CN114478430B - 一组芽孢噻唑类化合物及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一组芽孢噻唑类化合物,是命名为芽孢噻唑A‑N的14种化合物1‑14,其化学构式依次如式Ⅰ至式ⅩⅩ所示;该化合物是由枯草芽孢杆菌1A751‑Sfp/nrs突变菌株(Bacillus subtilis 1A751‑Sfp/nrs)经过液体发酵后,从其发酵产物中得到。本发明还公开了所述化合物在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用,实验证实本发明提供的芽孢噻唑A‑E(1‑5)对蛋白酪氨酸磷酸酶1B(PTP1B)有较强的选择性抑制活性,PTP1B已成为治疗Ⅱ型糖尿病和肥胖症的极具吸引力的靶点,本发明的化合物有望在糖尿病和肥胖症治疗方面提供新的应用途径。
Description
技术领域
本发明涉及一组芽孢噻唑类化合物,以及该芽孢噻唑类化合物的产生菌株、制备方法和其在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用。属于微生物技术及其制品与应用技术领域。
背景技术
噻唑环是天然产物中一类常见的基团,也是化合物中重要的药效官能团,因而备受关注。如批准上市的抗肿瘤药物博来霉素中的二联噻唑环被证实辅助化合物嵌入DNA。有研究发现,噻唑环的相连的数量与其活性呈正相关,化学合成的多联噻唑特别是三联噻唑被证明有良好的活性,但三联噻唑在自然界较为稀少,仅在coryneazolicin(Zhou,X.;Huang,H.;Chen,Y.,et al.J.Nat.Prod.2012,75,2251-2255)和抗肿瘤环肽marthiapeptide A(Takuma,M.;Kuroha,M.;Nagano,Y.,et al.J.Antibiot.2019,72,800-806)中有过报道。因此,发现和研究自然界中新型多联噻唑类化合物极具价值和迫切。
经检索,有关利用来自植物根际促生菌贝莱斯芽孢杆菌FZB42(Bacillusvelezensis FZB42)的nrs生物合成基因簇并对该基因簇进行克隆,之后构建nrs基因簇高表达的枯草芽孢杆菌突变菌株,从该菌株的液体发酵产物中分离多联噻唑类化合物,以及芽孢噻唑类化合物在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用还未见报道。
发明内容
针对现有技术的不足,本发明要解决的问题是提供一组芽孢噻唑类化合物,以及该芽孢噻唑类化合物的产生菌株、制备方法和其在制备蛋白酪氨酸磷酸酶(PTPs)尤其是蛋白酪氨酸磷酸酶1B(PTP1B)抑制剂中的应用。
本发明所述的一组芽孢噻唑类化合物,其特征在于:所述芽孢噻唑类化合物是命名为芽孢噻唑A的化合物1;或是命名为芽孢噻唑B的化合物2;或是命名为芽孢噻唑C的化合物3;或是命名为芽孢噻唑D的化合物4;或是命名为芽孢噻唑E的化合物5;或是命名为芽孢噻唑F的化合物6;或是命名为芽孢噻唑G的化合物7;或是命名为芽孢噻唑H的化合物8;或是命名为芽孢噻唑I的化合物9;或是命名为芽孢噻唑J的化合物10;或是命名为芽孢噻唑K的化合物11;或是命名为芽孢噻唑L的化合物12;或是命名为芽孢噻唑M的化合物13;或是命名为芽孢噻唑N的化合物14;其化学构式依次如式Ⅰ至式ⅩⅣ所示(结构式中的阿拉伯数字是化学结构中碳原子的标位):
上述芽孢噻唑类化合物的制备方法,其特征在于:所述芽孢噻唑类化合物是由枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs)经过液体发酵后,从其发酵产物中得到;其中,所述枯草芽孢杆菌1A751-Sfp/nrs突变菌株是通过ExoCET方法克隆nrs基因簇,构建得到p15A-spec-nrs质粒,然后以枯草芽孢杆菌1A751-Sfp(Bacillus subtilis 1A751-Sfp)为出发菌株,利用自然转化的方法将p15A-spec-nrs质粒转入该菌株中,使nrs生物合成基因簇通过双交换插入至amyE基因处得到;其中,所述nrs基因簇来源于贝莱斯芽孢杆菌FZB42(Bacillus velezensis FZB42),基因簇大小为19.8kb,共包含7个基因,分别是nrs0编码转运蛋白,该基因的核苷酸序列如SEQ ID No.1所示;nrsA编码硫酯酶,该基因的核苷酸序列如SEQ ID No.2所示;nrsB编码氧化酶,该基因的核苷酸序列如SEQ ID No.3所示;nrsC编码非核糖体肽合成酶,该基因的核苷酸序列如SEQ IDNo.4所示;nrsD编码聚酮合酶-内酰胺酶,该基因的核苷酸序列如SEQ ID No.5所示;nrsE编码调控蛋白,该基因的核苷酸序列如SEQ ID No.6所示;nrsF编码脂肪酰-AMP连接酶,该基因的核苷酸序列如SEQ ID No.7所示。
本发明所述芽孢噻唑类化合物在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用。
其中:所述芽孢噻唑类化合物优选是命名为芽孢噻唑A的化合物1;或是命名为芽孢噻唑B的化合物2;或是命名为芽孢噻唑C的化合物3;或是命名为芽孢噻唑D的化合物4;或是命名为芽孢噻唑E的化合物5;所述蛋白酪氨酸磷酸酶优选是蛋白酪氨酸磷酸酶1B(PTP1B)。
本发明还提供了一种上述芽孢噻唑类化合物的产生菌株,其特征在于:所述菌株是枯草芽孢杆菌1A751-Sfp/nrs突变菌株;该突变菌株是通过ExoCET方法克隆nrs基因簇,构建得到p15A-spec-nrs质粒,然后以枯草芽孢杆菌1A751-Sfp为出发菌株,利用自然转化的方法将p15A-spec-nrs质粒转入该菌株中,使nrs生物合成基因簇通过双交换插入至amyE基因处得到;其中,所述nrs基因簇来源于贝莱斯芽孢杆菌FZB42,基因簇大小为19.8kb,共包含7个基因,分别是nrs0编码转运蛋白,该基因的核苷酸序列如SEQ ID No.1所示;nrsA编码硫酯酶,该基因的核苷酸序列如SEQ ID No.2所示;nrsB编码氧化酶,该基因的核苷酸序列如SEQ ID No.3所示;nrsC编码非核糖体肽合成酶,该基因的核苷酸序列如SEQ ID No.4所示;nrsD编码聚酮合酶-内酰胺酶,该基因的核苷酸序列如SEQ ID No.5所示;nrsE编码调控蛋白,该基因的核苷酸序列如SEQ ID No.6所示;nrsF编码脂肪酰-AMP连接酶,该基因的核苷酸序列如SEQ ID No.7所示。
本发明公开了一组芽孢噻唑类化合物,以及该芽孢噻唑类化合物的产生菌株、制备方法和其在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用。其中所述芽孢噻唑类化合物是一类以二联或三联噻唑为骨架的新型天然产物,在原始宿主贝莱斯芽孢杆菌FZB42中产量极低,限制了对其进行制备和研究。本发明通过构建芽孢杆菌1A751-Sfp/nrs突变菌株,并用该菌液体发酵来生产芽孢噻唑类化合物,解决了上述化合物的制备难题。此外,实验证实:本发明所述芽孢噻唑类化合物能明显抑制蛋白酪氨酸磷酸酶,其中芽孢噻唑A-E对蛋白酪氨酸磷酸酶1B(PTP1B)有较强的选择性抑制活性。如芽孢噻唑C和E对PTP1B的抑制效果与Na3VO4(阳性对照)的相近,但选择性相对于Na3VO4(阳性对照)分别提高了超过11倍和9倍。PTP1B是胰岛素信号转导通路中的重要负性调控因子,已成为治疗Ⅱ型糖尿病和肥胖症的极具吸引力的靶点。然而PTP1B所属的蛋白酪氨酸磷酸酶家族其活性中心的序列高度相似,这为专一性识别和抑制PTP1B的抑制剂的开发造成了巨大挑战,目前还没有相关药物上市。本发明所获得的芽孢噻唑类化合物有望为蛋白酪氨酸磷酸酶抑制剂特别是PTP1B抑制剂的开发和应用提供新的途径,在糖尿病和肥胖症治疗方面有着重要应用前景,具备产生较好的社会效益和经济价值的潜力。
附图说明
图1:克隆得到带有nrs基因簇的p15A-spec-nrs质粒示意图。
其中:nrs0编码转运蛋白,nrsA编码硫酯酶,nrsB编码氧化酶,nrsC编码非核糖体肽合成酶,nrsD编码聚酮合酶-内酰胺酶,nrsE编码调控蛋白,nrsD编码脂肪酰-AMP连接酶。
图2:枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs)的液体发酵结果。
其中:Bacillus subtilis 1A751-Sfp/nrs是表达nrs基因簇的突变株,Bacillussubtilis1A751-Sfp/CK是阴性对照。
图3:芽孢噻唑A-L(1-12)的关键COSY和HMBC相关性。
图4:HPLC-MS分析水解的化合物芽孢噻唑A-D(1-4)中氨基酸的构型。
其中:A表示芽孢噻唑A(1)的L-FDLA衍生产物与标准氨基酸L-Ser/D-Ser的L-FDLA衍生物之间的比较,从而确定1中的Ser为L型;B表示芽孢噻唑B(2)和C(3)的L-FDLA衍生产物与标准氨基酸L-Thr/D-Thr/L-Allo-Thr/D-Allo-Thr的L-FDLA衍生物之间的比较,从而确定2和3中的Thr为L-Thr;C表示芽孢噻唑D(4)的L-FDLA衍生产物与标准氨基酸L-Ala/D-Ala的L-FDLA衍生物之间的比较,从而确定4中的Ala为L型。
图5:化合物芽孢噻唑F(6)、G(7)和I(9)衍生化的MPA酯的ΔδRS值。
图6:化合物芽孢噻唑A-E(1-5)、M(13)和N(14)的高分辨质谱和二级质谱的碎裂模式。
图7:L-13C-Leu喂养实验的结果分析。
其中:加粗部分为Leu整合至芽孢噻唑中的部分,“*”表示同位素标记的C原子。
具体实施方式
下面结合具体附图和实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已,应该说明的是,下述说明仅仅是为了解释本发明,并非对本发明作任何形式上的限制,凡是依据本发明的技术实质对实施方式所做的任何简单修改,等同变化与修饰,均属于本发明技术方案的范围内。
一般性说明:如下实施例所涉及的限制性内切酶均购自NEB公司,DNA聚合酶和DNA连接酶均购自Takara公司,质粒提取试剂盒和琼脂糖凝胶回收DNA片段试剂盒购自Qiangen公司,操作完全按照相应说明书进行。质粒构建中基因测序及引物合成由上海生工公司完成。质粒p15A-amyEF-Amp-ccdB-spec-amyER和枯草芽孢杆菌1A751-Sfp获取见文献(Liu,Q.;Shen,Q.;Bian,X.,et al.Sci.Rep.2016,6,1-10),菌株E.coli GB2005、E.coli GB05-dir、E.coli GB05-red获取见文献(Fu,J.;Bian,X.;Hu,S.,et al.Nat.Biotechnol.2012,30,440-446),质粒p15A-cm-tetR-tetO-hyg-ccdB和pBBR1-kan-hyg-ccdB获取见文献(Wang,H.;Li,Z.;Jia,R.,et al.Nat.Protoc.2016,11,1175-1190)。上述菌株均为科研实验常用工程菌株,可通过公开的保藏机构购买获得。实施例中的其他实验方法及试剂如无特殊说明,均为相关领域常规方法与市售试剂。
实施例1:nrs基因簇异源表达菌株枯草芽孢杆菌1A751-Sfp/nrs突变菌株的构建
1.1带有nrs基因簇的贝莱斯芽孢杆菌FZB42(Bacillus velezensis FZB42)基因组提取
