CN101397547B - 构建荚膜缺失的克雷伯氏菌的方法 - Google Patents
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Abstract
一种构建荚膜缺失的克雷伯氏菌的方法,属于生物化工技术领域。在PDO产生菌中,采用基因敲除的方法敲除荚膜主体基因的启动子或在启动子后整合终止子中止其转录。使得克雷伯氏菌荚膜部分或全部缺失。从而降低其发酵液粘度,降低PDO产物分离提取难度,同时降低了菌体的致病性,有利于克雷伯氏菌的PDO工业化生产应用。
Description
技术领域
本发明属于生物化工技术领域,特别提供了一种构建荚膜缺失的克雷伯氏菌的方法。
背景技术
荚膜是某些细菌在细胞壁外所包围的一层粘液性物质。其中多数细菌的荚膜由多糖组成。多糖的分子组成和构型多样,令其结构极为复杂,成为血清学分型的基础。少数细菌荚膜为多肽,如炭疽芽胞杆菌、鼠疫杆菌等。
荚膜对细菌有以下功能:①抗吞噬作用:荚膜因其亲水性及其空间占位、屏障作用,可有效抵抗寄主吞噬细胞的吞噬作用。②粘附作用:荚膜多糖可使细菌彼此间粘连,也可粘附于组织细胞或无生命物体表面,是引起感染的重要因素。③抗有害物质的损伤作用:处于细菌细胞最外层,荚膜犹如盔甲可有效保护菌体免受或少受多种杀菌、抑菌物质的损伤,如溶菌酶、补体等。④抗干燥作用:荚膜多糖为高度水合分子,含水量在95%以上,可帮助细菌抵抗干燥对生存的威胁。因此,荚膜是导致细菌致病性增强的重要因素。
1,3-丙二醇(简称PDO)是一种重要的化工原料,可作为有机溶剂应用于油墨、印染、涂料、润滑剂、抗冻剂等行业。1,3-丙二醇最主要的用途是作为聚酯和聚氨酯合成的单体,特别是与对苯二甲酸聚合生成的聚对苯二甲酸丙二酯(PTT),显示了比以1,2-丙二醇、丁二醇、乙二醇为单体合成的聚合物更优良的性能。目前全球每年消费数千万吨聚对苯二甲酸乙二酯(PET),而PTT的化学稳定性、生物可降解性等与PET相当,但耐污染性、韧性和回弹性及抗紫外性能等更优越。此外PTT纤维还具有耐磨、吸水性低、低静电等优点,可在地毯领域与尼龙竞争。它还可用于具有优良性能的无纺布、工程塑料、服装、家庭装饰、垫衬料、织物等方面。PTT被评为美国98年六大石化新产品之一,被认为将是PET的升级产品。
PTT的优越性能及市场潜力早在50年前就被人们所认识,只因原料1,3-丙二醇生产技术难度大、成本高而导致PTT很难大规模工业化生产,迄今为止,只有Dupont和Shell两家跨国公司采用传统的化学合成路线,以环氧乙烷或丙烯为原料生产仅供它们合成PTT自用的1,3-丙二醇。化学合成法的缺点是副产物多,选择性差,操作条件需高温高压,设备投资巨大,原料为不可再生资源,且环氧乙烷和另一路线的中间产物丙烯醛分别是易燃易爆或剧毒的危险品。由于发酵法生产1,3-丙二醇选择性高,操作条件温和,因此近年来受到特别的重视。
生物合成法生产1,3-丙二醇是利用微生物歧化甘油产生。自然界中可将甘油转化为1,3-丙二醇的微生物主要是厌氧或兼性厌氧菌,其中克雷伯氏肺炎杆菌(Klebsiellapneumoniae)、丁酸梭状芽胞杆菌(Clostridium butyricum)及弗氏柠檬酸菌(Citrobacterfreundii)具有较高的1,3-丙二醇转化率,而且对甘油和产物1,3-丙二醇具有较高的耐受力,因此具有较高的开发价值与应用前景。但其中克雷伯氏菌属的菌株大多具有荚膜,使其在工业生产应用中受到限制。有研究表明,克雷伯氏肺炎杆菌胞外毒性复合物成分:荚膜多糖(63%)、脂多糖(30%)和蛋白质(7%),因此去除荚膜有利于降低菌体致病性,同时缓解环境压力,有利于实现1,3-丙二醇的工业化生产。
另外,在对1,3-丙二醇分离提取的研究中发现,荚膜的存在使发酵液粘度变大导致过滤速度减慢,造成后提取的困难,这也是克雷伯氏菌发酵法生产1,3-丙二醇工业化的瓶颈之一。因此对克雷伯氏肺炎杆菌的荚膜基因进行敲除,可以大大简化1,3-丙二醇的分离提取步骤,降低后提取难度。
