CN114657155B - 一种新型的非核糖体多肽合成酶拆分表达方法 - Google Patents
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Abstract
本发明涉及生物技术领域,公开了一种非核糖体多肽合成酶拆分表达方法,是根据对NRPS结构域和三维结构的预测,在特殊拆分位点以Tn‑1‑Cn‑An‑Tn为单元对NRPS进行拆分表达,从而将一个NRPS基因的表达转换为以拆分单元为独立基因的多基因的共表达,以高效获得相应的非核糖体多肽产物。本发明不仅可用于巨型NRPS在真菌中的异源表达,提高非核糖体多肽产量,而且为通过组合生物合成的方法进行模块间组合,合成新型的非核糖体多肽提供理论依据和途径。对于利用合成生物学进行天然非核糖体多肽的挖掘、产量的提高以及非天然非核糖体多肽的创造等方面具有较高的实际应用价值。
Description
技术领域
本发明属于生物技术领域,涉及一种新型的非核糖体多肽合成酶拆分表达技术。
背景技术
非核糖体多肽类天然产物是现代药物创制的重要来源,如抗真菌药物棘白菌素、免疫抑制剂类药物环孢霉素等。然而,目前利用传统的筛选技术获得具有实用价值的新型非核糖体多肽的难度越来越大。随着高通量基因组测序技术、基因合成以及多基因装配等合成生物学技术的快速发展,为通过异源生物合成技术挖掘和生产有价值的非核糖体多肽提供了途径。
非核糖体多肽类化合物的核心骨架是由模块化的非核糖体多肽合成酶(nonribosomal peptide synthetase,NRPS)组装而成的。通常,NRPS是由多个模块构成的,每个模块包含三个核心功能结构域:腺苷化(adenylation,A)结构域、硫酯化(thiolation,T)结构域和缩合(condensation,C)结构域。此外,部分NRPS模块还会包含甲基化(methylation,MT)结构域、氧化(oxidation)结构域、杂环化(heterocyclization,Cy)结构域、异构化(epimerization,E)结构域等修饰结构域。在NRPS的C末端通常是负责终产物释放的硫酯酶(thioesterase,TE)结构域或末端缩合(terminal condensation,CT)结构域。绝大多数NRPS遵循共线性装配规则,即NRPS模块的数目、种类和排列顺序与其产物中氨基酸组成单元的数目、种类和排列顺序是一致的。NRPS按照固定的逻辑逐个行使各个模块的催化功能将氨基酸单体顺序装配成特定结构的非核糖体多肽。一般来说,非核糖体多肽结构越复杂,所需要的NRPS就越大。例如,棘白菌素B含有6个氨基酸单元,其NRPS EcdA则含有6个模块,并在C末端有一个CT结构域进行肽链的缩合环化和产物的释放,因此EcdA是由7260个氨基酸组成,其编码基因达21kb。含有11个氨基酸单元的环孢霉素A,其NRPS SimA由15228个氨基酸组成,编码基因高达45kb。我们对已测序的39个虫生真菌的基因组进行了次级代谢基因簇分析,共获得4个以上模块的NRPS基因112个,其中60%的次级代谢产物是未知的。然而,含有4个以上模块的NRPS也意味着他们的编码基因均在15kb以上。这样巨型的基因是很难扩增并通过常规的分子生物学方法进行异源表达的。
根据NRPS模块化的这种结构特点,研究者尝试以C-A、C-A-T、T-C-A或A-T-C等为交换单元进行非核糖体多肽装配线改造,在细菌NRPS中取得了成功。对于真菌NRPS结构域交换的研究则主要围绕环缩肽白僵菌素、类白僵菌素和恩镰孢菌素等的NRPS来开展。通过同源C-A-T以及C3等结构域的替换,形成的融合NRPS产生了多种新型的环缩肽类化合物。这些研究使我们看到了对巨型NRPS进行拆分表达的可能。
本发明以白僵菌素合成酶BbBEAS为模式NRPS,以结构域为节点对其进行了拆分与组合表达,通过比较其产物产量,获得了一种新型高效的双T式NRPS拆分表达技术。该发明不仅实现了巨型NRPS的拆分表达,提高了化合物产率,而且为通过组合生物合成的方法进行模块间组合,合成新型的非核糖体多肽提供了理论依据和途径。
发明内容
本发明的目的是提供一种新型的非核糖体多肽合成酶拆分表达方法。其是根据对NRPS结构域和三维结构的预测,在特殊拆分位点以Tn-1-Cn-An-Tn为单元对NRPS进行拆分,从而将一个NRPS基因的表达转换为以拆分单元为独立基因的多基因的共表达,以高效获得相应的非核糖体多肽产物。