将贝莱斯芽孢杆菌FZB42接种至LB固体平板中,30℃培养过夜。刮取菌体接种至LB培养基(50mL/250mL瓶)中培养过夜。8000rpm离心5min,收集菌体,用50mL无菌的ddH2O然后用无菌双蒸水清洗两次,再次离心收集菌体。菌体用8mL SET溶液(75mM NaCl,25mM EDTA,20mM Tris,pH 8.0)重悬。向重悬液中加入20mg溶菌酶,37℃孵育1h。再加入500μL的蛋白酶K溶液(20mg/mL)和1mL的SDS溶液(10%),轻轻颠倒混匀后于50℃中放置1h。待溶液变澄清,加入等体积的苯酚-氯仿-异戊醇(25:24:1),颠倒混匀。8000rpm离心30min后,用去尖的1mL枪头吸取500μL上清至新的2mL EP管中,再加入35μL的3M醋酸钠溶液(pH 7.5)和1.2mL无水乙醇,轻柔混匀,肉眼可见有絮状基因组DNA析出。将基因组DNA转移至含有1.5mL 70%乙醇的2mL EP管中,10000rpm离心1min,弃上清,于室温下干燥至DNA变透明。加入200μL ddH2O,放于4℃中溶解过夜以及保存。
1.2 nrs基因簇的直接克隆
取50μg上述制备的贝莱斯芽孢杆菌FZB42基因组用限制性核酸内切酶ApaLⅠ酶切,释放完整的nrs基因簇,释放的DNA片段大小为28.7kb。酶切体系为400μL,添加20μL ApaLⅠ,37℃酶切4h。用400μL的苯酚-氯仿-异戊醇酚抽提酶切体系一次,然后乙醇沉淀回收DNA,得到的DNA再溶解于12μL ddH2O中。使用引物nrs-HAF/nrs-HAR,以质粒p15A-amyEF-Amp-ccdB-spec-amyER为模板进行扩增,并对PCR产物进行切胶回收,获得带有60bp的nrs基因簇同源臂(HA)的线性克隆载体p15A-amyEF-spec-amyER-HA。
将上述制备的基因组酶切回收产物和克隆载体先在体外用T4 DNA聚合酶进行处理,反应体系(20μL):基因组酶切产物,10μg;克隆载体,200ng;10×NEB Buffer 2.1,2μL;T4 DNA聚合酶,0.13μL;ddH2O,补齐至20μL。反应程序:25℃消化60min;75℃酶失活20min;50℃退火30-60min。反应液用除盐膜透析40min,置于4℃保存。
转接40μL过夜培养的E.coli GB05-dir于1.3mL LB中,37℃950rpm培养2h;加入35μL阿拉伯糖来诱导重组酶的表达,之后置于37℃950rpm培养40min;9600rpm离心1min收集菌体,用1mL ddH2O清洗菌体两遍,得到电转感受态细胞;再将上述除盐液加入至感受态细胞中,用ddH2O补齐体积至30μL左右,混匀后加入到1mm电击杯中。将点转移电压设为1350V,电击;细胞于37℃复苏1h,涂布至含有壮观霉素(60μg/mL)的LB平板,37℃过夜培养。挑取单克隆,提取质粒并用Bstz17 I进行酶切鉴定,酶切正确的质粒进一步通过测序验证,将正确的质粒命名为p15A-spec-nrs-R。
上述引物序列如下(小写字母部分为同源臂):
nrs-HAF:
tgttcgtaaagacaccatctgctttttgtttcagcataataggcgtcagcgtatgctcgtCGAATGGCGATTTTCGTTCGTGnrs-HAR:
cggcccgcaaagcgacaaaaaccaacgttctgactgcattaagaagagaactgtaaaggaTCTTCATCATCATTGGCATACG
1.3 nrs基因簇的改造
克隆获得的DNA片段两侧分别带有2158bp和1565bp冗余序列,这些序列与枯草芽孢杆菌1A751-Sfp(Bacillus subtilis 1A751-Sfp)的基因组某些区域高度同源,将影响nrs基因簇在枯草芽孢杆菌1A751-Sfp中的定点插入,需要进一步改造上述的p15A-spec-nrs-R质粒,去除nrs基因簇两端的冗余序列。
使用nrs-km KO-F/R,以pBBR1-kan-hyg-ccdB为模板,PCR扩增得到两端带有50bp同源臂和AscⅠ酶切位点的kan-HA片段;转接40μL过夜培养的E.coli GB05-red于1.3mL LB中,37℃950rpm培养2h;加入35μL阿拉伯糖来诱导重组酶的表达,之后置于37℃950rpm培养40min;9600rpm离心1min收集菌体,用1mL ddH2O清洗菌体两遍,得到电转感受态细胞;将600ng的kan-HA片段与200ng的p15A-spec-nrs-R质粒共同电转至已诱导重组酶表达的E.coli GB05-red感受态细胞中;在重组酶的介导下kanR将替代一端的冗余序列,得到冗余序列分别被kanR替换的重组质粒;该质粒再通过AscⅠ酶切和DNA连接酶连接,去除kanR。另一端的冗余序列去除方法与上述方法类似,使用的片段改为cm-HA片段。通过引物nrs-cm KO-F/R,以p15A-cm-tetR-tetO-hyg-ccdB为模板,PCR扩增得到两端带有50bp同源臂和ApaLⅠ酶切位点的cm-HA。
利用Bstz17 I进行酶切鉴定去除两端的冗余序列的质粒,酶切正确的质粒进一步通过测序验证,将正确的质粒命名为p15A-spec-nrs。其中,所述nrs基因大小为19.8kb,共包含7个基因,分别是nrs0编码转运蛋白,该基因的核苷酸序列如SEQ ID No.1所示;nrsA编码硫酯酶,该基因的核苷酸序列如SEQ ID No.2所示;nrsB编码氧化酶,该基因的核苷酸序列如SEQ ID No.3所示;nrsC编码非核糖体肽合成酶,该基因的核苷酸序列如SEQ ID No.4所示;nrsD编码聚酮合酶-内酰胺酶,该基因的核苷酸序列如SEQ ID No.5所示;nrsE编码调控蛋白,该基因的核苷酸序列如SEQ ID No.6所示;nrsF编码脂肪酰-AMP连接酶,该基因的核苷酸序列如SEQ ID No.7所示。
上述引物序列如下(小写字母部分为同源臂,划线部分为酶切位点):
nrs-cm KO-F:
ctagtgacatgggtagagtcgcatgatacgtatgccaatgatgatgaagaGTGCACTGAAATAAGATCACTACCGGGCnrs-cm KO-R:
tataccatctctgtcttgtgtatgaattacctgatatttcttgtgagaatGTGCACATCTATCAACAGGAGTCCAAGCnrs-km KO-F:
ttacgtcttgaccgttccataattgtttcattgttccgctccatcattcaGGCGCGCCATGTCAGCTACTGGGCTATCTGnrs-km KO-R:
acgagcatacgctgacgcctattatgctgaaacaaaaagcagatggtgtctttacgaacaGGCGCGCCTCAGAAGAACTCGTCAAGAAGG
1.4枯草芽孢杆菌1A751-Sfp/nrs突变菌株的构建
以枯草芽孢杆菌1A751-Sfp(Bacillus subtilis 1A751-Sfp,基因型为his nprR2nprE18 sfp△aprA3ΔeglS102ΔbglT bglSRV)为出发菌株,先制备感受态细胞,方法如下:将枯草芽孢杆菌1A751-Sfp接种在LB中37℃培养1天;接种菌体于1.8mL GMⅠ培养基中,在30℃恒温震荡器上900rpm培养过夜;次日取1mL种子液转接到含有9mL的GMⅠ的250mL三角瓶中,37℃250rpm培养3.5h;再取上一步骤的5mL培养液转接到45mL GMⅡ培养基中,37℃125rpm培养1.5h;8000rpm离心5min,收集菌体,用5mL GMⅡ培养基悬浮细胞,即为感受态细胞。
将5-10μg上述构建的p15A-spec-nrs质粒的加入200μL枯草芽孢杆菌1A751-Sfp感受态细胞中,30℃培养1.5h后10000rpm离心1min,菌体全部涂布于含有壮观霉素(80μg/mL)的LB平板上。37℃培养过夜,次日通过菌落PCR检测转化子,检测引物为751-amyE-F/751-nrs-R1和751-nrs-F1/751-nrs-R1。p15A-spec-nrs质粒中,nrs基因簇的两端带有约1kb的同源臂,可以通过双交换的方式插入至枯草芽孢杆菌1A751-Sfp的amyE基因处。PCR验证正确的重组子命名为枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs)。
上述引物序列如下:
751-amyE-F:CTCCAGTCTTCACATCGGTTTG
751-nrs-F1:TCTGCTACTCTCTCTATATCATG
751-nrs-R1:AGGTGGAATCATACCTATTCCT
上述GMⅠ培养基配方为:0.5%葡萄糖;0.02%酪蛋白胨;0.1%酵母提取物;0.1%柠檬酸钠;0.02%MgSO4·7H2O;0.57%KH2PO4;1.34%K2HPO4。
上述GMⅡ培养基配方为:0.5%葡萄糖;0.004%酪蛋白胨;0.004%酵母提取物;0.1%柠檬酸钠;0.02%MgSO4·7H2O;0.58%KH2PO4;1.36%K2HPO4;0.051%MgCl2;0.037%CaCl2。
1.5枯草芽孢杆菌1A751-Sfp/nrs(Bacillus subtilis 1A751-Sfp/nrs)的液体发酵
将枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs)接种到添加了含有壮观霉素(80μg/mL)的LB固体平板中,30℃培养过夜。刮取菌体接种至LB液体培养基(50mL/250mL,含有60μg/mL的壮观霉素)中30℃培养过夜,得到发酵种子液。按2%(v/v)的接种量转接到装有50mL BPY发酵培养基的250mL三角瓶中,共发酵20L。在30℃,200rpm培养12h后加入2%(v/v)大孔树脂XAD-16。之后继续发酵3天后提取化合物。8000rpm离心5min收集XAD-16和菌体,再加入1L甲醇重悬XAD-16和菌体,在30℃,180rpm萃取3h,重复萃取三次。将合并甲醇再旋转蒸干,得粗浸膏,共20g。
上述BPY培养基组成:0.5%牛肉提取物;1%蛋白胨;0.5%NaCl;1%葡萄糖。用氢氧化钠调节pH至7.0。
实施例2:芽孢噻唑类化合物的分离纯化和结构鉴定
2.1芽孢噻唑类化合物的分离纯化
将实施例1制得的粗浸膏用少量甲醇溶解,取上清用Sephadex LH20凝胶柱初步分离,以甲醇为溶剂,得到两个含有目的化合物的组分(Fr.1和Fr.2)。利用中压MPLC进一步分离,仪器配备的分离柱型号为YMC-pack ODS-A,20×250mm,5μm,使用流速为8mL/min,流动相A相为添加了0.1%三氟乙酸的Milli-Q H2O,B相为乙腈。
Fr.1的洗脱程序为:0-5min,35%B;5.1-25min,35-60%B;25.1-65min,60-90%B;65.1-75min,100%B;75.1-85min,35%B,得到Fr.1.1。
Fr.2的洗脱程序为:0-5min,45%B;5.1-20min,45-70%B;20.1-60min,70-100%B;60.1-70min,100%B;70.1-80min,45%B,得到Fr.2.1-Fr.2.3。
