CN110628802A - 一种高产埃博霉素d的纤维堆囊菌及其构建方法和应用 - Google Patents
一种高产埃博霉素d的纤维堆囊菌及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种高产埃博霉素D的纤维堆囊菌及其构建方法和应用。首次公开利用TALEN技术构建epoK基因被敲除的重组纤维堆囊菌So ce M4菌株,经试验证明,运用TALEN技术敲除纤维堆囊菌So ce M4菌株中的epoK基因能显著地提高埃博霉素D的产量,同时降低埃博霉素B的产量,从而实现纤维堆囊菌中埃博霉素的定向生物合成。本发明首次公开利用TALEN技术提高纤维堆囊菌生产埃博霉素的产量,将TALEN技术应用于纤维堆囊菌生产埃博霉素D产量的提高在促进纤维堆囊菌基因工程改造技术发展的同时促进埃博霉素的生物合成,扩大埃博霉素D在抗肿瘤方面的应用,对于我国生物医药发展有十分重要的意义。
Description
技术领域
本发明涉及生物化学和分子生物学领域,具体涉及一种高产埃博霉素D的纤维堆囊菌及其构建方法和应用。
背景技术
埃博霉素是一种具有光谱高效抗肿瘤活性的十六元大环内酯类化合物。由于其良好的水溶性、更高效的抗肿瘤活性和更低的毒副作用,是有望取代紫杉醇的抗肿瘤药物。目前埃博霉素B衍生物伊沙匹隆已经用于临床治疗晚期乳腺癌,而埃博霉素D也已经用于临床III期试验治疗乳腺癌。但埃博霉素较低的产量限制了其广泛应用。埃博霉素的细胞毒性导致其异源表达产量较低,而纤维堆囊菌遗传操作体系的匮乏限制了其代谢工程改造。TALE是来自于植物致病细菌黄色单胞杆菌属的一类具有高度特异性的DNA结合蛋白(SanjanaNE,Cong L,Zhou Y,Cunniff MM,Feng G,Zhang F.A TAL effector toolbox for genomeengineering.Nature Protocols,2012,7:171-192.),其特异性是由其重复单元中的重复可变区(Repeat Variable Diresidue,RVD)决定的。TALE元件主要包括TALEN元件和TALE-TF元件,TALEN元件由于基因敲除效率高、几乎无脱靶作用等优点已经在哺乳动物细胞、干细胞和植物等真核细胞的基因组编辑方面有着十分广泛的应用,而由于相应载体的缺乏,TALEN在纤维堆囊菌中的应用较少,尚未见到利用TALEN技术提高纤维堆囊菌埃博霉素产量的报道。而埃博霉素D已经被证实具有治疗乳腺癌的巨大潜力。因此,将TALEN技术应用于纤维堆囊菌埃博霉素D产量的提高有助于促进纤维堆囊菌埃博霉素生物合成代谢工程改造,降低埃博霉素D的生产成本,促进其在抗肿瘤方面的应用。
发明内容
本发明的目的是提供一种高产埃博霉素D的纤维堆囊菌及其构建方法和应用。
本发明采取的技术方案如下:
一种高产埃博霉素D的纤维堆囊菌的构建方法,包括以下步骤:
从靶向epoK基因中选取TALEN的左臂序列和右臂序列作为靶标序列,根据TALEN左右臂靶标序列设计TAL重复单元,分别得到靶向epoK基因左臂的TALE重复单元和右臂的TALE重复单元,将靶向epoK基因左臂的TALE重复单元和右臂的TALE重复单元分别插入至Ptalen L48和Ptalen R36载体,并测序验证,得到重组TALEN-Ptalen L48载体和重组TALEN-Ptalen R36载体;将重组TALEN-Ptalen L48载体和重组TALEN-Ptalen R36载体进行酶切,用适用于纤维堆囊菌的P43启动子替换pgpd启动子,构建重组TALEN骨架载体epoK-TALEN-L48-P43和epoK-TALEN-R36-P43,将epoK-TALEN-L48-P43和epoK-TALEN-R36-P43同时采用电转化导入至纤维堆囊菌感受态细胞中,筛选阳性克隆,得到epoK基因被敲除的重组纤维堆囊菌菌株。