本发明提供的技术方案是:
一种NRPS拆分单元,其序列为Tn-1-Cn-An-Tn,其中T为硫酯化结构域、A为腺苷化结构域、C为缩合结构域,n为NRPS中模块的序号。
一种非核糖体多肽合成酶拆分表达方法,通过双T式NRPS拆分方式将一个NRPS基因的表达转换为以拆分单元为独立基因的多基因的共表达,以高效获得相应的非核糖体多肽产物。其中所述NRPS拆分单元具有下述序列:
Tn-1-Cn-An-Tn。若为起始模块,则从起始密码子至T1为起始拆分单元;若为最后一个模块,则从前一个模块的T至终止密码子为终止拆分单元;若模块中含有甲基化(methylation,MT)结构域、杂环化(heterocyclization,Cy)结构域、异构化(epimerization,E)结构域等修饰结构域,则拆分单元中也添加相应修饰结构域。拆分单元中结构域组成应与相应模块中结构域组成一一对应。
所述的拆分表达技术适合于NRPS在真菌中的异源表达,真菌具体为酵母、丝状真菌,如酿酒酵母。
所述拆分位点是指Tn-1-Cn-An-Tn的N端起始于An-1sub与Tn-1之间的linker区,C端终止于Cn+1的1~10氨基酸。
本发明还提供一种所述拆分表达NRPS的重组菌。
本发明还提供一种拆分单元在异源表达中的应用,所述拆分单元具有下述序列:Tn-1-Cn-An-Tn。若为起始模块,则从起始密码子至T1为起始拆分单元;若为最后一个模块,则从前一个模块的T至终止密码子为终止拆分单元;若模块中含有甲基化(methylation,MT)结构域、杂环化(heterocyclization,Cy)结构域、异构化(epimerization,E)结构域等修饰结构域,则拆分单元中也添加相应修饰结构域。拆分单元中结构域组成应与相应模块中结构域组成一一对应。
上述的应用,所述异源表达宿主是真菌,具体为酵母、丝状真菌,如酿酒酵母。
具体而言,本发明的实施方案如下。
1)NRPS拆分单元的确定。Tn-1-Cn-An-Tn。若为起始模块,则从起始密码子至T1为起始拆分单元;若模块中含有MT、Cy、E等修饰结构域,则拆分单元中也添加相应修饰结构域。
2)NRPS拆分位点的确定。Tn-1-Cn-An-Tn的N端起始于An-1sub与Tn-1之间的linker区,C端终止于Cn+1的1~10氨基酸。
3)根据目标NRPS的基因序列与拆分位点设计引物,并在引物末端添加相应的起始密码子、终止密码子以及载体同源片段。通过PCR对拆分单元进行扩增,进一步通过无缝克隆进行连接与克隆,获得拆分单元的表达载体。
4)通过转化将获得的表达载体转入宿主细胞中。
5)发酵检测阳性转化子中相应非核糖体多肽的产量。
本发明的NRPS拆分表达技术不仅可用于巨型NRPS在真菌中的异源表达,提高非核糖体多肽产量,而且为通过组合生物合成的方法进行模块间组合,合成新型的非核糖体多肽提供理论依据和途径。对于利用合成生物学进行天然非核糖体多肽的挖掘、产量的提高以及非天然非核糖体多肽的创造等方面具有较高的实际应用价值。
附图说明
图1非核糖体多肽合成酶拆分表达技术示意图。
图2白僵菌素合成酶BbBEAS结构域示意图。
图3白僵菌素合成酶BbBEAS单模块拆分表达对白僵菌素产量的影响。
图4白僵菌素合成酶BbBEAS双T式拆分的位点对白僵菌素产量的影响。其中,(A)T1-C2-A2-T2a-T2b-C3拆分单元中T1的N端起始位点的选择。粉色为未拆分BbBEAS,绿色代表起始位点在A1sub区,蓝色代表起始位点在A1sub与T1的linker区,黄色代表起始位点在T1区。(B)C1-A1-T1拆分单元中T1的C端终止位点的选择。粉色为未拆分BbBEAS,黄色和蓝色代表终止位点在C2区,绿色代表终止位点在T1与C2的linker区,紫色代表终止位点在T1区。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
表达载体:YEpADH2p-TRP、YEpADH2p-URA。(Xie L,Zhang L,Wang C,Wang X,XuYM,Yu H,Wu P,Li S,Han L,Gunatilaka AAL,Wei X,Lin M,Molnar I,XuY.2018.Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis.Proc Natl Acad Sci USA 115:E4980-E4989.)