目前对克雷伯氏菌野生菌株的基因工程改造主要集中在以下几个方面:
(1)通过基因工程方法强化表达还原途径中限速酶(如甘油脱水酶,1,3-丙二醇氧化还原酶)
Zeng等[Sun JB.,Heuvel J.,Soucaille P.,Qu Y.,and Zeng A.P.Comparative GenomicAnalysis of dha Regulon and Related Genes for Anaerobic Glycerol Metabolism in Bacteria.Biotechnol.Prog.200319:263-272]构建了含有编码甘油脱水酶和1,3-丙二醇氧化还原酶基因的质粒,将其插入野生菌种中,结果证明这两个酶的活性得到了大幅度提高.但是在实际的发酵过程中,该工程菌并未产出高浓度的1,3-丙二醇。黄日波等采用一种全新的甘油脱水酶基因的克隆方法,并将其表达在大肠杆菌里,最终可得到30-35g/l 1,3-丙二醇,1,3-丙二醇对甘油得率约为40%。[黄日波等,产气荚膜梭菌甘油脱水酶基因及其1,3-丙二醇的生产方法中国专利申请号:200610019452.X]
(2)敲除无益副产物编码基因,阻断副产物代谢途径;
张延平[张延平,刘铭,曹竹安.醛脱氢酶基因敲除的K.pneumoniae重组菌的构建.中国生物工程杂志,2005,25(12):34~38]等利用同源重组技术对K.pneumoniae M5al中产乙醇途径的乙醛脱氢酶基因进行了敲除,获得两株工程菌。在厌氧条件下进行批次发酵实验,结果表明乙醇的生成浓度降低了43%~53%,1,3-丙二醇合成浓度提高了27%~42%,但1,3-丙二醇的最终浓度只有16g/L。杨光分别构建了K.pneumoniae M5al乙酸、乙醇和乳酸代谢途径缺失的基因工程菌,甘油转化率有所提高,但1,3-丙二醇终浓度和生产强度反而有所下降[杨光.1,3-丙二醇产生菌肺炎克氏杆菌的分子育种[D].北京:中国农业大学,2003]。
(3)在1,3-丙二醇生产菌中构建辅酶再生系统;
黄志华等[黄志华,张延平,曹竹安等.甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析.微生物学报,2007,47(1):64~68]从C.boidinii基因组中获取了具有还原型辅酶I(NADH2)再生功能的甲酸脱氢酶基因,构建了甲酸脱氢酶重组质粒,首次在1,3-丙二醇生产菌株K.pneumoniae中构建了NADH2再生系统,重组质粒转入K.pneumoniae后菌株1,3-丙二醇合成浓度达到78.6g/L,比出发菌株YMU2提高了12.5%。黄志华[黄志华,张延平,曹竹安.在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH2再生系统.中国生物工程杂志,2006,26(12):75~80]等将甲酸脱氢酶重组质粒转入醛脱氢酶失活菌Klebsiella pneumoniae DA 21HB后,重组菌合成1,3-丙二醇的浓度达到75.06g/L,比出发菌株DA 21HB提高了19.2%。
(4)在E.coli中构建利用葡萄糖合成1,3-丙二醇的基因工程菌
杜邦公司和Genencor国际有限公司在构建以葡萄糖为底物的生物催化剂方面申请了多项专利保护[Bulthuis B A,Gatenby A A,Haynie S L,et al.Method for theProduction of Glycerol by Recombinant Organisms[P].United States Patent:6358 716,2002-05-19.Diaz-Torres M,Dunn-Coleman N S,Chase M W,et al.Methodfor the