申请/专利权人：灿盛制药荷兰公司;格罗宁根大学

申请日：2017-04-13

公开（公告）日：2022-10-18

公开（公告）号：CN109563528B

主分类号：C12P15/00

分类号：C12P15/00;C12P17/02;C12P35/00;C12P37/00;C12N1/15

优先权：["20160414 EP 16165262.3","20160630 EP 16177069.8"]

专利状态码：有效-授权

法律状态：2022.10.18#授权;2019.05.07#实质审查的生效;2019.04.02#公开

摘要：本发明涉及改善由非核糖体肽合成酶催化的次生代谢物的产生的方法，该方法包括在真核宿主中使真核非核糖体肽合成酶和MbtH样蛋白接触。本发明进一步涉及包含作为非杂合体的真核非核糖体肽合成酶和原核MbtH的组合物，以及包含非核糖体肽合成酶和允许表达MbtH样蛋白的多核苷酸的真核宿主细胞。

主权项：1.一种改善由非核糖体肽合成酶催化的次生代谢物的产生或导致所述次生代谢物的途径中发生的前体的产生的方法，该方法包括在真核宿主中使所述非核糖体肽合成酶与MbtH样蛋白接触，其特征在于所述非核糖体肽合成酶来自真核起源并且不是杂合体，其中所述MbtH样蛋白的序列如SEQIDNO:1、3、4、6或7所示，并且其中所述真核宿主是产黄青霉。