所述的纤维堆囊菌为纤维堆囊菌So ce M4、纤维堆囊菌So ce 90、纤维堆囊菌So0157-2或纤维堆囊菌ATCC 15384。
优选,所述的纤维堆囊菌为纤维堆囊菌So ce M4。
所述的TALEN的左臂序列的核苷酸序列为5'-GAGACGAAGCCTGCTTT-3';右臂序列的核苷酸序列为5'-CCTCCGCGTACCCAGGCG-3'。
本发明还请求保护由上述的高产埃博霉素D的纤维堆囊菌的构建方法构建得到的重组纤维堆囊菌菌株。以及所述的重组纤维堆囊菌菌株在生产埃博霉素D中的应用。
本发明还提供一种利用TALEN技术提高纤维堆囊菌生产埃博霉素D产量的方法,包括以下步骤:将上述得到的重组纤维堆囊菌菌株在G52培养基中发酵生产得到埃博霉素D。优选,所述的纤维堆囊菌为纤维堆囊菌So ce M4。
与现有技术相比,本发明具有以下有益效果:
目前,埃博霉素D已经被证实具有广谱高效的抗肿瘤活性,目前处于晚期乳腺癌卵巢癌治疗临床试验III期阶段。由于埃博霉素D结构复杂,人工合成成本较高,而异源表达由于其对宿主的毒性导致产量较低。因此需要通过对纤维堆囊菌进行代谢工程改造,以提高埃博霉D的产量。本发明首次公开利用TALEN技术提高纤维堆囊菌生产埃博霉素的产量,将TALEN技术应用于纤维堆囊菌生产埃博霉素D产量的提高在促进纤维堆囊菌基因工程改造技术发展的同时促进埃博霉素的生物合成,扩大埃博霉素D在抗肿瘤方面的应用,对于我国生物医药发展有十分重要的意义。
附图说明
图1为靶向epoK基因重组TALEN载体(重组TALEN-Ptalen L48载体和重组TALEN-Ptalen R36载体)构建测序验证图。
图2为重组TALEN骨架载体(epoK-TALEN-L48-P43和epoK-TALEN-R36-P43)的构建。其中,图A为epoK-TALEN-L48质粒图谱,图B为epoK-TALEN-R36质粒图谱,图C为P43启动子验证图,泳道1为DNA marker,2-5分别为epoK-TALEN-L48替换P43启动子扩大培养菌液为模板获得的PCR产物,6-10分别为epoK-TALEN-R36替换P43启动子扩大培养菌液为模板获得的PCR产物。
图3为重组TALEN骨架载体(epoK-TALEN-L48-P43和epoK-TALEN-R36-P43)导入纤维堆囊菌So ce M4的鉴定图。其中M:marker,泳道1-2为epoK-TALEN-L48-P43导入纤维堆囊菌So ce M4鉴定图;泳道3-4为epoK-TALEN-R36-P43导入纤维堆囊菌So ce M4鉴定图。
图4为重组纤维堆囊菌So ce M4中epoK基因成功敲除的验证图。其中M:marker,阴:阴性对照,1#、2#、3#为重组纤维堆囊菌So ce M4(△epoK So ce M4),野生为野生纤维堆囊菌So ce M4(野生So ce M4)。
图5为野生型纤维堆囊菌So ce M4(野生So ce M4)和重组纤维堆囊菌So ce M4(△epoK So ce M4)中埃博霉素产量分析对比图。其中图5A为野生So ce M4中埃博霉素D的检测色谱图,图5B为野生So ce M4中埃博霉素B的检测色谱图,图5C为△epoK So ce M4中埃博霉素D的检测色谱图,图5D为△epoK So ce M4中埃博霉素B的检测色谱图,图5E为野生So ce M4和△epoK So ce M4中埃博霉素D和埃博霉素B的产量对比图。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1
纤维堆囊菌So ce M4中epoK基因的扩增及TALEN载体的构建:
根据已知的纤维堆囊菌epoK基因设计引物,对纤维堆囊菌So ce M4的epoK基因进行了扩增。从扩增得到的epoK基因中选取TALEN的左臂靶标序列(5'-GAGACGAAGCCTGCTTT-3')和右臂靶标序列(5'-CCTCCGCGTACCCAGGCG-3')作为靶标序列。采用Fast TALENassembly kit(上海斯丹赛生物工程有限公司,货号:2801-005),根据TALEN的左右臂靶标碱基序列设计TAL重复单元,分别得到靶向epoK基因左臂的TALE重复单元和右臂的TALE重复单元,将靶向epoK基因左臂的TALE重复单元连接至左臂载体PtalenL48,得到重组TALEN-Ptalen L48载体,将靶向epoK基因右臂的TALE重复单元连接至右臂载体Ptalen R36,得到重组TALEN-Ptalen R36载体,并进行了测序验证,氨基酸测序结果证实了针对靶向epoK基因的重组TALEN-Ptalen L48载体和重组TALEN-Ptalen R36载体的成功构建(图1)。
将构建成功的重组TALEN-Ptalen L48载体用HindIII、SpeI进行酶切,重组TALEN-Ptalen R36载体用AscI、SpeI进行酶切,以去除pgpd启动子,然后将适用于纤维堆囊菌的P43启动子用相应的限制性内切酶进行酶切,分别插入至酶切后的TALEN-PtalenL48和TALEN-PtalenR36载体,以使该载体适用于纤维堆囊菌,构建重组TALEN骨架载体(epoK-Ptalen-L48-P43和epoK-Ptalen-R36-P43),并通过菌液PCR和测序验证P43启动子成功插入PtalenL48和PtalenR36载体(图2),得到epoK-TALEN-L48-P43和epoK-TALEN-R36-P43。
实施例2
纤维堆囊菌So ce M4菌株中重组TALEN载体的导入和epoK基因的敲除:
将含有靶向左臂靶标序列和右臂靶标序列的重组TALEN骨架载体(epoK-TALEN-L48-P43和epoK-TALEN-R36-P43)同时采用电转化导入至纤维堆囊菌So ceM4感受态细胞中,用含有卡那霉素的G52平板进行筛选。用扩增ColE1复制子的方式验证重组TALEN载体成功导入至纤维堆囊菌So ce M4中。以扩大培养的菌液为模板,采用如SEQ ID NO.1和SEQ IDNO.2所示的核苷酸序列(F:5'-TGAGATCCTTTTTTTCTGCG-3',R:5'-TTTCCATAGGCTCCGCCCCC-3')为引物进行PCR扩增,菌液PCR扩增成功获得了ColE1复制子,证实了重组TALEN骨架载体(epoK-TALEN-L48-P43和epoK-TALEN-R36-P43)的导入(图3)。
在epoK靶向敲除基因两端各设计25bp左右的引物,提取野生纤维堆囊菌So ce M4(野生So ce M4)和重组纤维堆囊菌So ce M4(重组So ce M4)的RNA进行逆转录获得cDNA,以获得的cDNA为模板,使用epoK靶标序列附近两侧序列(SEQ ID NO.3和SEQ ID NO.4)为引物(F:5'-TCAGAGTGAGACGAAGCCTGCTTT-3',R:F:5'-AACGGGTCCTCCGCGTACCCAGGC-3')对靶向敲除片段进行扩增,并以不加基因组模板为阴性对照。结果表明,以野生So ce M4菌株cDNA为模板,可以扩增得到70bp左右的目的片段,而以重组So ce M4菌株的cDNA为模板,则不能扩增得到任何片段。结合测序结果验证了epoK基因的成功敲除(图4),由此得到epoK基因被敲除的重组So ce M4菌株(△epoK So ce M4)。
实施例3
野生So ce M4和重组So ce M4菌株(△epoK So ce M4)中埃博霉素产量分析:
将野生So ce M4和△epoK So ce M4菌株进行发酵,加入大孔吸附树脂,用甲醇对大孔吸附树脂上的产物进行洗脱。将所得产物分别上样至HPLC-MS,对比分析产物中埃博霉素B和埃博霉素D的产量。结果表明,野生So ce M4菌株中埃博霉素D的产量为4.35±0.16mg/L,△epoK So ce M4菌株中埃博霉素D的产量为5.82±0.25mg/L。而野生So ce M4菌株中埃博霉素B的产量为7.64±1.18mg/L,△epoK So ce M4菌株中埃博霉素B的产量为5.06±0.16mg/L(图5)。以上结果说明运用TALEN技术敲除纤维堆囊菌So ce M4菌株中的epoK基因能显著地提高埃博霉素D的产量,同时降低埃博霉素B的产量,从而实现纤维堆囊菌中埃博霉素的定向生物合成。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东省微生物研究所(广东省微生物分析检测中心)
<120> 一种高产埃博霉素D的纤维堆囊菌及其构建方法和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tgagatcctt tttttctgcg 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tttccatagg ctccgccccc 20
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcagagtgag acgaagcctg cttt 24
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aacgggtcct ccgcgtaccc aggc 24
Claims (9)
1.一种高产埃博霉素D的纤维堆囊菌的构建方法,其特征在于,包括以下步骤:
从靶向epoK基因中选取TALEN的左臂序列和右臂序列作为靶标序列,根据TALEN左右臂靶标序列设计TAL重复单元,分别得到靶向epoK基因左臂的TALE重复单元和右臂的TALE重复单元,将靶向epoK基因左臂的TALE重复单元和右臂的TALE重复单元分别插入至PtalenL48和Ptalen R36载体,并测序验证,得到重组TALEN-Ptalen L48载体和重组TALEN-PtalenR36载体;将重组TALEN-Ptalen L48载体和重组TALEN-Ptalen R36载体进行酶切,用适用于纤维堆囊菌的P43启动子替换pgpd启动子,构建重组TALEN骨架载体epoK-TALEN-L48-P43和epoK-TALEN-R36-P43,将epoK-TALEN-L48-P43和epoK-TALEN-R36-P43同时采用电转化导入至纤维堆囊菌感受态细胞中,筛选阳性克隆,得到epoK基因被敲除的重组纤维堆囊菌菌株。
2.根据权利要求1所述的高产埃博霉素D的纤维堆囊菌的构建方法,其特征在于,所述的纤维堆囊菌为纤维堆囊菌So ce M4、纤维堆囊菌So ce 90、纤维堆囊菌So0157-2或纤维堆囊菌ATCC 15384。
3.根据权利要求2所述的高产埃博霉素D的纤维堆囊菌的构建方法,其特征在于,所述的纤维堆囊菌为纤维堆囊菌So ce M4。
4.根据权利要求1所述的高产埃博霉素D的纤维堆囊菌的构建方法,其特征在于,所述的TALEN的左臂序列的核苷酸序列为5'-GAGACGAAGCCTGCTTT-3'。
5.根据权利要求1所述的高产埃博霉素D的纤维堆囊菌的构建方法,其特征在于,所述的TALEN的右臂序列的核苷酸序列为5'-CCTCCGCGTACCCAGGCG-3'。
6.一种按照权利要求1-5任一所述的高产埃博霉素D的纤维堆囊菌的构建方法构建得到的重组纤维堆囊菌菌株。
7.权利要求6所述的重组纤维堆囊菌菌株在生产埃博霉素D中的应用。
8.一种利用TALEN技术提高纤维堆囊菌生产埃博霉素D产量的方法,其特征在于,包括以下步骤:将权利要求1得到的重组纤维堆囊菌菌株在G52培养基中发酵生产得到埃博霉素D。
9.根据权利要求8所述的利用TALEN技术提高纤维堆囊菌生产埃博霉素D产量的方法,所述的纤维堆囊菌为纤维堆囊菌So ce M4。
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