通过半制备型HPLC进行纯化,仪器配备的分离柱型号为Agilent ZORBAX SB-C18,9.4×250mm,5μm或YMC-pack ODS-A,10×250mm,5μm,流速为3mL/min,A相改为添加了0.1%三氟乙酸的Milli-Q H2O。Fr.1.1的分离程序为:0-40min,35%B;40.1-45min,100%B;45.1-50min,100%B。Fr.2.1的为:0-4min,41%B;4.1-35min,41-48%B;35.1-40min,100%B;40-45min,41%B。Fr.2.2的分离程序为:0-55min,62%B;55.1-59min,100%B;59.1-63min,62%B。Fr.2.3的分离程序为:0-65min,64%B;65.1-69min,100%B;69.1-73min,64%B。共纯化得到14个产物,命名为芽孢噻唑A-N(1-14):1(2mg),2和3(4mg),4(2mg),5(5mg),6(2mg),7(2mg),8(2mg),9(7mg),10(2mg),11和12(3mg),13(0.5mg),14(0.5mg)。
2.2芽孢噻唑类化合物的结构鉴定
进一步地对上述分离到的芽孢噻唑类化合物进行高分辨质谱、核磁、旋光、红外光谱的检测。
仪器型号:核磁测试为Bruker AVNEO 600MHz和Agilent 500MHz DD2核磁系统;高分辨质谱用Bruker Impact HD microTOF Q III质谱仪;红外光谱测试为Bruker Tensor-27分光光度计;旋光测试为Rudolph AUTOPOL IV自动旋光计。
本发明所述芽孢噻唑类化合物的理化性质及波谱数据如下:
芽孢噻唑A(1):白色粉末;[α]27 D-14(c 0.1,MeOH:CH2Cl2=1:1);UV(MeOH)λmax226nm;IR(KBr)vmax 3429,2924,1709,1365,1232,1209,803cm-1;HRESI/MS:m/z 495.1187[M+H]+(计算值C21H27N4O4S3,495.1189).1H和13C-NMR数据见表1。
芽孢噻唑B(2):白色粉末;[α]27 D-23(c 0.13,MeOH:CH2Cl2=1:1);IR(KBr)vmax3437,2924,1709,1365,1226,1093,736cm-1;HRESIMS m/z 509.1355[M+H]+(计算值C22H29N4O4S3,509.1345).1H和13C-NMR数据见表2。
芽孢噻唑C(3):白色粉末;HRESIMS m/z 509.1357[M+H]+(计算值C22H29N4O4S3,509.1345).1H和13C-NMR数据见表2。
芽孢噻唑D(4):白色粉末;HRESIMS m/z 479.1233[M+H]+(计算值C21H27N4O3S3,479.1240).1H和13C-NMR数据见表1。
芽孢噻唑E(5):白色粉末;[α]27 D+9(c 0.11,CH2Cl2);IR(KBr)vmax 3407,2921,1683,1208,1142,1093,802cm-1;HRESIMS m/z 408.0860[M+H]+(计算值C18H22N3O2S3,408.0869).1H和13C-NMR数据见表3。
芽孢噻唑F(6):白色粉末;[α]27 D-10(c 0.1,CH2Cl2);IR(KBr)vmax 3413,2925,1683,1540,1204,1143,802cm-1;HRESIMS m/z 424.0820[M+H]+(计算值C18H22N3O3S3,424.0818).1H和13C-NMR数据见表3。
芽孢噻唑G(7):白色粉末;[α]27 D-11(c 0.15,EtOAc);IR(KBr)vmax 3412,2930,1685,1557,1206,838,802cm-1;HRESIMS m/z 424.0819[M+H]+(计算值C18H22N3O3S3,424.0818).1H和13C-NMR数据见表4。
芽孢噻唑H(8):白色粉末;[α]27 D+8(c 0.13,EtOAc);IR(KBr)vmax 3412,2930,1683,1651,1208,1143,802,725cm-1;HRESIMS m/z 424.0817[M+H]+(计算值C18H22N3O3S3,424.0818).1H和13C-NMR数据见表4。
芽孢噻唑I(9):无色粉末;[α]27 D+10(c 0.1,MeOH:CH2Cl2=1:1);IR(KBr)vmax3395,2922,1647,1597,1261,1099,800cm-1;HRESIMS m/z 341.0989[M+H]+(计算值C15H21N2O3S2,341.0988).1H和13C-NMR数据见表5。
芽孢噻唑J(10):无色粉末;[α]27 D-25(c 0.12,CH2Cl2);IR(KBr)vmax 3393,2922,1647,1595,1460,1261,1059,800,741cm-1;HRESIMS m/z 341.0993[M+H]+(计算值C15H21N2O3S2,341.0988).1H和13C-NMR数据见表5。
芽孢噻唑K和L(11和12):无色粉末;HRESIMS m/z 341.0993[M+H]+(计算值C15H21N2O3S2,341.0988).1H和13C-NMR数据见表6。
芽孢噻唑M(13):白色粉末;HRESIMS m/z 495.1191[M+H]+(计算值C21H27N4O4S3,495.1189)。
芽孢噻唑N(14):白色粉末;HRESIMS m/z 479.1227[M+H]+(计算值C21H27N4O3S3,479.1240)。
表1化合物1和4在DMSO-d6中的1H(600MHz)和13C(150MHz)NMR数据
a表示重叠信号。
表2化合物2和3在DMSO-d6中的1H(600MHz)和13C(150MHz)NMR数据
a表示重叠信号;b因为化合物2-3量太少而产生的重叠或未为观察到该信号。
表3化合物5和6在DMSO-d6中的1H(500MHz)和13C(125MHz)NMR数据
a表示重叠信号。
表4化合物7和8在DMSO-d6中的1H(500MHz)和13C(125MHz)NMR数据
a表示重叠信号。
表5化合物9和10在DMSO-d6中的1H(500MHz)和13C(125MHz)NMR数据
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a表示重叠信号。
表6化合物11和12在DMSO-d6中的1H(500MHz)和13C(125MHz)NMR数据
a表示重叠信号。
根据HRESIMS推测芽孢噻唑A(1)的分子式为C21H26N4O4S3。化合物1的低场1H NMR谱显示在δH 8.39(H-3,H-6)和8.26(H-9)处有三个尖锐的单峰,并且与δC 125.1,118.8和117.7处有相关的13C NMR共振,表明1有三个2,4-二取代的噻唑基团,HMBC相关性进一步证实了这一点。H-11到H-17及H-15/H-18的连续1H-1H COSY相关性确定了烷基侧链的存在,并根据关键HMBC相关性推测该烷基连接在C-10。其余三个碳信号(δc 171.8,61.3,54.7)根据COSY相关性鉴定为丝氨酸(Ser),且由HMBC判断其与C-1相连接(图3)。芽孢噻唑B(2)和C(3)具有相同的分子式C22H28N4O4S3。将它们的1D NMR数据与1的进行比较发现,2和3中C-1相连的为Thr。用同样的方式推导了化合物4-12的结构。
此外,通过Marfey水解确定化合物1、2、3、4中氨基酸的构型。方法为:取1、2、3、4(0.5mg)于0.5mL 6 N HCl中90℃水解7-8h,水解产物冻干后溶解于200μL ddH2O中,同时取1mg氨基酸标准品溶解于200μL ddH2O中;向上述溶液中依次加入25μL NaHCO3(1N)和200μLL-FDLA(1%,丙酮)溶液,混匀,于40℃下反应1h后,用10μL HCl(2M)淬灭反应;衍生后的产物通过HPLC-MS检测,流动相为添加了0.1%甲酸的Milli-Q H2O和乙腈。化合物1、2、3及相应的标准氨基酸衍生产物的洗脱程序为4-34min,5%-55%B;化合物4及相应的标准氨基酸衍生产物的洗脱程序为4-27min,30%-68%B。经检测,化合物1中的丝氨酸(Ser)、化合物2和3中的苏氨酸(Thr)、4中的丙氨酸(Ala)的绝对构型均为L-型(图4)。
进一步通过Mosher法确定了化合物6、7和9的绝对构型。方法为:将上述化合物(1-2mg)溶解在1mL二氯甲烷中,并与4-二-甲氨基吡啶(DMAP)、二环己基碳二亚胺(DCC)及(R)-或(S)-α-甲氧基-α-苯乙酸(MPA)混合。在室温下反应22h,产生的Mosher衍生物利用半制备型HPLC纯化,然后根据1H NMR化学位移来确定其构型。经检测和计算,化合物6的C-14、7的C-15和9的C-13的绝对构型别为S、S和R型(图5)。因为来源同一个基因簇,推测化合物10的C-12的构型与7的C-15相同,为S型;推测化合物8的C-16的构型与9的C-13相同,为R型。
化合物13和14的产量低,主要通过高分辨质谱和同位素标记实验推测了它们的结构。13和14分别与1和4显示出相同的质子化HRESIMS峰和二级质谱碎裂模式,表明它们是相应的异构体(图6)。此外,同位素喂养实验表明亮氨酸(Leu)衍生的脂肪酸链可以掺入13和14结构中,说明它们带有anteiso-脂肪酸链(图7)。同位素喂养实验的方法为:转移1.2mL在BPY(50mL/250mL)培养基中发酵过夜的枯草芽孢杆菌1A751-Sfp/nrs菌液,至无菌的2.0mLEP管中,管盖上用针头扎孔;加入50μL L-[1,2-13C2]-Leu水溶液(20mg/mL,过滤除菌,-20℃保存),阴性对照加入50μL无菌水;置于30℃,950rpm振荡培养24h和48h后再加入50μL L-[1,2-13C2]-Leu溶液;继续培养12h后,加入200μL XAD-16,孵育过夜;离心收集菌体和XAD-16,用1mL甲醇萃取,萃取液真空蒸干后重新溶解在200μL甲醇中,得到粗提物;利用HPLC-MS检测分析粗提物。
实施例3:芽孢噻唑类化合物的蛋白酪氨酸磷酸酶抑制活性测试
测试样品为上述实施例5中分离精制的化合物1-10。精密称取适量样品,用DMSO配制成所需浓度的溶液,供测活性。
将三种纯化蛋白酪氨酸磷酸酶(PTP1B、SHP1和TCPTP)与测试化合物于96孔板中30℃预孵育15-30min,加入反应缓冲液(50mM HEPES,100mM NaCl,1mM EDTA,1mM二硫苏糖醇)后继续孵育30min,加入2mM磷酸对硝基苯酯作为底物,将体系置于37℃下反应1h,加入50μL3M NaOH终止反应。以Na3VO4为阳性对照,DSMO为阴性对照。对硝基苯磷酸盐可以被蛋白酪氨酸磷酸酶脱磷酸生成对硝基苯酚,在405nm处测量对硝基苯酚的吸光度可以确定其产生量,进而确定蛋白酪氨酸磷酸酶的抑制活性。选择显示出良好抑制率的化合物(在50μg/mL时抑制率>50%)进一步测定IC50值。以Na3VO4作为阳性对照。所有实验一式三份进行。
活性结果见表7。化合物1-5对能有效抑制PTP1B,且选择性比Na3VO4更强。特别是化合物3和5对PTP1B的抑制作用分别是对TCPTP的23倍和19倍。PTP1B是胰岛素信号转导通路中的重要负性调控因子,已成为治疗Ⅱ型糖尿病的极具吸引力的靶点,PTP1B抑制剂是研发抗Ⅱ型糖尿病药物的重要方向。然而PTP1B所属的蛋白酪氨酸磷酸酶家族其活性中心的序列高度相似,特别是TCPTP与PTP1B的有接近80%的序列是相同的,这为专一性识别和抑制PTP1B的抑制剂的开发造成了巨大挑战,目前还没有相关药物和抑制剂上市。由于化合物1-5表现出的高效性和选择性,将有进一步开发成为治疗PTP1B相关疾病的先导药物和相关制剂的潜力。
表7化合物1-10对蛋白酪氨酸磷酸酶的抑制活性(IC50/μM)