Recombinant Production of 1,3-Propanediol[P].United States Patent:6 136 576,2000-10-24.Emptage M,Haynie S L,Laffend L A,et al.Process forthe Biological Production of 1,3-Propanediol with High Titer.United StatesPatent:6 514 733,2003-08-21.],他们以E.coli K12为出发菌株,成功构建了1株产量高、生产过程为好氧的工程菌.利用此基因工程菌株进行发酵,在补料批式发酵实验中得到1,3-丙二醇浓度135g/L,但其缺点是该菌发酵需依赖辅酶B12,因此,生产成本较高。
(5)在甘油产生菌中构建合成1,3-丙二醇的基因工程菌
Cameron等[Cameron DC,Altaras NE,Hoffman ML et.al.Metabolic Engineeringof Propanediol Pathways.Biotechnol.Prog.1998,14:116-125]在酿酒酵母(Saccharomyces cerevisia)中表达来自克雷伯氏肺炎杆菌中的这两个酶的基因。在厌氧条件下以5g/L的葡萄糖为碳源,同样添加维生素B12的培养基进行发酵,但是经过48小时培养在发酵液中没有检测到1,3-丙二醇。
目前还未见构建荚膜缺失的克雷伯氏菌用于1,3-丙二醇发酵的报道。
发明内容
本发明的目的是提供一种构建荚膜缺失的克雷伯氏菌的方法。实现了构建荚膜缺失的克雷伯氏菌用于1,3-丙二醇发酵。
在1,3-丙二醇产生菌中,采用基因敲除的方法敲除荚膜主体基因的启动子(图1)或在启动子后整合终止子中止其转录(如图2)。使得克雷伯氏菌荚膜部分或全部缺失。从而降低其发酵液粘度,降低1,3-丙二醇产物分离提取难度,同时降低了菌体的致病性,有利于克雷伯氏菌的1,3-丙二醇工业化生产应用。
其具体工艺步骤包括工艺1或工艺2:
1.敲除荚膜主体基因启动子
A.克雷伯氏肺炎杆菌等具有荚膜的克雷伯氏菌属基因组DNA的提取并纯化。
B.以纯化的基因组样品为模板,设计引物,分别进行PCR(聚合酶链式反应)扩增实验,获得启动子上游3’端同源臂(R臂)和下游5’端同源臂(L臂)部分序列,转化大肠杆菌。
C.分别筛选阳性克隆并进行酶切、回收、连接到有多克隆位点及抗性基因的载体,转化大肠杆菌。
D.筛选阳性克隆并进行酶切,回收、连接自杀性载体,构建重组质粒PMD-cps3(如图3),然后转化大肠杆菌。
E.筛选含有重组质粒的阳性克隆,并将其与野生型宿主菌株进行双亲交换实验,根据抗性标记,筛选目的菌株。
本发明所述的有多克隆位点是指具有3~30个克隆位点。
2.在荚膜基因启动子后整合终止子
A.克雷伯氏肺炎杆菌等具有荚膜的克雷伯氏菌属基因组DNA的提取并纯化。
B.以纯化的基因组DNA样品为模板,对荚膜基因启动子后的部分序列设计引物,进行PCR(聚合酶链式反应)扩增实验,转化大肠杆菌,筛选阳性克隆。
C.根据克雷伯氏菌的荚膜基因终止子序列,设计终止子,以B步骤中筛选的阳性克隆为模板,设计引物,进行PCR扩增实验,连接终止子并转化大肠杆菌。
D.筛选阳性克隆,连接自杀性载体,构建重组质粒PMD-cps4(如图4),然后转化大肠杆菌。
E.筛选含有重组载体的阳性克隆,将其与野生型宿主菌株进行双亲交换实验,使用自杀载体上面的抗性基因作为筛选标记,筛选出目的菌株。
本发明所述的用于构建基因工程菌的野生菌株包括克雷伯氏肺炎杆菌等克雷伯氏菌属具有荚膜的菌株。所构建的基因工程菌发酵底物为甘油、甘油发酵液、生物柴油副产物粗甘油或肥皂行业的副产物粗甘油。
本发明的有益效果:
所构建的基因工程菌荚膜部分或全部缺失,发酵液粘度降低,过滤速度显著提高,降低了1,3-丙二醇产物分离提取难度,也降低了提取成本;另一方面由于荚膜的缺失或部分缺失降低了菌体的致病性,有利于1,3-丙二醇的工业化生产应用。