全文数据：真核NRPS催化过程中的MBTH样蛋白技术领域本发明涉及改善由非核糖体肽合成酶催化的次生代谢物的产生的方法，该方法包括在真核宿主中使真核非核糖体肽合成酶和MbtH样蛋白接触。本发明进一步涉及包含作为非杂合体的真核非核糖体肽合成酶和原核MbtH的组合物，以及包含非核糖体肽合成酶和允许表达MbtH样蛋白的多核苷酸的真核宿主细胞。背景技术次生代谢物是通过初生代谢物合酶的修饰在微生物中产生的化合物。它们不像初生代谢物那样在生长、发育和繁殖中发挥作用，但许多通过作为抗生素和通过产生色素在生态功能包括一种或多种防御机制中发挥作用。如今，许多次生代谢物具有很高的社会价值，并且在发酵过程中通常以工业规模进行生产。在工业微生物学中具有重要作用的次生代谢物的一些实例包括阿托品、博来霉素和抗生素如杆菌肽、红霉素、青霉素和万古霉素。与任何其他产生过程的情况一样，产生次生代谢物的工业发酵也是产量改进计划的持续主题。这提高了单位生产率，降低了成本，并且在许多情况下改善了产品的分离和纯化，从而最终提高了产品质量。除了提高发酵过程产量的众多明显策略如调整营养成分、优化pH和温度等条件、基因改造微生物途径和完善下游加工之外，通过实施可以进一步提高生产率的新技术，仍有对于进一步提高的持续需要。MbtH样蛋白是小8-10kD蛋白，其具有类似于结核分枝杆菌的MbtH的特别保守的序列基序。尽管最近的研究表明其在肽的生物合成中起作用，但MbtH样蛋白的功能在很大程度上仍然是未知的。编码MbtH样蛋白的基因mbtH样基因通常存在于非核糖体肽合成酶NRPS基因簇中。非核糖体肽NRP是一类重要的次生代谢物。GenBank中存放有许多mbtH样基因。为了鉴定MbtH样蛋白，一项BLASTP研究显示，同源物由放线菌门、壁菌门和变形菌门的成员编码，而非古菌R.H.Baltz,J.Ind.Microbiol.Biotechnol.[工业微生物学及生物技术杂志]201138,1747-1760。在天然存在的真核NRPS基因簇中没有关于mbtH样基因的报道，它们的功能仅与原核NRPS相关。然而，在WO2013113646中，描述了MbtH样蛋白在青霉菌中制备半合成β-内酰胺抗生素中的用途。然而，在这种情况下，NRPS是非天然杂合体包括真核和原核模块，并且所讨论的MbtH样蛋白仅对由NRPS的原核模块催化的腺苷酸化反应产生积极影响。这证实了MbtH样蛋白仅作用于原核NRPS的预断。然而，真核生物尤其是像曲霉属、青霉属和木霉属的真菌是工业产生过程中使用的一类重要的微生物。来自真核生物的若干种经济上有吸引力的次生代谢物例如，β-内酰胺抗生素、黄青霉素chrysogenins、酪青霉毒素roquefortins、环孢菌素和棘白菌素。它们属于真菌非核糖体肽。上述对进一步提高次生代谢物的发酵生产率的一般需求同样适用于由真核NRPS催化的那些过程。具体实施方式在本发明的上下文中，与模块组合使用的术语“异源”是指其中结构域例如腺苷酸化或缩合结构域来自不同模块的模块。这些不同的模块可以来自相同的酶或可以来自不同的酶。术语“杂合体”是指NRPS，该NRPS包含来自真核起源和原核起源的模块。通常，杂合NRPS是通过遗传构建获得的，而非天然存在的。不是杂合体的NRPS仅包含来自真核起源的模块或仅包含来自原核起源的模块。术语“模块”定义了催化单元，该催化单元能够在产物通常是肽中掺入一个肽结构单元通常是氨基酸，并且可以包括用于修饰的结构域如差向异构化和甲基化。术语“非核糖体肽”或“NRP”是指肽次生代谢物，通常由微生物如细菌和真菌产生。NRP由NRPS合成。NRP通常具有环状和或支化结构，可含有非蛋白原氨基酸包括D-氨基酸，携带修饰如N-甲基和N-甲酰基基团，或被糖基化、酰化、卤化或羟基化。通常进行氨基酸相对于肽“骨架”的环化，产生噁唑啉和噻唑啉，它们可以被进一步氧化或还原。有时，对丝氨酸进行脱水，产生脱氢丙氨酸。NRP通常是链接在一起或环化或甚至分支的相同序列的二聚体或三聚体。NRP通常是毒素、铁载体或色素。非核糖体肽抗生素，细胞抑制剂和免疫抑制剂可作商业用途。