注:选择性表示TCPTP IC50/PTP1B IC50。
序 列 表
<110> 山东大学
<120> 一组芽孢噻唑类化合物及其制备方法与应用
<141> 2022-02-19
<160> 7
<210> 1
<211> 1668
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrs0的核苷酸序列
<222>(1)…(1668)
<400> 1
ttgaatttca aaaaaataat catgttatct gcattattat taggtacatt catttcagta 60
atggacacca ctatagtaaa tatcgcctta cctaagatgc tggattattt ttcgagtaac 120
ctttctcaag tagcatgggt ggctacaggt tataccttag cttttgcggt tttattagta 180
ggtgcatcaa agctggcaga ccattttgga agaaaaaaag cttttatgtt tggattagca 240
ctttttattt tgacatcagt tattgcatgt ttgagcactt caattgaaat gcttgtcaca 300
attagagtga ttcagggatt atctgctgct ttcattgtac ctgttacaat gcctatagct 360
ttagctatcg tacctcaaaa taaaaaagga atgatcattg gaatctgggg tgctttctca 420
ggattagcag ctacattagg acctgtactt ggaggattat taactgaaaa ttttaattgg 480
caagctatct tcttcataaa tattccttta ggtctgattt ctcttttttt agcagcattt 540
tttattactg agagttatga tgaaacagtg agtaaaaaaa ttgattacat ggggattctt 600
attttatccg gagctctctt ctgtttaacc tttggttttg caaaagtatc tgacttaggc 660
tggtcatcac cattaattat ctctcttttt gtaatctctg tattattatt ctgtttgttt 720
ttctttattg aatctaaaat taaaaacccg atgattccct tatccatgct gcagatacgt 780
acgtttagtt tcagttcttt caccctcttt atgcagggcc ttggattaac aagcggtacc 840
cttattatta ctttactcct cacgaattta atgggaaaaa ctgaattaga agcggggtta 900
attgtttcta cattagctct atcatctatg tttacctctg tactttctgg aaagctatct 960
gataaactcg ggggcatttg gctttcgggt ctaggtatgc tcggtttaac aatcacaaca 1020
tatttatatg gatacatacg atacgacagt tctataacac cagtaattat tctgttatgt 1080
ttgaccggct tagctttagg gcttgtgata ggaccagcaa tgagttcagg gatacggctt 1140
attcctcctg agaaagtagg aattgcctca ggtattctta atatgatgcg gacagttgga 1200
caagcgctag gcattgccat acttacatct gtgcttacaa tgaatataaa tgatcacaca 1260
gctactgcga aaaaagaagc cattaaaatt gtggaaagca atcaggtttt tgataaaaat 1320
gcaaagaacg agataataac tcacttaaag cattcagaca gtaaaggttt cgatcgaagc 1380
aaaagtattg ctgaattaaa caaaaaagag gaagaaattc tctctataac cccggatccg 1440
tataaagaaa aagtacaaaa aagcttcaat atccaaaaga aggaagttat ggctatccag 1500
aaaaaaattt caaatttata taatgaaaaa ataagtgaca gttttaataa tacattcaaa 1560
gtaggcagtg taattcttat tttaggaatt atttttgcat tgttcagtga tatttctcca 1620
aaacgtttaa aacaacaaca gaaaaaccag gtagatatta cgtcttga 1668
<210> 2
<211> 705
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsA的核苷酸序列
<222>(1)…(705)
<400> 2
atgacccatc caattaaact tttttgcatt ccgcatgctg gaggattgtc aagtatttat 60
agtgaatgga gtaactcagt tggaccagaa attgaactaa tacctgttga actagcagga 120
agaggctcta gattttctga aaatccctac aaaacaataa atcaggctat agatgatatt 180
acagaattaa tattaagaaa atgtgataat aagtgctatg cgatttacgg ccatagtatg 240
ggtgcatata tatcgtatga agtaatactt aaattaatga aggttacaga taatcttcct 300
cagcatttat ttgtatctgg aaaaggcgca ccttatttaa ctaaggaaac atttataagc 360
ggtcttcctg ataaacaagt tattcaatgg ttaaacaaat tagggggaat tcccaaggaa 420
cttatcgaga attacgaact aatgaaacta tttcttccag taataaggaa tgattataaa 480
ctaatagaaa gttacaacca caacatcaaa aaaaaaatat caaatgttac gttaactgtt 540
ttaacgggca agtttgacga atcaataact ataggagaag ttgaagggtg gaaagaatat 600
tcagaaaagg aggttgatgt ataccagatt agcggagggc atttttttat taatgaatcc 660
gttacagaag ttatatcaat tattaaagat aaattattgg tttaa 705
<210> 3
<211> 984
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsB的核苷酸序列
<222>(1)…(984)
<400> 3
gtgaaaaaat acttttgggc tcctcatgtt cactggagaa atgaaaataa ccatattatt 60
attaatgatt ttcaatttag agggaaagta gttgaacttt ttcctgatat ttattttcat 120
tttcaaaaag gcatttcttt aagcgaaata tatgttattt ttaaagactt taatccaaag 180
attttaaaaa tgtttctaaa agatttaata cgtaatagag ttttagtttg caatgtaagc 240
acaccgacag agcttttcga aacgcaaaac aaatttgttt atcaagaata tgacgatgag 300
ttttttttat acaaagaaaa tatagaatct tataagaaga aacaattaac taggtttatg 360
tcaaagaata ctcttcaatc aattgatttg gaggaatgct cagatttgcc ctctttagtt 420
caaaatagaa aaagtactag gaattttgat actagtaaaa tgatgaattt ttctaagttt 480
agtcggttgt taaatgtctt taaacagtct ccaattcaag agggtattaa atatttctac 540
ccaagtgcag gaggactgta tcctgtcaac atatatttgt atattaaaga tgggagagtc 600
gaaaatcttg aggaagggat atatttttat aatcctcaca gccactctat caatttactt 660
aatagagtaa aactggctga aaaaaaatat cattatttta taaaccacag tgcatttaat 720
acttcgaatt tctctatctt tttcaccctt gattgtgatg taatcatgcc caaatataag 780
ggtgcggggt attactatgg tattcttgaa tgtggtgtta tgtcagctac attggctaat 840
atagcagaat attttgatgt tggatcgtgc tccataggag aaatagactt cgataaaatt 900
aaagatgaat ttaatttgaa tagcaatgaa atctatttac attcggtaga ggtaggacta 960
aagaataagg aggtaaatca ttga 984
<210> 4
<211> 10236
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsC的核苷酸序列
<222>(1)…(10236)
<400> 4
ttgattaata acacgctagt aacgagtaat aaatttaaag agatttctaa ctttataata 60
aaaaaaattc gtgaattaag taaagatgat tcagttgatt taagaactaa tatttttgat 120
tatcaaatat cttctattaa tatattaaaa ttacttggag ccattgaaga tacatataat 180
ataaatattg aattttatga ttatattgac aacccagatg tactgtcatt ttgtcaagtt 240
gtttatgaaa agtttcagtt attagataat aaaaaagagt ataatatatt acatttatta 300
agcgatgaag aaaatgccta taaagaattt gatcttacaa atgttcaagt tgcttattta 360
tctggaagag acaagcaatt tgaactgggg ggaacttcca cccatgcttt tgtagaattt 420
gaggctgaac tagatattga aaaattcaat aaaagtgtac aagctgtgat agagcgtcaa 480