附图说明
图1为双交换法敲除荚膜示意图。
图2为单交换法敲除荚膜示意图。
图3为单交换法克隆载体PMD-cps3的构建。
图4为双交换法克隆载体PMD-cps4的构建。
图5为构建的强终止子示意图。
图6为单交换法克隆序列示意图。
图7野生型克雷伯氏肺炎杆菌菌株HR521和改造的基因工程菌KCM1电镜照片。
图8野生型克雷伯氏肺炎杆菌菌株HR526和改造的基因工程菌KCM2电镜照片。
具体实施方式
本发明的目的是提供一种构建荚膜缺失的克雷伯氏菌的方法。在1,3-丙二醇产生菌中,采用基因敲除的方法敲除荚膜主体基因的启动子(图1)或在启动子后整合终止子中止其转录(如图2)。使得克雷伯氏菌荚膜部分或全部缺失。下面再举具体实施例对本发明予以进一步说明。
实例1:
(1)野生菌株:克雷伯氏肺炎杆菌HR521
(2)整合终止子的基因工程菌的构建
①提取克雷伯氏肺炎杆菌HR521基因组DNA并纯化。以纯化的基因组样品为模板,设计引物1:ACTCCCAATTGTGACCGAAATCC和引物2:
GAGCCACTGGTTCCAGAACTTCA,进行PCR(聚合酶链式反应)扩增实验,PCR产物CPS3经测序,结果如下:
ACTCCCAATTGTGACCGAAATCCCGTAAACTTAACGCCGCCAAAAATAATGGCCT
GACCAATTATTCATCCGCGGGTCGATAAAAATTAAGTCGTTCAGGTAGTCAGTGCG
CTGGTAGCTGTTAAGCCAGGGGCGGTAGCGTCGCTGAAGCCGCTATCTGTAGAGC
ACACGGGACGATTGTGAAACGGCTGCTTTATCGCCTGACCTGAGGTAAGCATCCC
TATTAGATGCGTATCATAATCACTAGGTATCCTTCGAAGTTGGTCAGGATAGGCTGC
TGATATTGCCTGTGCAAAAACTTTCCCTACTCTTCCATAGCCTGGACGATAAAATT
CGCCTACATTGTGAGCTGGGCAGATGTTATGAGGGGTACAAGCAGCTTAGGGTAA
ATGTACTTGCCTCGTCGGTGTTGCACAGTGAAGTCGACTGGTGCCGCGAGCGCTT
ACTATCTTGGTATTCCCCTCATTCTCATTGAGAGACGACAGCGGGGCTGAAAATG
GATCTTGTACAATGATAAAAATTGCGCGCATTGCTATGACGCTGGGTTTGCTTACC
TCCCTGGGAGCCCAGGCTTACGCGGCCGGGTTAGTGGTAAATGACAACGACTTGC
GTAACGACCTGGCCTGGCTTTCCGATCGCGGGGTCATCCATCTGAGCCTGTCGAC
CTGGCCGCTGAGCCAGGAAGAGATCGCCCGAGCACTGAAAAAGGCCAAGCCTTC
CTATTCTTCAGAGCAAGTGGTGCTGGCCCGTATCAACCAGCGACTGTCTGCCTTA
AAAGCCGATTTCCGGTTCACCGGCTACACTTCAACCGACCAGCCGGGCACTCCGC
AGGGGTTTGGTCAGACACAGCCGGCGGATAACTCGTTAGGCCTGGCGTTCAACA
ACAGCGGCGAGTGGTGGGATGTCCACCTTCAGGGCAACGTCGAAGGGGGAGAG
CGGATCAGCAACGGGTCGCGCTTCAACGCCAACGGCGCCTACGGCGCGGTGAAG
TTCTGGAACCAGTGGCTC
②把PCR产物基因片段纯化后与克隆载体(如:pMD18-T-simple vector)连接,转化大肠杆菌(如DH5α)感受态细胞,并筛选阳性克隆。