NRP的实例是抗生素如放线菌素、杆菌肽、头孢菌素C、达托霉素、短杆菌肽、青霉素G、青霉素V、泰斯巴汀teixobactin、短杆菌酪肽、万古霉素、双效菌素A、抗真菌药如棘白菌素或阿库来菌素、抗生素前体如ACV三肽、细胞抑制剂如博来霉素、埃博霉素、免疫抑制剂如环孢素、氮贮存聚合物如紫杉醇、植物毒素如AM-毒素、HC-毒素、色素如靛蓝、铁载体如肠杆菌素，美克奇林AmyxochelinA和毒素如微囊藻毒素、结节蛋白、蓝藻毒素。术语“非核糖体肽合成酶”或“NRPS”是指在细菌和真菌的细胞质中发现的一类模块化多结构域酶，其合成多种高度多样化的肽，并且与核糖体不同，该酶不依赖于信使RNA。NRPS被组织在多亚基簇中，每个亚基又由模块组成，这些模块能够进行一个链延伸循环。典型的模块由腺苷酸化A结构域、肽基载体蛋白PCP结构域和缩合C结构域组成。A结构域约550个残基和C结构域约450个残基负责加载具有同源氨基酸的PCP结构域并分别催化上游氨酰基或肽基PCP与下游肽基PCP之间的肽键的形成。在整个过程中，生长的肽链与磷酸泛酰巯基因子辅因子共价连接，磷酸泛酰巯基因辅因子本身通过专用的Ppan转移酶Pptase与保守的丝氨酸连接。术语“次生代谢物”是指不直接参与生物体的正常生长、发育或繁殖的化合物。次生代谢物通常局限于系统发育组内的狭窄组的物种，并且通常在防御系统中起重要作用。人类使用次生代谢物作为色素、调味剂和药物。次生代谢物的实例是生物碱如阿托品、可卡因、可待因、吗啡、河豚毒素、天然酚类如多酚、单萜类化合物如香叶基二磷酸酯、柠檬烯、蒎烯、二萜类化合物如阿非迪霉素、牻牛儿基牻牛儿基二磷酸酯、海松二烯、紫杉醇、NRP、色素如黄酮、真菌毒素如罗克福特和抗生素如β-内酰胺类如6-氨基青霉烷酸、7-氨基去乙酰氧基头孢烷酸、己二酰基-7-氨基去乙酰氧基头孢烷酸、头孢菌素C、青霉素G或青霉素V、链霉素、四环素。在本发明的第一方面，公开了一种改善由NRPS催化的次级或导致所述次生代谢物的途径中发生的前体的产生的方法，该方法包括在真核宿主中使代谢物所述NRPS与MbtH样蛋白接触，其特征在于所述NRPS来自真核起源并且不是杂合体。出人意料地发现，在真核宿主中引入的MbtH样蛋白积极地影响NRPS依赖性中间体和其他NRPS依赖性次生代谢物的产生水平。本发明通过一方面选择覆盖多种不同来源和等级的同源性的MbtH蛋白，另一方面结合全部是真核的一系列NRPS即不是杂合体，证明了成功的结果。表1总结了在两种不同菌株中用三种不同的NRPS和一系列MbtH样蛋白研究的许多实例，清楚地表明至少在次生代谢物的途径中的一个点上，所研究的NRPS和MbtH样蛋白结果的任何组合具有积极的效果。表1表4-9中在生产率方面由MbtH样蛋白的存在影响的平均代谢物数量的概述+：代谢物数量在生产率方面提高10％；+-：代谢物数量在生产率方面+-10％；-：代谢物数量在生产率方面降低10％在第一实施例中，真核宿主是真菌或酵母，因为在工业中它们是常规使用的真核微生物。合适的实例是曲霉属、克鲁维酵母属、青霉属、毕赤酵母属、酵母属、木霉属和耶氏酵母属，并且优选产黄青霉、构巢曲霉、黑曲霉、巴斯德毕赤酵母、乳酸克鲁维酵母、酿酒酵母或解脂耶氏酵母。在本发明的第二实施例中，如上所定义的次生代谢物是优选β-内酰胺、色素或真菌毒素。更优选地，β-内酰胺是6-氨基青霉烷酸、7-氨基去乙酰氧基头孢烷酸、己二酰基7-氨基去乙酰氧基头孢烷酸、头孢菌素C、青霉素G或青霉素V，色素是黄青霉素，并且真菌毒素是酪青霉毒素。在第三实施例中，真核宿主菌株是高产菌株。已经发现，例如在产黄青霉的一些菌株中高水平的青霉素产生是由于青霉素基因簇的扩增的串联重复序列的存在综述于MartinF.2000J.Bacteriol[细菌学杂志]182:2355-2362。次生代谢物的高水平产生可以是扩增的生物合成基因簇的结果，因此真核宿主是关于目标次生代谢物簇的多拷贝株。在第四个实施例中，优选的MbtH样蛋白是R.H.BaltzJ.Ind.Microbiol.Biotechnol.[工业微生物学及生物技术杂志]201138,1747-1760。更优选的MbtH样蛋白是以下这些：其包含不变氨基酸N17、E19、Q21、S23、W25、P26、P32、G34、W35、L48、W55、T56、D57、R59和P60，也适合地指具有氨基酸密码NXEXQXSXWP-X5-PXGW-X12-L-X6-WTDXRPSEQIDNO:17。