cacatgctac gtgcagtttt tttatcaaat ggtaagcaaa aaatacttga acacgtaccg 540
aattataaat tagctattca agatgtgagt tatcttaaaa aatcagacca agaagacaaa 600
atattagaag caagaaaagt aactaacgaa cgaatcttcg atccatacca atggccattg 660
tttgaattta aagctttaaa aatcagtgat aaactatata gaatattctt tgatatcgat 720
atgttaatag cagatggagc tagtatccag cttttattta aagagatatc tgaattgtac 780
aatggaaaga ttcaagagcc tttagaagta tcctatagag attatatgtt agcttataac 840
aaattaaagg gaaatcataa atatcaggat gacaaggaat attggttaaa taaacttaaa 900
agttttcctt caacaccagc tctgccagta aaagtagatt tagcatgtat aaaaaaacct 960
acttttaaaa gaaagaaaaa aattattgag aaaagaaatt ggaatcaatt tgtgaattta 1020
tgctctcgga agaatattag gcctagtata ctacttttgt ctatttattc aaaagtgttg 1080
tctaaatgga gtaatcagca aaatattgcg ttaaacacta cattatttaa aagaagacct 1140
ttgcataagc agattaataa cgttataggc gattttacag aaattatgat atttgatgta 1200
tgcttcaata atttaaatca tttttgggaa gaagtaaaaa atattcagga taacttcttt 1260
aaatgcttat cacataatga ttttgatgga atagagttta ccagagaata ctgtgaattt 1320
aatgatatag atagaaggaa agctgttttt ccgattgttt ttacaagtat gttatttgaa 1380
aatgaattaa actatttaga ttctctagga gtttttatag agagtatcag ccagacatca 1440
caagtttatc tggatcatca agtgatagag ctcaatgatg gagtcatgct tagttgggac 1500
tatgtttcag aggcttttga aaatgacgta attgagtcta tgtttgagga gtatacccac 1560
attattgacc aattaacgtc cggtgagatg atgtcttctg aagatacagc actggaacaa 1620
gcaattactg attataatca aactgagaag gccatcaagc cggatacttt atatcacttg 1680
tttgcgagac aaacaataaa aacacctgag aatattgcga ttgagtgtca aggaagagca 1740
gttacctatc aagagttaca agtcatgtcc aaccgtatat ctagtttttt ggaagagaag 1800
ggaattcagc caaatgagta tgttggggtt attgttgatc gtgagatcga gacaatcgcc 1860
agtatcctcg cagtgttaaa aattggagct gcctatatcc caattaatcc ggaatttcca 1920
aaagaaagac aatcatatat tctgaaagat ggtgattgta aagttgtatt aacagctgaa 1980
ctggtaaaaa ctattgtgtc aagctataaa gagtctaaaa gagaaagtgt ggcagtgccg 2040
gaacaaatag cttatgcgat atacacttca ggaagtacgg gaaaacctaa aggtgtaatt 2100
ataaagaatg aggctgtaac taatactata ttagacatca atgaaaaata cagtgtgaat 2160
gaaacagacc ggtttatcgg attatcatct atgtcctttg atttatcaat atatgatatt 2220
tttggtgctt tttctgcagg agcaacatta gtaatgatcg aagatcagag agatattaaa 2280
aagattcatg acatcgtaaa agaggaaaaa attacagtct ggaactccgt tcccatgatc 2340
atggaaatgt tagtaaacta tatggatgaa acagaaaaca aatctaacgc tggagttatt 2400
aactatgacg agcttcctga attaagactg gtattattaa gtggggattg gatacctgtt 2460
catcttcctg agaggattaa ggatcacttc gttgaaagtg aagtcataag cttaggcggg 2520
gctactgaag cgtctatttg gtcgatttat tatccaatta aaacagtcaa ggctgaatgg 2580
accagcattc cttacggtta tccattatca aatcaaactt actatgtttt aaactatgaa 2640
aatgaccctt gtccaatcgg agtgaaaggt gaacttttca tagggggaaa aggtgtagcg 2700
gaaggctatt taaatgataa agaaaaaact gaagcgtcat ttattgatca cccagatttc 2760
ggaagaattt atcgtacggg ggatatgggg gtcttgacgc aagagggtta tattgagttt 2820
ttgggacgga aagatcatca gattaaagtt cgtggatatc gcgtggagtt aggtgaaata 2880
gaaagtgtaa tattagagca ccgacaggtg agaaatgcgg tagtcattaa tcaaaaggat 2940
gccagaaatc aagatgtgtt atatgcgtac gtagcggggc accaatcctt accgccaact 3000
gatctgaaag agtttttaag cctgaaagtg ccggaatata tgatacctag ctatatcgta 3060
cagattgaag aggttccttt aacaagtaac ggaaaagttg atcgtaaaaa actactggct 3120
ctagacgtta cagatcaagc atcgattgga aggaaaatca aagagcctcg aacagaaatt 3180
gagagagatt tagttggtat ctggaaaagc gttctgaaaa cagatgaaat cagcatagat 3240
gacaactttt ttgaattagg cggcaattcc atattgatca ttaatttaat tactgctatt 3300
gaggatcgtt tatttattaa attagatttt aaaaatttta tatccaacag taccattgag 3360
aaactagcaa aagtaatcag tgtctctgaa agaaaaagta aaacaaatag taatgtgatg 3420
gaaattcaag ctgatatcaa gcataaaaat acggaattcg atttaactaa cgttcaaatg 3480
gcttatctaa tgggaagaga taaacaattt gaacttggtg gcacctcaac gcatgcttat 3540
ttagaaattg agactgtgct gaatattgaa aaactcaaca aaagtttaca aaaagtaata 3600
gatcatcaag acatgctgcg cgcggtcatt cttccgaatg gaaggcaaaa gattcttcaa 3660
caagtccctc agtatgaaat atctgtacag gatattagtc aattaagtga tatagaacaa 3720
gagaagaaaa ttgcagaaga aagagacaga acatcacatc atatctttaa tccaaatgag 3780
tggcctttat ttgaattcaa agccttgaaa attcaagggg ataaacatag attatttttc 3840
gacatcgata tgttaatagc tgatggagcc agcatccagc ttttatttaa acagatatct 3900
gaattgtata atggaaaaga tgaaatggta ccattggaaa taagttatag agattatatg 3960
ttagcatacc aaaaagtcaa gaccactcaa aaatttgaaa ttgaaaaagc ctattgggta 4020
agtcagatcc aaaatttctc aaaagcactt gcggtgccct ttaaagaaag attagatgca 4080
ataagcaagc caactttcaa aagaaaatct aaaataattg acaagcttct atgggaaaca 4140
ctttgtaaaa aagctgcaag ctataatatt acacctgcaa taatattatt tacagcctac 4200
gcaaaagtgt tgtcgaagtg gagtaaccaa caaaacttag cggtaaatac aacagtattt 4260
aatagacagc ctctccataa acaagttaat gatataatag gtgattttac atcgctgatt 4320
atgttaaaag cgagttttga gaaaaaaatg aacttttggg aggaggcaaa aaaggttcag 4380
gttactttca tggaagcgct tgaacataga gactatgatg gagttgaatt tataagagaa 4440
tatgctagat ataataatct aaagcacaaa agtgcgacaa tgccagtagt gttcaccagt 4500
atgttatttg gaaacatgtt taatcttaat aataatgatc tattgaatat tggtgagtta 4560
aaaatggcag tcagccagac atcacaagtt tatctggatc atcaagtgat agagctcaat 4620
gatggagtca tgcttagttg ggactatgtt tcagaggctt ttgaaaatga tgtaattgag 4680
