊样的结构，生物膜厚度为24.6μm；而敲除菌中细菌和多糖的关联依旧并不紧密，生物膜厚度为5.81μm。
SEQUENCE LISTING
<110> 南京工业大学
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atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta tcgttatctt 780
ttacgtccag atatttatta cgtggcgacg cgtgaagttc ctatactttc tagagaatag 840
gaacttcgcg actcatagaa ttatttcctc ccgttaaata atagataact attaaaaata 900
gacaatactt gctcataagt aacggtactt aaattgttta ctttggcgtg tttcattgct 960
tgatgaaact gatttttagt aaacagttga cgatattctc gattgaccca ttttgaaaca 1020
aagtacgtat atagcttcca atatttatct ggaacatctg tggtatggcg ggtaagtttt 1080
attaagacac tgtttacttt tggtttagga tgaaagcatt ccgctggcag cttaagcaat 1140
tgctgaatcg agacttgagt gtgcaagagc aaccctagtg ttcggtgaat atccaaggta 1200
cgcttgtaga atccttcttc aacaatcaga tagatgtcag acgcatggct ttcaaaaacc 1260
acttttttaa taatttgtgt gcttaaatgg taaggaatac tcccaacaat tttatacctc 1320
tgtttgttag ggaattgaaa ctgtagaata tcttggtgaa ttaaagtgac acgagtattc 1380
agttttaatt tttctgacga taagttgaat agatgactgt ctaattcaat agacgttacc 1440
tgtttactta ttttagccag tttcgtcgtt aaatgccctt tacctgttcc aatttcgtaa 1500
acggtatcgg tttcttttaa attcaattgt tttattattt ggttgagtac tttttcactc 1560
gttaaaaagt tttgagaata ttttatattt ttgttcatac cagcaccaga agcaccagca 1620
tctcttgggt taattgaggc ctgagtataa ggtgacttat acttgtaatc tatctaaacg 1680
gggaacctct ctagtagaca atcccgtgct aaattgtagg actgcccttt aataaatact 1740
tctatattta aagaggtatt tatgaaaagc ggaatttatc agattaaaaa tactttctct 1800
agagaaaatt tcgtctggat tagttactta tcgtgtaaaa tctgataaat ggaattggtt 1860
ctacataaat gcctaacgac tatccctttg gggagtaggg tcaagtgact cgaaacgata 1920
gacaacttgc tttaacaagt tggagatata gtctgctctg catggtgaca tgcagctgga 1980
tataattccg gggtaagatt aacgacctta tctgaacata atgccatatg aatccctcct 2040
aatttatacg ttttctctaa caacttaatt atacccacta ttattatttt tatcaatata 2100
gaagttccta tactttctag agaataggaa cttcacgcgt tgggaaatgg caatgatagc 2160
gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat cgtcacgtga ttcataaaca 2220
caagtgaatg tcgacagtga atttttacga acgaacaata acagagccgt atactccgag 2280
aggggtacgt acggttcccg aagagggtgg tgcaaaccag tcacagtaat gtgaacaagg 2340
cggtacctcc ctacttcacc atatcatttt ctgcagcccc ctagaaataa ttttgtttaa 2400
ctttaagaag gagatataca tatatggcta gatcgtccat tccgacagca tcgccagtca 2460
ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcactc gtagtagtct 2520
gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact aaaattaaaa 2580
attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag aaagaatcag 2640
taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt atcttttacg 2700
tccagatatt tattacgtgg cgtatcaaaa tttatattcc aataaaggag cttccacaaa 2760
aggaatatta gatgatacag cggatggctt tagtgaagaa aaaataaaaa agattattca 2820
atctttaaaa gacggaactt actatcctca acctgtacga agaatgtata ttgcaaaaaa 2880
gaattctaaa aagatgagac ctttaggaat tccaactttc acagataaat tgatccaaga 2940
agctgtgaga ataattcttg aatctatcta tgaaccggta ttcgaagatg tgtctcacgg 3000
ttttagacct caacgaagct gtcacacagc tttgaaaaca atcaaaagag agtttggcgg 3060
cgcaagatgg tttgtggagg gagatataaa aggctgcttc gataatatag accacgttac 3120
actcattgga ctcatcaatc ttaaaatcaa agatatgaaa atgagccaat tgatttataa 3180
atttctaaaa gcaggttatc tggaaaactg gcagtatcac aaaacttaca gcggaacacc 3240
tcaaggtgga attctatctc ctcttttggc caacatctat cttcatgaat tggataagtt 3300
tgttttacaa ctcaaaatga agtttgaccg agaaagtcca gaaagaataa cacctgaata 3360
tcgggagctc cacaatgaga taaaaagaat ttctcaccgt ctcaagaagt tggagggtga 3420
agaaaaagct aaagttcttt tagaatatca agaaaaacgt aaaagattac ccacactccc 3480
ctgtacctca cagacaaata aagtattgaa atacgtccgg tatgcggacg acttcattat 3540
ctctgttaaa ggaagcaaag aggactgtca atggataaaa gaacaattaa aactttttat 3600
tcataacaag ctaaaaatgg aattgagtga agaaaaaaca ctcatcacac atagcagtca 3660
acccgctcgt tttctgggat atgatatacg agtaaggaga tctggaacga taaaacgatc 3720
tggtaaagtc aaaaagagaa cactcaatgg gagtgtagaa ctccttattc ctcttcaaga 3780
caaaattcgt caatttattt ttgacaagaa aatagctatc caaaagaaag atagctcatg 3840
gtttccagtt cacaggaaat atcttattcg ttcaacagac ttagaaatca tcacaattta 3900
taattctgaa ctccgcggga tttgtaatta ctacggtcta gcaagtaatt ttaaccagct 3960
caattatttt gcttatctta tggaatacag ctgtctaaaa acgatagcct ccaaacataa 4020
gggaacactt tcaaaaacca tttccatgtt taaagatgga agtggttcgt gggggatccc 4080
gtatgagata aagcaaggta agcagcgccg ttattttgca aattttagtg aatgtaaatc 4140
cccttatcaa tttacggatg agataagtca agctcctgta ttgtatggct atgcccggaa 4200
tactcttgaa aacaggttaa aagctaaatg ttgtgaatta tgtgggacgt ctgatgaaaa 4260
tacttcctat gaaattcacc atgtcaataa ggtcaaaaat cttaaaggca aagaaaaatg 4320
ggaaatggca atgatagcga aacaacgtaa aactcttgtt gtatgctttc attgtcatcg 4380
tcacgtgatt cataaacaca agtgaatgtc gagcacccgt tctcggagca ctgtccgacc 4440
gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga 4500
tcatggcgac cacacccgtc ctgtggatcg ccaagccgcc gatggtagtg tggggtctcc 4560
ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 4620
gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 4680
cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 4740
cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt 4800
ttctacaaac tcttcctgtc gtcatatcta caagccatcc ccccacagat acgggcgcgc 4860
cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa gtgatagtca 4920
aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat agaatttagt 4980
atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat acttgttatg 5040
tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa ataagtcctc 5100
agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat gtgctaccaa 5160
cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc gacatacaag 5220
agaaacatta actatatata ttcaatttat gagattatct taacagatat aaatgtaaat 5280
tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt acgcaaaggc 5340
ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt tatgaaaatg 5400
aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca aggtattagc 5460
aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg tagctaagta 5520
gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata aattaattta 5580
tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt aaagatactg 5640
aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc tattgcagta 5700
aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat tgttcaaaga 5760
tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa gaacagaaaa 5820
aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat gtaaagaaga 5880
gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac acagtatgca 5940
ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact cgcattttca 6000
taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa cccacaataa 6060
aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca agctaggata 6120
tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg ctattaagat 6180
gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt ttgtttgttc 6240
atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta tatcctttct 6300
aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta ttatgatatt 6360
atgtttttct attaaaataa attaagtata tagaatagtt taataatagt atatacttaa 6420
tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat aagcggccgg 6480
ccagtgggca agttgaaaaa ttcacaaaaa tgtggtataa tatctttgtt cattagagcg 6540
ataaacttga atttgagagg gaacttagat ggtatttgaa aaaattgata aaaatagttg 6600
gaacagaaaa gagtattttg accactactt tgcaagtgta ccttgtacct acagcatgac 6660
cgttaaagtg gatatcacac aaataaagga aaagggaatg aaactatatc ctgcaatgct 6720
ttattatatt gcaatgattg taaaccgcca ttcagagttt aggacggcaa tcaatcaaga 6780
tggtgaattg gggatatatg atgagatgat accaagctat acaatatttc acaatgatac 6840
tgaaacattt tccagccttt ggactgagtg taagtctgac tttaaatcat ttttagcaga 6900
ttatgaaagt gatacgcaac ggtatggaaa caatcataga atggaaggaa agccaaatgc 6960
tccggaaaac atttttaatg tatctatgat accgtggtca accttcgatg gctttaatct 7020
gaatttgcag aaaggatatg attatttgat tcctattttt actatgggga aatattataa 7080
agaagataac aaaattatac ttcctttggc aattcaagtt catcacgcag tatgtgacgg 7140
atttcacatt tgccgttttg taaacgaatt gcaggaattg ataaatagtt aacttcaggt 7200
ttgtctgtaa ctaaaaacaa gtatttaagc aaaaacatcg tagaaatacg gtgttttttg 7260
ttaccctaag tttaaactcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7320
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7380
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7440
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7500
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7560
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7620
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7680
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7740
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7800
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7860
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 7920
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7980
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 8040
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 8100
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcagggcccc 8160
ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac 8220
aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg 8280
caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc 8340
gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt 8400
aacagatgag ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc 8460
tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc 8520
cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt 8580
cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg 8640
cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac 8700
gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat 8760
gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg 8820
tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc 8880
tggtgaagta catcaccgac gagcaaggca agaccgatcg ggccc 8925
Claims (8)
1.一种调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，该方法是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列。
2.根据权利要求1所述的调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，所述的丙酮丁醇梭菌保藏编号为CGMCC No.5234。
3.根据权利要求1所述的调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，所述的Spo0A基因为CA_C2071，其核苷酸序列如SEQ ID No.：1所示。
4.根据权利要求1所述的调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，利用二型内含子基因敲除技术将SEQ ID No.：2所示的内含子序列插入Spo0A基因的第242bp和第243bp之间。
5.根据权利要求4所述的调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，所述二型内含子基因敲除技术的操作方法如下：
(1)分析SEQ ID No：1所示的序列，得到内含子的核苷酸序列和基因的插入位点；
(2)内含子序列构建到二型内含子基因敲除质粒中，得到重组质粒；
(3)将步骤(2)得到的重组质粒转化丙酮丁醇梭菌，筛选得到Spo0A基因失活的菌株。
6.根据权利要求5所述的调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，所述的所述二型内含子基因敲除质粒为PMTL007C-E2质粒，该质粒的核苷酸序列如SEQ ID No：5所示。
7.权利要求1～6任一所述调控丙酮丁醇梭菌胞外聚合物合成的方法构建得到的丙酮丁醇基因工程菌。
8.权利要求7所述丙酮丁醇基因工程菌在EPS催化体系中的应用。
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