在上述注释中，字母D、E、G、L、N、P、Q、R、S、T、W和X是指公知的氨基酸单字母密码其中X表示一个未指定的氨基酸，X5表示5个未指定的氨基酸，X6表示6个未指定的氨基酸，并且X12表示12个未指定的氨基酸。在优选的实施例中，所述MbtH样蛋白包含氨基酸密码NXEXQXSXWP-X5-PDGW-X12-L-X6-WTDXRP或NXEXQXSXWP-X5-PAGW-X12-L-X6-WTDXRP或NXEXQXSXWP-X5-PGGW-X12-L-X6-WTDXRP或NXEXQXSXWP-X5-PQGW-X12-L-X6-WTDXRP，其中X5选自由以下组成的列表：AFAEV、AFAAV、AFAEI、TFAEV、TFAAV、TFAEI、VFAEV、VFAAV和VFAEISEQIDNO:18-SEQIDNO:53。在更优选的实施例中，所述MbtH样蛋白包含氨基酸密码NXEXQXSLWP-X5-PDGW-X12-L-X6-WTDXRP或NXEXQXSLWP-X5-PAGW-X12-L-X6-WTDXRP或NXEXQXSLWP-X5-PGGW-X12-L-X6-WTDXRP或NXEXQXSLWP-X5-PQGW-X12-L-X6-WTDXRP，其中X5选自由以下组成的列表：AFAEV、AFAAV、AFAEI、TFAEV、TFAAV、TFAEI、VFAEV、VFAAV和VFAEISEQIDNO:57-SEQIDNO:92。值得注意的是，在R.H.BaltzJ.Ind.Microbiol.Biotechnol.[工业微生物学及生物技术杂志]201138,1747-1760和在WO2013113646中错误地提到了氨基酸密码NXEXQXSXWP-X5-PXGW-X13-L-X7-WTDXRP，其中实际上这指的是并且应该是NXEXQXSXWP-X5-PXGW-X12-L-X6-WTDXRP。优选地，本发明的MbtH样蛋白是从来自游动放线菌Actinoplanesteichomyceticus的替考拉宁生物合成簇中获得的Tcp13SEQIDNO:1或Tcp17SEQIDNO:2Sosio等人.,Microbiology[微生物学]2004150,95-102，或是从未培养的土壤细菌中可获得的Veg生物合成簇中鉴定的MbtH样同源物由GenBank:EU874252SEQIDNO:3被称为VEG8的nt33826-34035编码BanikJ.J.和BradyS.F.,Proc.Natl.Acad.Sci.USA[美国科学院院刊]2008105,17273-17277或是从未培养的土壤细菌中可获得的Teg生物合成簇中鉴定的MbtH样同源物由GenBank:EU874253SEQIDNO:4被称为TEG的nt33949-33158编码BanikJ.J.和BradyS.F.,Proc.Natl.Acad.Sci.USA[美国科学院院刊]2008105,17273-17277或是来自Amycolatopsisbalhimycina的balhimycine生物合成簇鉴定的MbtH样同源物SEQIDNO:5其被称为BPSRecktenwald等人.,Microbiology[微生物学]2002148,1105-1118,Stegman等人.,FEMSMicrobialLett.[微生物学函电]2006262,85-92或是在淡紫灰链霉菌Streptomyceslavendulae的补体生物合成簇中鉴定的MbtH样同源物SEQIDNO:6其被称为COMChiu等人.,Proc.Natl.Acad.Sci.USA[美国科学院院刊]200198,8548-8553或是在天蓝色链霉菌Streptomycescoelicolor的钙依赖性抗生素CDA生物合成簇中鉴定的MbtH样同源物SCO0489SEQIDNO:7其被称为CDAIHojati等人.Chem.&Biol.[化学与生物学]20029,1175-1187或MbtH样蛋白，其氨基序列与所述序列的同一性百分比为至少70％，更优选至少80％，甚至更优选至少90％，最优选至少95％。这种具有至少70％同一性百分比的多肽模块也称为同源序列或同源物。