tcaatgtttg aggaatatac ccacattatt gaccaattaa cgtccggtga gatgatgtct 4740
tctgaagata cagcactgga acaagcaatt actgattata atcaaactga gaaggccatc 4800
aagccggata ctttatatca cttgtttgcg agacaaacaa taaaaacacc tgagaatatt 4860
gcgattgagt gtcaaggaag agcagttacc tatcaagagt tacaagttat gtccaaccgc 4920
atatctagtt ttttggaaga gaagggaatt cagccaaatg agtatgttgg ggttattgtt 4980
gatcgtgaaa tcgagacaat cgccagtatc ctcgcagtgt taaaaattgg agctgcctat 5040
atcccaatta atccggaatt tccaaaagaa agacaatcat atattgtgaa agatggtaat 5100
tgtaaagttg tattaacagc tgaactggta aaaactattg tgtcaagcta taaagagtct 5160
aaaagagaaa gtgtggcagt gccggaacaa atagcttatg cgatatacac ttcaggaagt 5220
acgggaaaac ctaaaggtgt aattataaag aatgaggctg taactaatac tatattagac 5280
atcaatgaaa aatacagtgt gaatgaaaca gaccggttta tcggattatc atctatgtcc 5340
tttgatttat caatatatga tatttttggt gctttttctg caggagcaac attagtaatg 5400
atcgaagatc agagagatat taaaaagatt catgacatcg taaaagagga aaaaattaca 5460
gtctggaact ccgttcccat gatcatggaa atgttagtaa actatatgga tgaaacagaa 5520
aacaaatcta acgctggagt tattaactat gacgagcttc ctgaattaag actggtatta 5580
ttaagtgggg attggatacc tgttcatctt cctgagagga ttaaggatca cttcgttgaa 5640
agtgaagtca taagcttagg cggggctact gaagcgtcta tttggtcgat ttattatcca 5700
attaaaacag tcaaggctga atggaccagc attccttacg gttatccatt atcaaatcaa 5760
acttactatg ttttaaacta tgaaaatgac ccttgtccaa tcggagtgaa aggtgaactt 5820
ttcatagggg gaaaaggtgt agcagaaggc tatttaaatg ataaagaaaa aactgaagcg 5880
tcatttattg atcacccaga tttcggaaga atttatcgta cgggggatat gggggtcttg 5940
acgcaagagg gttatattga gtttttggga cggaaagatc atcagattaa agttcgtgga 6000
tatcgcgtgg agttaggtga aatagaaagt gtaatattag agcaccgaca ggtgagaaat 6060
gcggtagtca ttaatcaaaa ggatgccaga aatcaagatg tgttatatgc gtacgtagcg 6120
gggcaccaat ccttaccgcc aactgatctg aaagagtttt taagcctgaa agtgccggaa 6180
tatatgatac ctagctatat cgtacagatt gaagaggttc ctttaacaag taacggaaaa 6240
gttgatcgta aaaaactact ggctctagac gttacagatc aagcatcgat tggaaggaaa 6300
atcaaagagc ctcgaacaga aattgagaga gatttagttg gtatctggaa aagcgttctg 6360
aaaacagatg aaatcagcat agatgacaac ttttttgaat taggcggcaa ttccatattg 6420
atcattaatt taattactgc tattgaggat cgtttattta ttaaattaga ttttaaaaat 6480
tttatatcca acagtaccat tgagaaacta gcaaaagtaa tcagtgtttc tgaaagaaaa 6540
agtaaaacaa atagtaatgt gatggaaatt caagctgata tcaagcataa aaatacggaa 6600
ttcgatttaa ctaacgttca aatggcttat ctaatgggaa gagataaaca atttgaactt 6660
ggtggcacct caacgcatgc ttatttagaa attgagactg tgctgaatat tgaaaaactc 6720
aacaaaagtt tacaaaaagt aatagatcat caagacatgc tgcgcgcggt cattcttccg 6780
aatggaaggc aaaagattct tcaacaagtc cctcagtatg aaatatctgt acaggatatt 6840
agtcaattaa gtgatataga acaagagaag aaaattgcag aagaaagaga cagaacatca 6900
catcatatct ttaatccaaa tgagtggcct ttatttgaat tcaaagcctt gaaaattcaa 6960
ggggataaac atagattatt tttcgacatc gatatgttaa tagctgatgg agccagcatc 7020
cagcttttat ttaaacagat atctgaattg tataatggaa aagatgaaat ggtaccattg 7080
gaaataagct atagagatta tatgttagca taccaaaaag tcaagaccac tcaaaaatat 7140
gaaaatgata aagcctattg gttaagtcag ctcgaagatt ttccaaaagc acctgcggtg 7200
ccattaaaag aaagattaga tgcaataagc aagccaactt tcaaaagaaa atctaaaata 7260
attgacaagc ttctatggga aacactttgt aaaaaagctg caagctataa tattacacct 7320
gcaataatat tatttacagc ctacgcaaaa gtgttgtcga agtggagtaa ccaacaaaac 7380
ttagcggtaa atacaacagt atttaataga cagcctctcc ataaacaagt taatgatata 7440
ataggtgatt ttacatcgct gattatgtta aaagcgagtt ttgagaaaaa aatgaacttt 7500
tgggaggagg caaaaaaggt tcaggttact ttcatggaag cgcttgaaca tagagactat 7560
gatggagttg aatttataag agaatatgct agatataata atctaaagca caaaagtgcg 7620
acaatgccag tagtgttcac cagtatgtta tttggaaaca tgtttaatct taataataat 7680
gatctattga atattggtga gttaaaaatg gcagtcagcc agacatcaca agtttatctg 7740
gatcatcaag tgatagagct caatgatgga gtcatgctta gttgggacta tgtttcagag 7800
gcttttgaaa atgatgtaat tgagtcaatg tttgaggaat atacccacat tattgaccaa 7860
ttaacgtccg gtgagatgat gtcttctgaa gatacagcac tggaacaagc aattactgat 7920
tataatcaaa ctgagaaggc catcaagccg gatactttat atcacttgtt tgcgagacaa 7980
acaataaaaa cacctgagaa tattgcgatt gagtgtcaag gaagagcagt tacctatcaa 8040
gagttacaag ttatgtccaa ccgcatatct agttttttgg aagagaaggg aattcagcca 8100
aatgagtatg ttggggttat tgttgatcgt gaaatcgaga caatcgccag tatcctcgca 8160
gtgttaaaaa ttggagctgc ctatatccca attaatccgg aatttccaaa agaaagacaa 8220
tcatatattg tgaaagatgg taattgtaaa gttgtattaa cagctgaact ggtaaaaact 8280
attgtgtcaa gctataaaga gtctaaaaga gaaagtgtgg cagtgccgga acaaatagct 8340
tatgcgatat acacttcagg aagtacggga aaacctaaag gtgtaattat aaagaatgag 8400
gctgtaacta atactatatt agacatcaat gaaaaataca gtgtgaatga aacagaccgg 8460
tttatcggat tatcatctat gtcctttgat ttatcaatat atgatatttt tggtgctttt 8520
tctgcaggag caacattagt aatgatcgaa gatcagagag atattaaaaa gattcatgac 8580
atcgtaaaag aggaaaaaat tacagtctgg aactccgttc ccatgatcat ggaaatgtta 8640
gtaaactata tggatgaaac agaaaacaaa tctaacgctg gagttattaa ctatgacgag 8700
cttcctgaat taagactggt attattaagt ggggattgga tacctgttca tcttcctgag 8760
aggattaagg atcacttcgt tgaaagtgaa gtcataagct taggcggggc tactgaagcg 8820
tctatttggt cgatttatta tccaattaaa acagtcaagg ctgaatggac cagcattcct 8880