③以②步骤中筛选的载体为模板,根据设计的强终止子AGTCCGGCCATTTGGCCGGACTTTTTTTT(如图5所示)3’端部分序列设计引物3:TTGGCCGGACTTTTTTTTGCTGTACAATAGTTCCTGTTC和引物4:GAATTCGAGCCACTGGTTCCAGAACTT进行PCR扩增实验。把PCR产物基因片段纯化后与克隆载体连接(如图6所示),转化大肠杆菌感受态细胞,并筛选阳性克隆载体。
④以其为模板,根据设计的强终止子5’端部分设计引物5:GAATTCAGTCCGGCCATTTGGCCGGACTTTTTTTTG,并同时利用步骤D中引物4,进行PCR扩增实验,目标产物序列两端均为EcoRI核酸内切酶位点。把PCR产物基因片段纯化后与克隆载体连接,转化大肠杆菌感受态细胞,并筛选阳性克隆。
⑤将③步骤中筛选的载体进行EcoRI单酶切,回收、连接同样EcoRI单酶切之后得到的自杀性载体(如pGPKm或pGP704)片段,然后转入大肠杆菌(如SM10)感受态细胞,并筛选阳性克隆载体。
⑥含有重组质粒大肠杆菌与野生型宿主菌株进行双亲交换实验,使用自杀载体(如pGPKm)上面的卡那霉素抗性基因作为筛选标记,筛选出单交换法敲除ORF3基因并将一强终止子整合入基因组的工程菌株。
(3)基因工程菌特性
①电镜观察
将野生型克雷伯氏肺炎杆菌菌株HR521和改造的基因工程菌KCM1进行电镜观察。由电镜照片(图7)可以看出改造的基因工程菌KCM1菌株荚膜明显变薄。
②过滤速度及发酵液粘度比较
分别测定野生型克雷伯氏肺炎杆菌菌株HR521和改造的基因工程菌KCM1发酵液粘度和过滤速度。由表1数据可知,与野生型菌株相比较,改造的基因工程菌KCM1的发酵液粘度降低了34%,发酵液过滤速度提高了89%。
表1HR521和KCM1菌株的粘度和过滤速度比较
菌种 | HR521 | KCM1 |
粘度(厘泊) | 1.7782 | 1.3249 |
过滤速度(毫升/分钟) | 1.121 | 2.125 |
③发酵
将野生型克雷伯氏肺炎杆菌菌株HR521和改造的基因工程菌KCM1进行发酵,并比较其发酵结果。
A.培养基:
表2培养基组成
*铁溶液的配制:每升水中加入FeSO4·H2O 5.0g,37%的浓盐酸4ml。
B.发酵方式:将野生菌HR521和所构建的基因工程菌KCM1在固体培养基上培养24h,将菌种接入含有30g/L甘油的种子培养基中(250ml三角瓶,装液量100ml),培养温度37℃,摇床转速150rpm,有氧培养24h。以5%的接种量接入含初始甘油为30g/l的发酵培养基。发酵采用5L发酵罐,发酵温度37℃。发酵过程中通入0.5vvm空气,搅拌转速200rpm。
C.发酵结果:
表3野生型菌HR521和基因工程菌KCM1发酵结果比较
菌种 | 转化率(%) | 1,3-丙二醇(g/L) | 2,3-丁二醇(g/L) | 丁二酸(g/L) | 乙酸(g/L) | 乳酸(g/L) | 乙醇(g/L) |
HR521 | 45.2 | 9.73 | 4.06 | 0.27 | 0.23 | 0.09 | 1.19 |
kCM1 | 47.9 | 10.37 | 4.77 | 0.34 | 0.19 | 0.08 | 1.51 |
由表3结果可知,与野生型菌株HR521相比较,基因工程菌KCM11,3-丙二醇、2,3-丁二醇和丁二酸浓度分别提高7%、17%和26%,乙酸浓度降低了17%。
实例2:
(1)野生菌株:克雷伯氏肺炎杆菌HR526
(2)敲除荚膜基因主体启动子
①提取克雷伯氏肺炎杆菌HR526基因组DNA并纯化。以其为模板,设计引物6:GGGGTCTGGGTATTCGTG和引物7:TGCCCTGGAGGTGGACAT,进行PCR(聚合酶链式反应)扩增实验,目标产物CPS4序列包含双交换敲除的L臂、R臂以及将被置换敲除的启动子全序列CPS4。CPS4序列测序结果如下:
GGGGTCTGGGTATTCGTGAGCTGGATTACACCGGCTTCAGCGGCCATTCCGCGC
TCTCCGCGGCCTTCTGGCCTATCTTCCTGTGGCTGCTCAGCGCCCGTTTCTCCGTC
GGTCTGCGTAAAGCGGCCGTTATTACCGGCTATGTTCTGGCCGCCGTGGTGGGCT
ATTCGCGGCTGGTCATCCATGCGCATTCCGTCTCGGAGGTGATTGCCGGCCTGCT
GCTGGGCGCTGCTGGCAGCGCTTTGTTCCTGGTGTTGCAAAAACGTACCCCTGAT
CCGGAAAGCGTGAATATCTCATGGGGCGGTGTTGCATGCCTGGTGATGGTTCCG
CTTATCCTTCTACATAGCGGCAGCAAAGCGCCGACTCAGTCCCTGCTGGGACAA
ATCGCCACCGCGGTGGGGCCGCTGGATAAACCCTTTACGCGTACCGATCTCCAC
AAGCAGGCCTGGTAATCACCATTTGTTTTACAGGGGAAATTAAGTTTTCGCCGGT
TAATTAGACAAATAAGAAAATATTCTACTATATATCCGGTAATTGATAATTCATA
TTTATGAAATAATGAAGTGCTACTACATTGTTATTGCATATTTGTCTGGATATAG
CTTTGATCAGACGTTTCTTAAATGCGCTGAGACGTTACCTGATGCACATTCAATG
AATATCCAGACACTGGAAACTATTAGAAATTGTAGTTGACTATAACGCCATCAT
CTGATTTTTGCATGATGATGAGCTAAGTAAAAAGATGTTTTTTTACAGTAAGTTA
GCTGCGTGTGTGATGTTGTAGCAGTACCGGGAAGATTTCTAACCCTGGAAATCA
GTGAATAATCTTAATTGGTGACCCGCTTATTTTTTGTCAGTAGAACGACAGGCGG
CATTAATTGGCAGATGTTCGGTAACAACACTCCCAATTGTGACCGAAATCCCGT
GAACTTAACGCCGCCAAAAATAATGGCCGGACCAATTATTCATCCGCGGGTCGA
TAAAAATTAAGTCGTTCAGGTAGTCAGTGCGCTGGTAGCTGTTAAGCCAGGGGC
GGTGGCGTCGCTGAAGCCGCTATCTGTAGAGCACACGGGACGATTGTGAAACGG
CTGCTTTATCGCCTGACCTGAGGTAAGCATCCCTATTAGATGCGTATCATAATCA
CTAGGTATCCTTCGAAGTTGGTCAGGATAGGCTGCTGATATTGCTTGTGCAAAA
ACTTTCCCTACTCTTCCATAGCCTGGACGATAAAATTCGCCTACATTGTGAGCTG
GGCAGATGTTATGAGGGGTACAAGCAGCTTAGGGTAAATGTACTTGCCTCGTCG
GTGTTGCACAGTGAAGTCGACTGGTGCCGCGAGCGCTTACTATCTTGGTATTCCC
CTCATTCTCATTGAGAGACGACAGCGGGGCTGAAAATGGATCTTGTACAATGAT
AAAAATTGCGCGCATTGCTATGACGCTGGGTTTGCTTACCTCCCTGGGAGCCCA
GGCTTACGCGGCCGGGTTAGTGGTAAATGACAACGACTTGCGCAGCGACCTGGC
CTGGCTTTCCGATCGCGGGGTCATCCATCTGAGCCTGTCGACCTGGCCGCTGAGC
CAGGAAGAGATCGCCCGAGCACTGAAAAAGGCCAAGCCTTCCTATTCTTCAGAG