酿酒酵母BJ5464:MATαura3-52his3-Δ200leu2-Δ1trp1pep4::HIS3prb1Δ1.6Rcan1GAL。美国模式培养物集存库(ATCC,网址为www.atcc.org/),ATCC编号为208288。
酶与试剂盒:
高保真DNA扩增MIX、无缝克隆试剂盒购自诺唯赞公司;
DNA loading buffer和DNA marker购自康为世纪公司;
DNA纯化胶回收试剂盒购自康宁公司;
冷冻酵母转化试剂盒购自ZYMO RESEARCH生物公司;
大肠杆菌DH5α感受态购自康为世纪公司;
LC-MS检测用试剂为色谱纯,其它试剂均为国产分析纯产品。
培养基:
大肠杆菌培养基为LB培养基(1%蛋白胨、0.5%酵母提取物、1%NaCl,pH7.0)。SC--Trp/-ura缺陷培养基(1%葡萄糖、6.7%DifcoTM Yeast Nitrogen Base w/o AminoAcids、-Trp/-ura DO Supplement)。YPD低糖培养基(1%酵母膏、2%Peptone蛋白胨、1%葡萄糖)。若制固体培养基,加入2%琼脂粉。
实施例1、NRPS拆分单元的确定
以营养缺陷型酿酒酵母BJ5464-NpgA为底盘细胞,以本研究团队前期报道的白僵菌素NRPS BbBEAS(氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.2所示)为例(Xu YQ,Orozco R,Wijeratne EMK,Gunatilaka AAL,Stock SP,MolnarI.2008.Biosynthesis of the cyclooligomer depsipeptide beauvericin,a virulencefactor of the entomopathogenic fungus Beauveria bassiana.Chem Biol15(9):898-907.),以结构域为节点对BbBEAS进行2段式拆分表达,通过LC-MS检测转化子中白僵菌素的产量,筛选高效的拆分单元。
实验方法
(1)BbBeas及其拆分片段异源表达盒的构建
根据BbBeas及其结构域的序列信息,其氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.2所示。其中,1-924bp编码C1结构域,1507-2979bp编码A1结构域,2716-2979bp编码A1sub结构域,3121-3330bp编码T1结构域,3391-4287bp编码C2结构域,4792-6012bp编码A2结构域,6286-6951bp编码MT结构域,7684-7887bp编码T2a结构域,7951-8175bp编码T2b结构域,8314-9159bp编码C3结构域。
据此设计特异性引物(请见表1),并在引物末端添加相应的起始密码子、终止密码子以及载体同源片段。按照无缝克隆试剂盒说明书进行操作,将这些片段与表达载体相连,最终获得含有BbBeas或其拆分片段异源表达盒的表达载体(请见表2)。
(2)酵母转化
按照冷冻酵母转化试剂盒说明书,将异源表达载体转入酿酒酵母中。通过PCR对获得的酵母转化子进行验证。将获得的阳性酵母转化子划线培养在新的SC--Trp/-ura缺陷培养基上,于30℃培养箱中培养2天左右。
(3)发酵培养
采取二步法发酵技术,首先将适量的酵母转化子菌体接种至10mL SC--Trp/-ura液体缺陷培养基中,30℃、220rpm培养16h左右,再加入等体积含有15mM D-Hiv和L-Phe的YPD低糖培养基,继续培养48h;用乙酸乙酯提取发酵产物,乙酸乙酯与发酵液的比例为1:1;旋转蒸发后获得干燥萃取物,1mL甲醇复溶萃取物。
(4)LC-MS检测
取1mL上述所得发酵产物经高速离心和0.22μm滤膜过滤后,用LC-MS检测。