tacggttatc cattatcaaa tcaaacttac tatgttttaa actatgaaaa tgacccttgt 8940
ccaatcggag tgaaaggtga acttttcata gggggaaaag gtgtagcaga aggctattta 9000
aatgataaag aaaaaactga agcgtcattt attgatcacc cagatttcgg aagaatttat 9060
cgtacggggg atatgggggt cttgacgcaa gagggttata ttgagttttt gggacggaaa 9120
gatcatcaga ttaaagttcg tggatatcgc gtggagttag gtgaaataga aagtgtaata 9180
ttagagcacc gacaggtgag aaatgcggta gtcattaatc aaaaggatgc cagaaatcaa 9240
gatgtgttat atgcgtacgt agcggggcac caatccttac cgccaactga tctgaaagag 9300
tttttaagcc tgaaagtgcc ggaatatatg atacctagct atatcgtaca gattgaagag 9360
gttcctttaa caagtaacgg aaaagttgat cgtaaaaaac tactggctct agacgttaca 9420
gatcaagcat cgattggaag gaaaatcaaa gagcctcgaa cagaaattga gagagattta 9480
gttggtatct ggaaaagcgt tctgaaaaca gatgaaatca gcatagatga caactttttt 9540
gaattaggcg gtaattcaat ttcactttca aatatataca ctaaaataga aaaaaaatat 9600
tcagatttga atatagttga tttattcaag tatcaaaaaa ttagtgaatt atcacatttt 9660
attagtgtta tgaaagagga gaataagaat ttaaaattaa aatcagtaaa attgcctcca 9720
aaaattcttg ctaatagaaa agataataaa aatgaaaaga caataatttt agaaaaacag 9780
ttaaaaatta agtttaataa cttagaagta agtaataaag taataaatac atttttatat 9840
ttatttagca actttataga aaacgatgaa tattatgtat cccttttaaa atcaagctat 9900
atagatttgg aaaaaataaa tatagaagag tatagtgatt taaccgagtt tataaaaaac 9960
atagaggaaa ataagataaa aagaaagaca gtatccaaaa taaatttgcg tagcaacctg 10020
ggtttagaga gagataatgc tcatattaat gttttaataa tagataaaca aaaagtcgaa 10080
aataatatta gcgaatctta tgaatttata tttagttatt tagttaatga taacaatata 10140
aatctcgaat taatatttaa aaataataag ttgcgggaaa tctcagtaga aaagtttttt 10200
aatacttata taaatattat ttctgattta atataa 10236
<210> 5
<211> 2745
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsD的核苷酸序列
<222>(1)…(2745)
<400> 5
atgtttaatg tttttgaaca actagataaa aacgaaatta agttcactaa aaaagaaata 60
gaaaccaaag tcgctataat tggaataagc tcgagattac ctaaatcaaa taactttaga 120
gatttttggg gtaatttggt taatggagtt gattgtattg gagaaataga tagccagaga 180
aaaaaggatt gcgattattt ttttgagaat aaaggtatag atacgaaaaa aataaactat 240
cagcaaatgg cttttgtaaa cgatgtggat aaatttgatt acaaatattt taatattcct 300
tatcaagaag catctttgat ggaccctcgt caaagattat ttttagagtc aagtattcat 360
gcattattgg atgcaggata cccagaaaaa aaagttaacg gaagtaatat tggtgtatac 420
gttggtagca gtaatagtga ttcaatatat aattatgata aggttatata tgattcatct 480
attcgaaatc ctgtaagtat tgttaataat cttccttcaa tgattgctag tcgggtttct 540
tattatttag atttaaaagg acctagtatg actctagata cttcttgttc ctcctctctt 600
gtagcagttc acgaagcatg taaagctata attcataatg aatgtgagat ggctattgca 660
gggggatgta acttaaatct tattcctctt cagcatgaag taaggctcgg cattgaatcg 720
agtactttta gaacgagggc ttatgacaat gactcagatg gaacaggatt tggagaaggt 780
attattacat ttattttaaa acctttattc agtgctttaa gagataatga taacatttat 840
tctgtgatag aaggaagtag tgttagtaat caagggtttt cttttggggt tgctactcca 900
agcattgaag gacaggaaaa ccttatttcg gaaaatatta gcaagtccaa tatagatatc 960
aatgacattt cgtttataga agggcatgga acaggaactg caattggaga tgtaatagag 1020
atagaagcaa tcaatcgtgc tttctccgaa attaacaaaa aaaattattt gcctatgggg 1080
tctgtaaaaa ataatatagg tcatttatta agtgcatccg gagctgctgg attattaaaa 1140
agcgtgcttg cattaaaaaa tcaaatgttt cccccaacga taaattataa tactcctaat 1200
aaaaatatag attttagtaa ctcacctgtg tttattaata ctgagaaagt agagttacat 1260
aaaaaagaat tgctgttagg atgtataagt tcttttggta tgagcggaac taattgccat 1320
atgattattt ctaattacga caaaaaaaga actattccaa atgaggattg tttagagcct 1380
caaatattca cattttctgc aaacgatttg gatactttaa cttttctatt aagtgattta 1440
tataattttc ttcttgaaga gaatctgatg ctactaagtg atattggata tacattgaat 1500
tgcagaagag atcatttagc aaatagagtg gctataatag catcatgtaa ggaagattta 1560
ttagaaaaat taaaagaagc attaaataag attcaacaag gaaataaaat tattaaagat 1620
agtgaaattt attttaacgt tagtgatata acctctaatg taaaagaatt gtctttagga 1680
gatgagtctc aagacgattt aaggcaaatt tgcagaaggt atattatggg cggtgaaata 1740
agttgggaca tgtattataa agaatataat caagttgttt ctttgcctgt tttcaccttt 1800
aagaaaactc ggtgctggta tagtgaagaa aaaatagact ttgtcttaaa tgacgatgtt 1860
attttaaatg aaaaagaaaa aattgtagtt gaaacatggg caaaactatt tggcctaaat 1920
aaaatagact tttctcaaga tatttaccaa gtaggtggag attccttgag cttggtaagt 1980
atgtatcatg aaatagagtc gaaaataccc aatatttcat tagaagtgtt tatagaaaaa 2040
gaaacattaa atgaaattct agattacgtt aatgaagcat cacaaaaaaa taatgaaaca 2100
gatgaattaa tttttgaaaa gaataattgt aaagtttata aattctcgaa tggaataaat 2160
aattcaaaca cgtatttaat ttgtaatcgt cataagagta tccttattga ttctggagtt 2220
agtttagaat atttaacaga gttctgtgaa agattgaaat tgaatcgaat agattatatt 2280
gttcttacac atgctcactt tgatcatact tattctgcaa atgaaataaa agaaaaattc 2340
aattgtaatt tcataattca tgaaagagaa ataaattttg cgagagatat cataaataat 2400
ggacttgccc ttcttgggaa aagttttacg tttgaaagcg atgatatcct aattaaagat 2460
aaatatacag ttttaaacat tggagatatt agcctcaaga taattctcag tccgggtcat 2520
acaacaggta gcgtatgcgt gctttatgac aattttttat ttacaggtga tactattttt 2580
aaagataaaa aagcaaaacc cgataaaaaa acaggaaatg aagattattt aaagaaatcc 2640
atttcttatt taattaataa tactgaagat aatactataa tatgccctgg tcatgaagat 2700
attacaacaa tttcatttgt gaaaaaaaga atttatgatt tttga 2745
<210> 6
<211> 1113