CAAGTGGTGCTGGCCCGTATCAACCAGCGACTGTCTGCCTTGAAAGCCGATTTC
CGGTTCACCGGCTACACTTCAACCGACCAGCCGGGCACTCCGCAGGGGTTTGGT
CAGACACAGCCGGCGGATAACTCGTTAGGCCTGGCGTTCAACAACAGCGGCGA
GTGGTGGGATGTCCACCTCCAGGGCA
并将PCR产物基因片段纯化后与带有氨苄抗性基因的克隆载体pMD18-T-simple vector连接,转化大肠杆菌DH5α。
②筛选阳性克隆并以其为模板,根据L臂序列设计引物8:
TCTAGAGGTCTGGGTATTCGTGAG和引物9:GGATCCGTCACAATTGGGAGTGTT,根据R臂设计引物10:GGATCCGAGCTGGGCAGATGTTAT和引物11:GAATTCGTGGACATCCCACCACTC,分别进行PCR扩增实验,获得目标产物L臂序列cps5和R臂序列cps6,并分别转化大肠杆菌DH5α。筛选阳性克隆,并将阳性克隆进行酶切,回收、连接到有多克隆位点的载体pMD18-T-vector片段中,转化大肠杆菌DH5α,筛选阳性克隆pMD-CPS5-CPS6。
③将pMD-CPS5-CPS6进行BamHI单酶切,连接到含有卡那霉素抗性基因的载体Puc4K上,筛选具有氨苄抗性和卡那抗性的阳性克隆PMD-cps4。将PMD-cps4进行EcoRI和XbaI双酶切,回收、连接到同样经EcoRI和XbaI双酶切处理的自杀性载体pGPCm片段,然后转入大肠杆菌SM10感受态细胞。
④筛选阳性克隆,将含有重组质粒的大肠杆菌与野生型宿主菌株进行双亲交换实验,利用重组质粒中L臂与R臂之间的抗性基因作为筛选标记,筛选出双交换法敲除荚膜基因启动子的工程菌株。
(3)基因工程菌特性
①电镜观察
将野生型克雷伯氏肺炎杆菌菌株HR526和改造的基因工程菌KCM2进行电镜观察。由电镜照片(图8)可以看出改造的基因工程菌KCM2菌株荚膜显著变薄。
②过滤速度比较
表4HR526和KCM2菌株的粘度和过滤速度比较
分别测定野生型克雷伯氏肺炎杆菌菌株HR526和改造的基因工程菌KCM2发酵液粘度和过滤速度。由表4数据可知,与野生型菌株相比较,改造的基因工程菌KCM2的发酵液粘度降低了27%,发酵液过滤速度提高了90.3%。
③发酵
将野生型克雷伯氏肺炎杆菌菌株HR526和改造的基因工程菌KCM2进行发酵,并比较其发酵结果。
A.培养基:
同实例1
B.发酵方式:同实例1
C.发酵结果:
表5野生型菌HR526和基因工程菌KCM2发酵结果比较
由表3结果可知,与野生型菌株HR526相比较,基因工程菌KCM21,3-丙二醇、丁二酸浓度分别提高11%和33%,乙酸浓度降低了34.8%。
Claims (2)
1.一种构建荚膜缺失的克雷伯氏菌的方法,其特征在于,在具有荚膜的克雷伯氏属菌株中,通过在启动子后ORF3的部分序列后整合终止子,阻断克雷伯氏菌荚膜形成,降低其发酵液粘度,降低PDO产物分离提取难度,同时降低了菌体的致病性。
2.按照权利要求1所述的方法,其特征在于,在启动子后ORF3的部分序列后整合终止子,包括以下步骤:
(1)具有荚膜的克雷伯氏菌属菌株基因组DNA的提取并纯化;
(2)以纯化的基因组DNA样品为模板,对荚膜基因启动子后的ORF3的部分序列设计引物,进行PCR扩增实验,转化大肠杆菌,筛选阳性克隆;
(3)根据克雷伯氏菌的荚膜基因终止子序列,设计终止子,以步骤(2)中筛选的阳性克隆为模板,设计引物,进行PCR扩增实验,连接终止子并转化大肠杆菌;
(4)筛选阳性克隆,连接自杀性载体,构建重组质粒PMD-cps3,然后转化大肠杆菌;
(5)筛选含有重组载体的阳性克隆,将其与野生型宿主菌株进行双亲交换实验,使用自杀载体上面的抗性基因作为筛选标记,筛选出目的菌株。
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