液相色谱分析仪器为Agilent公司1290Infinity II超高效液质联用色谱仪,色谱柱为Agilent ZORBAX Eclipse Plus C18RRHD column(1.8μm,50mm x 2.1mm);流动相总流速为0.35mL min-1;流动相为流动相A和流动相B的混合物,流动相A为0.1%(体积比)甲酸水溶液、流动相B为0.1%(体积比)甲酸乙腈;总洗脱时间为10分钟;洗脱过程为:0~4min,流动相B占流动相的体积比由10%线性上升至50%,4~8min流动相B占流动相的体积比由50%线性上升至95%,8~9.3min流动相B占流动相的体积比为95%,9.3~10min流动相B占流动相的体积比由95%线性下降至10%;柱温40℃,样品的进样量为5μL,检测波长为210nm。柱后流出液不经分流直接进入质谱检测。质谱分析仪器为Agilent G6125B单四极杆质谱,质谱检测条件为正离子模式,扫描分子量范围为300-1000m/z。
实验结果及分析:
通过LC-MS检测发现,不同拆分单元会影响白僵菌素的产量(请见表2,图3)。在T2a和T2b之间进行拆分(即SPU7),白僵菌素产量可与拆分前持平,说明T2a和T2b之间是最好的拆分位置,但T2a-T2b在NRPS中并不是普遍存在的,因此T2a和T2b之间不能作为通用的拆分位置,但2个T结构域可能有利于白僵菌素的合成;在T1与C2之间拆分(即SPU3),白僵菌素产量达到拆分前的28%;在T2b与C3之间拆分(即SPU8),白僵菌素产量达到拆分前的83%,可见T与C之间为较好的拆分位置;然而在C与A之间拆分(如SPU1和SPU4),白僵菌素产量极低,表明C与A之间不适合进行拆分。因此,我们推测在T1与C2、A1与T1之间进行拆分,构建C1-A1-T1和T1-C2-A2-T2a-T2b-C3两个异源表达盒可能会提高白僵菌素的产量。
实施例2、NRPS拆分位点的确定
以营养缺陷型酿酒酵母BJ5464-NpgA为底盘细胞,在不同的拆分位点将BbBEAS拆分成C1-A1-T1和T1-C2-A2-T2a-T2b-C3两个拆分单元,通过LC-MS检测转化子中白僵菌素的产量,筛选高效的拆分位点。
实验方法:
(1)C1-A1-T1和T1-C2-A2-T2a-T2b-C3异源表达盒的构建。
根据BbBeas及其结构域的序列信息(请见序列表),设计特异性引物(请见表1),并在引物末端添加相应的起始密码子、终止密码子以及载体同源片段。按照无缝克隆试剂盒说明书进行操作,将这些片段与表达载体相连,最终获得含有BbBeas或其拆分片段异源表达盒的表达载体(请见表2)。
(2)酵母转化:同实施例1中酵母转化步骤。
(3)发酵培养:同实施例1中发酵培养步骤。
(4)LC-MS检测:同实施例1中LC-MS检测步骤。
实验结果及分析:
通过LC-MS检测发现,不同拆分位点会显著影响白僵菌素的产量(请见表2,图4)。在T1-C2-A2-T2a-T2b-C3的16种T1的N端起始位点中(即SPU3-X系列菌株),多数位点的白僵菌素产量能够达到拆分前的50%以上。起始位点位于A1sub与T1之间linker区时(即SPU3-0、5、7、8、11-13、15、16、19-21),白僵菌素的产量普遍高于起始位点在A1sub区或T1时的产量,其中SPU3-12和SPU3-19中白僵菌素的产量能够分别达到拆分前的130%和101%。我们还在此基础上检测了C1-A1-T1的3种T1的C端终止位点(即SPU3-12-X系列菌株),发现终止位点位于C2时(即SPU3-12),白僵菌素的产量显著高于终止位点在T1与C2之间linker区(即SPU3-12-1)或T1(即SPU3-12-2)时的产量。因此,在对BbBEAS进行双T式拆分表达时,拆分位点的选择对于白僵菌素的产量是至关重要的。根据上述实验中高效拆分位点所处位置,双T式拆分表达NRPS的拆分单元为Tn-1-Cn-An-Tn。其中Tn-1-Cn-An-Tn的N端起始于An-1sub与Tn-1之间的linker区,C端终止于Cn+1的1~10氨基酸。
表1本发明中用到的引物列表
表2本专利中构建的质粒与重组菌列表
<110> 中国农业科学院生物技术研究所
<120> 一种新型的非核糖体多肽合成酶拆分表达方法