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsE的核苷酸序列
<222>(1)…(1113)
<400> 6
atgaggtgtt tagatgtgaa gttggtgatt agtgacagtg tttcggattt ttttgataag 60
atttcttctc tgcttttctt aaaacatgaa ggattcttca aagattatag cattaaaatg 120
ggtataaaat tagataatga atattttgta ttactagatg atcttaaaag aataaaagag 180
ttagatagtg agtttttaaa tttttatttt ggcagctttt gcgactacaa agatgttagg 240
aatgcaaata ttttttgttt agccacaacc ttttttaaaa taccacaaat ttatacgaaa 300
gatttttttg aaattattga ttatttgtct gcagttacag aagaagagat aagaaaaaat 360
attttagagt ctctaattta tttacaagga gatcttaaaa ctgaaacatc tacccaacag 420
tatttaaatg atagggaaag tttatttgat ctacttaata atctaaatat caagcccgaa 480
tacaagtgga tcatatttaa ggtaatgaaa tctcctaaaa cttatgtaga ccaattttgc 540
aataaattaa tgttggtgaa tggttatctt aacgaggcga tttataattt aattaaattt 600
agagatgagt ggataaatca attaaaaaac ataaatatgg attttccaaa aaagataatc 660
agtacgttaa aaggtaagga atacttatct tgtaattata ttaatatata tccaacactt 720
ttaccttaca ctgcaactat tacgaaaagt gaagataaca tttatgttgg actggggtat 780
aaagttgatg aaatttttga aaatacattg gagaaagcag agattaatca catcattgat 840
tttttgaagc ttttatcgga taaaagtaag ttcaagatat taatcttact tagtcataaa 900
aaaatgtatg ctaatgagat tgctgaacag ctaaatttaa ccaacgcaac gatttcacac 960
catatgagaa ttttgacatt gcaaggttta gttgagagta cgagaaatca aaataaaaca 1020
tactattatc ttaattcttc taaatttcat tctcttctaa atggattatt agggaaattt 1080
aagtctgctt taattttaga gaaagaggag taa 1113
<210> 7
<211> 1650
<212> DNA
<213>贝莱斯芽孢杆菌FZB42 (Bacillus velezensis FZB42)
<221> nrsF的核苷酸序列
<222>(1)…(1650)
<400> 7
gtgtataaaa cagtttatga ttttatagtt gatgctgaaa atcgagatga taaaggaatt 60
acatttataa atggatccga taatgaagta tatatttctt atgctcaatt atttgaaaaa 120
tccagaaaag tattaggtgt tttacaaaaa aagggtttaa gaaagaaaga tgaactaatt 180
atacaaattg aggataatga acaatttatt aaagtttttt gggcgtgcat attaggtgga 240
atcataccta ttcctttaag tgtggggtat aatgaagagc ataaacaaaa aatacttcgt 300
atatggagta ttcttaataa tccttattgt atttctgatc aaagaacatt acattcactt 360
gagaaagttg cagatgcaga aacttattca tcaattgtaa aacaatcaat ttatttagat 420
gaaattaaga attctaatga agtgggtgaa ttaatttccc ctaaaccaga agaaattgct 480
tttattcaat tctcttctgg tacaactgga gatcctaaag gtgtaatatt aacacataaa 540
aatcttatta ccaatatatc agcattaaat gaggcttggg agacatcgaa atccgattcc 600
tcactaagct ggatgccttt aactcatgat atgggcctca ttgctattca cttagcatca 660
acgtataaaa aaattcaaca atatataatt ccaacctccg tttttatccg aagaccgact 720
ttgtggttat tgaaaacaca tcaacaccgt gttacacaat tatactcccc taactttgga 780
tataagtttt tgttggataa ctataaaaaa aatcaaatct ataactggga tttaacttgt 840
gttagattat tagctaatgg agcagaacca atttctactt ctttgtgcca gagattttta 900
gaagaaatga aacaattcgg attaaaatat gaaaccctaa atactgtata tggtttagct 960
gaggctacag ttgctgttag tgtcccaaaa ttaggaaact cttttgttcc tgtaaccctt 1020
aatagggaag ctttagaagt aggtcaaaaa attcaaaaaa ctgttgaaga aaaaaaggga 1080
ataacttttg ttgaagttgg cccggctgta cgtcattgtt ctttccgtat atgtgattct 1140
aataattacg ttctggatga tgattttata ggtcatgtgc aaataaaggg ggaaaacgta 1200
acgtctgggt attacaataa tgaatctgct accaaagacg ctataactgc agatggatgg 1260
ttaaatacag gagaccttgg gtttatacat aataatagtt tggttattac cggaagaaca 1320
aaggatatca ttataaagaa tggccaaaat tattattcac atgatataga gagagtagca 1380
gagaatgtaa aggatattga attaggtaat gttgtggcat gcggggtccc gaatgaaaca 1440
aaggcggaag aagatcttgt gatatttgta aaatgtaagc aaagttacga tattacaaca 1500
gcagagaagg aattgaaaag agaattaaca aaacaattgg gtttgaatgt acatcaagtt 1560
atacctgtta ggaaaatttt caaaactaca agcggtaaag tgcaacgttc taaaatgaag 1620
gatttttata aagagttaat tctgtcttga 1650
Claims (4)
1.一组芽孢噻唑类化合物,其特征在于:所述芽孢噻唑类化合物是命名为芽孢噻唑A的化合物1;或是命名为芽孢噻唑B的化合物2;或是命名为芽孢噻唑C的化合物3;或是命名为芽孢噻唑D的化合物4;或是命名为芽孢噻唑E的化合物5;其化学构式依次如式Ⅰ至式V所示:
2.权利要求1所述芽孢噻唑类化合物的制备方法,其特征在于:所述芽孢噻唑类化合物是由枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs)经过液体发酵后,从其发酵产物中得到;其中,所述枯草芽孢杆菌1A751-Sfp/nrs突变菌株是通过ExoCET方法克隆nrs基因簇,构建得到p15A-spec-nrs质粒,然后以枯草芽孢杆菌1A751-Sfp(Bacillus subtilis 1A751-Sfp)为出发菌株,利用自然转化的方法将p15A-spec-nrs质粒转入该菌株中,使nrs生物合成基因簇通过双交换插入至amyE基因处得到;其中,所述nrs基因簇来源于贝莱斯芽孢杆菌FZB42(Bacillus velezensis FZB42),其大小为19.8kb,共包含7个基因,分别是nrs0编码转运蛋白,该基因的核苷酸序列如SEQ ID No.1所示;nrsA编码硫酯酶,该基因的核苷酸序列如SEQ ID No.2所示;nrsB编码氧化酶,该基因的核苷酸序列如SEQ ID No.3所示;nrsC编码非核糖体肽合成酶,该基因的核苷酸序列如SEQ ID No.4所示;nrsD编码聚酮合酶-内酰胺酶,该基因的核苷酸序列如SEQ ID No.5所示;nrsE编码调控蛋白,该基因的核苷酸序列如SEQ ID No.6所示;nrsF编码脂肪酰-AMP连接酶,该基因的核苷酸序列如SEQ ID No.7所示。
3.权利要求1所述芽孢噻唑类化合物在制备蛋白酪氨酸磷酸酶(PTPs)抑制剂中的应用,其中所述芽孢噻唑类化合物是命名为芽孢噻唑A的化合物1;或是命名为芽孢噻唑B的化合物2;或是命名为芽孢噻唑C的化合物3;或是命名为芽孢噻唑D的化合物4;或是命名为芽孢噻唑E的化合物5;所述蛋白酪氨酸磷酸酶是蛋白酪氨酸磷酸酶1B(PTP1B)。
4.一种权利要求1所述芽孢噻唑类化合物的产生菌株,其特征在于:所述菌株是枯草芽孢杆菌1A751-Sfp/nrs突变菌株(Bacillus subtilis 1A751-Sfp/nrs);该突变菌株是通过ExoCET方法克隆nrs基因簇,构建得到p15A-spec-nrs质粒,然后以枯草芽孢杆菌1A751-Sfp(Bacillus subtilis 1A751-Sfp)为出发菌株,利用自然转化的方法将p15A-spec-nrs质粒转入该菌株中,使nrs生物合成基因簇通过双交换插入至amyE基因处得到;其中,所述nrs基因簇来源于贝莱斯芽孢杆菌FZB42(Bacillus velezensis FZB42),其大小为19.8kb,共包含7个基因,分别是nrs0编码转运蛋白,该基因的核苷酸序列如SEQ ID No.1所示;nrsA编码硫酯酶,该基因的核苷酸序列如SEQ ID No.2所示;nrsB编码氧化酶,该基因的核苷酸序列如SEQ ID No.3所示;nrsC编码非核糖体肽合成酶,该基因的核苷酸序列如SEQ ID No.4所示;nrsD编码聚酮合酶-内酰胺酶,该基因的核苷酸序列如SEQ ID No.5所示;nrsE编码调控蛋白,该基因的核苷酸序列如SEQ ID No.6所示;nrsF编码脂肪酰-AMP连接酶,该基因的核苷酸序列如SEQ ID No.7所示。
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