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atggagccgctcaaaaatgtcaatactgggcagccatgctcaactgttccgtttccagtgtctgacgagacagttgagcatttgaatggtctatatgaggaaatcaaccgccgttttggcttggacagggatgccattgagactatcctcccatgtacacccttccagtatgatgtgcttgattgcgctgccaatgatgcaagacacgccgtcggtcatgccatgtacgaaatatcgcaacatgttcatgtccaacgcttcatcgctgcttggagagagactgtgcggcgcactccagccttgcgcgcctgcacctttacatcaacgaccggggagtcgtttcagctggtactgagagagagctttgtgctttcgcgcatatactggtcttcttcttctagcttacaggcagctgttttgaaggatgagacgacggcggccattgctgggccgcgttgcaatcgacttgtccttcttgaagacccagatacaaggaaacaactgctgatttgggtatttcatcttgcactcgtggacagcaccgttcaggaacccattctccggcgggttctggcggcgtacaagagtgaagacgaccagctagacagccttccgctcacaccagactcttctggaggttccgactcggactctcccagcacgctcaagatgcctcgggcttttgaccaggaaaaagccacgcaattttggcagcgccagttgagcggtctcgatgcttccgtctttcctcctctgtcttctcacctgactacgcccaaggccgatgccaagattgagcactacatatcatggccggcctctgctgcccagcacaggtggtcatccactactgtgtgtcaagctgctcttgcggttctcctgtctcgctacagtcactcttccgaggcgctctttggcgtcgtgaccgagcaagtgtgcatgtttgagggacagcgacttctcattaacggtccgacccgctcagtggtgccttttcgcgtacattgtggcccggaacagtctgtcacggacctcttgaaaagcattgcttccgacaatcacgacatgcgccagtttgcgcatgtcggcctttgcaacataagccgcatcggagatgatcagtctgccgcttgcaggtttcagactgtgctgagcgtcagcaatcgccgctcctccgaagacgctgcctcgggcgaggttctccagattctgcaagagtcagagggctttgcgccttgtgcagaccgtgctcttttgctgcgttgtgagacgagccgtcaaggagcgctgttggtcgcaagatatgaccaaggcgtgattgagccccctcagatggctcgctttctgagacagctgggatggttgatggagcagttgcagagtgcggcagacgatgctctttcggtgaaacaactagacattgtgactcgcgaggaccgcgcagagattgacagttggaattctgatgccttggaggtacaggaaagcctgcttcacagtgcgtttgtgaaacgggctgctgagtcccctagcgatcctgcagtactctcttgggatggagcatggacttattccgaactggataatgtctcctcgcgactggctgcacacatcagatctctcgacttgagtcacgagcagttgatagtgcccgtctactttgaaaaatcgaaatgggttgttgcctcgatattggcggtgctcaaggccggtcacgcctttacactcatcgaccccaaagatccgccagcccggacaactcggatagtccagcagacatccgccaaggttgccctcacgtccaagctccatcaagacaccgtgcaggccatcattggtcgctgcattgtggtcgacgatgatttcgttcagtcgctaggctcagcgagtcaatgtcaagaaaagtccgagttgacagtaaagccgcacaatctagcctacgccatcttcacctcggggagtacaggagaccccaagggcatcatgattgagcatcaagcgtttgcttcttgtgttgccaaattcggccccgcgctcatccctcataatgcgcgtgctcttcaattcgcatcccacgggttcggtgcctgtctgctggagattttgcctactctactacgcggtggctgcgtctgcattccctccgaccttgaccgcatgcacaacatccccgactttattaggcggtataacgttaattggatgatggctacaccgtcgtatatgacgacttttaaaccagaagatgttccgggactccagactctcatcctggttggagaacaaatgtcagcatctgtcaacgccacctgggcctctcgacttggactctttgacggctacggccaaagcgaaagctgctccatctgtttcattggcaagattagtccagtttccgaggcaaacaacattggccgagcagtaggcgcacattcttggatcgtgcaccctgatgatcctgatcgtttggctcccgtcggtgccgttggcgagcttttgatagagagtcctgggatcgcgcgcggctacattgccgctccagcaacggacagaaatccctttctcgagacagctcccgcttggtatgctccgaggcagcctccaactggcgtcaagttttatagaacaggcgatctcgcgcgctacgcagctgacggtacagtggtgtgtcttggtcgtatagactcacaggtcaagatccgaggccagcgcgtggagatgggcgcagtggaaacgcgtctgcgacaacaagtaccaagcgatataaccgttgtggccgaagccgtcaagcgctcaggttcatctggcagcacggtcataacggcctttttgatagactcgtcggacaagaacaattccagtgctgcatccgctaaagatgcacgaattttggatcaaacggcaacccaagagatgaatgcaaagttgtgtcaggttcttcctccgcattccgttccgtcttgctacatctgcatgcacgctcttccacgcactgccaccggaaaggtcgaccgaaagacgttgcgctctatcggcagcaagctcctagagcaacaagcttacaagaaatctcccgagacaatgcaaaagtcaaagtcagcagagacactggaaactggtccagaagccagactcaaagaggtttggttgcagagtttcaatcttgaacctgcttctccaaagtgtggtgcaagcttctttgagctgggcggagattcaatcaccgccattaaaatggttaacatggcaagagctgctggattagagctgaaagtctctgacattttccaaaatcccacccttgctcgtcttcaggccgtgatgagcggcgattctacgccctcgaccatcacgacgccctttgccaccattccggcgtcgacttgggacggacccgttgagcagtcttactctcaaggtcgattgtggttcctcgaccagctggatattggagctgtatggtacctgataccttatgccgttcgcatgcggggagctctcaacattgacgctctacgtgctgctctgctggcgttggagcagcgacacgagaccctgcggacgacctttgagaaccaaaacggtgtaggagtgcagattgttcaccaaagacttgccaaggagctgaaaattatcgatgcgtcgtcccacggcgatgacggctacctccagccacttgagcaggagcagaccactccattcgatctgacttgtgaggcgggctggagggcatcactcatctgcgtcggcgaggaccatcatgtcttgtctattgtcatgcatcacattgtctccgatggctggtccattgacgtgctgcggcaagaactaggccagctctacgcagcggttttgcatggcgacgaggatcctctgtcggccgtgagcccgctccccatacagtatcgagacttttccatgtggcagagacgtcaacaggtcgccgagcatgacagacagcttcaatactggcggaaacagctcgcagactgctcgccggccaagctgcccaccgatttcccccgaccacccttgctgtccggcgacgctggcagcgtaccggtggagatttcgggcgagctgttccaaaagctgcacaggttctgcaacgtgaccagcacgaccccgtttgccgtgcttctggccgcgtttcgtgctgcgcattaccgactcaccggggtcgacgacgccgtcgtgggcacgcccattgccaaccggaaccggcccgagctggagcgcctgattggtttctttgtcaacacgcaatgtatgcgcatcaccgtggacgatgatgatacatttgagggcttggtacgccaagtccgtaggacaacgactgaggcttttgaaaacgaagatgtcccctttgaacgcgtcgtgtccgccatgctaccggcaggaggaggatccagagatttgtcccagacgcccctggcacagctcatctttgccgtgcactcgcaagaaaatctaggcaagtttgagctagaaggtctcgagtcggaacctgttgcgaacaaggcatatacgcgctttgacgctgaatttcacctgttccaaactcgtgacggattaaacggctacttgaactttgccgcggaattgttcaagctagagacgatgcaaaatgtcgtcagcgtcttcttacagattctacgccatggactggagcagcctaaatccttgatatccgttttgccgcttactgacgggttaaaggagctcgacagcatgggcctcttgaagattcatcgggggcttgaatatcagcgagactctagcctagtcgacatcttccgcagccaggttgctacttgtcctgatacaattgccgtcattgactcatcagcacgtctgacgtatgctcagctggaccatcagtccaacctactcgaggcctggattcgccgcaaaggcttgccggccgaatcattggttggcgtgctttcaccgcggtcctgcgagacaatcatcgcctttcttggtattctcaaagcaaa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Claims (3)
1.一种非核糖体多肽合成酶拆分表达方法,其特征在于,通过双T式NRPS拆分表达方式将一个NRPS基因的表达转换为以拆分单元为独立基因的多基因的共表达,以高效获得相应的非核糖体多肽产物,其中所述NRPS拆分单元具有下述序列:C1-A1-T1和T1-C2-A2-T2a-T2b-C3,T1-C2-A2-T2a-T2b-C3的T1的N端起始位点位于A1sub与T1之间linker区,C1-A1-T1的T1的C端终止位点位于C2;其中,若为起始模块,则从起始密码子至T1为起始拆分单元;若为最后一个模块,则从前一个模块的T至终止密码子为终止拆分单元;若模块中含有甲基化结构域、杂环化结构域、异构化结构域,则拆分单元中也添加相应修饰结构域;且拆分单元中结构域组成与相应模块中结构域组成一一对应;
其中T为硫酯化结构域、A为腺苷化结构域、C为缩合结构域,数字为NRPS中模块的序号;其中,拆分的NRPS原始序列如SEQ ID NO.1所示的氨基酸序列;
所述非核糖体多肽合成酶拆分表达方法包括如下步骤:
1) 根据目标NRPS的基因序列与拆分位点设计引物,并在引物末端添加相应的起始密码子、终止密码子以及载体同源片段;通过PCR对拆分单元进行扩增,进一步通过无缝克隆进行连接与克隆,获得拆分单元的表达载体;所述引物的序列为: 5′-TGACGACAAGCTTCATatgCGCTCTATCGGCAGCAAG-3′;
2)通过转化将获得的表达载体转入宿主细胞中;
3)发酵检测阳性转化子,用于表达所述的非核糖体多肽。
2.如权利要求1所述的拆分表达方法,其特征在于,所述拆分表达适合于NRPS在真菌中的异源表达。
3.如权利要求2所述的拆分表达方法,其特征在于,所述真菌是酿酒酵母。
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