在第二方面，本发明提供了一种组合物，其包含不是杂合体的真核生物NRPS和原核生物MbtH。真核NRPS和MbtH样蛋白的优选组合是NRPSN-5-氨基-5-羧基戊酰基-L-半胱氨酰-D-缬氨酸合酶PcbABUniProtKB-P26046ACVS2_PENCH、SEQIDNO:54和COM、PcbAB和CDAI、PcbAB和TCP13、PcbAB和TEG、PcbAB和VEG8、NRPSPc21g12630蛋白ChyAUniProtKB-B6HLP9B6HLP9_PENRW、SEQIDNO:55和COM、ChyA和CDAI、ChyA和TCP13、ChyA和TEG、ChyA和VEG8、NRPS酪青霉毒素美利戈林meleagrin合成蛋白ARoqAUniProtKB-B6HJU6.1ROQA_PENRW、SEQIDNO:56和COM、RoqA和CDAI、RoqA和TCP13、RoqA和TEG或RoqA和VEG8。因此，优选的组合是SEQIDNO:54和SEQIDNO:1、SEQIDNO:54和SEQIDNO:3、SEQIDNO:54和SEQIDNO:4、SEQIDNO:54和SEQIDNO:6、SEQIDNO:54和SEQIDNO:7、SEQIDNO:55和SEQIDNO:1、SEQIDNO:55和SEQIDNO:3、SEQIDNO:55和SEQIDNO:4、SEQIDNO:55和SEQIDNO:6、SEQIDNO:55和SEQIDNO:7、SEQIDNO:56和SEQIDNO:1、SEQIDNO:56和SEQIDNO:3、SEQIDNO:56和SEQIDNO:4、SEQIDNO:56和SEQIDNO:6、SEQIDNO:56和SEQIDNO:7、或与任何一者或两者至少90％同源的序列。在第三方面，本发明提供了真核宿主细胞，其包含来自真核起源的不是杂合体的NRPS和允许表达MbtH样蛋白的多核苷酸。优选地，MbtH样蛋白是本发明第一方面的蛋白。优选地，宿主细胞是真菌或酵母。合适的实例是曲霉属、克鲁维酵母属、青霉属、毕赤酵母属、酵母属、木霉属和耶氏酵母属，并且优选产黄青霉、构巢曲霉、黑曲霉、巴斯德毕赤酵母、乳酸克鲁维酵母、酿酒酵母或解脂耶氏酵母。优选地，宿主细胞包含具有SEQIDNO:17的MbtH样蛋白。在第四方面，本发明提供了制备本发明第三方面的宿主细胞的方法。这可以根据本领域技术人员已知的方法实现，例如靶向或随机整合由合适的启动子、目的基因和终止子组成的表达盒。在整个说明书中，以下三字母密码和一字母密码用于氨基酸：同源性和同一性术语“同源性”或“百分比同一性”在本文中可互换使用。为了本发明的目的，这里定义为了确定两个氨基酸序列或两个核酸序列的同源性百分比，对齐序列以达到最佳比较目的。为了优化两个序列之间的比对，可以在比较的两个序列中的任何序列中引入缺口。这种比对可以在被比较的序列的全长上进行。或者，可以在较短的长度上进行比对，例如超过约20、约50、约100或更多的核酸碱基或氨基酸。同一性是报告的比对区域上两个序列之间相同匹配的百分比。可以使用数学算法完成序列的比较和两个序列之间的百分比同一性的确定。本领域技术人员将意识到若干种不同的计算机程序可用于比对两个序列并确定两个序列之间的同源性Kruskal,J.B.1983AnoverviewofsequencecomparisonInD.SankoffandJ.B.Kruskal[D.Sankoff和J.B.Kruskal中的序列比较概述],ed.,Timewarps,stringeditsandmacromolecules:thetheoryandpracticeofsequencecomparison[时间扭曲，字符串编辑和大分子：序列比较的理论和实践],pp.1-44AddisonWesley。两个氨基酸序列之间的同一性百分比可以使用用于比对两个序列的Needleman和Wunsch算法确定。Needleman,S.B.和Wunsch,C.D.1970J.Mol.Biol.[分子生物学杂志]48,443-453。该算法可以比对氨基酸序列和核苷酸序列。Needleman-Wunsch算法已在计算机程序NEEDLE中实现。出于本发明的目的，使用来自EMBOSS包的NEEDLE程序版本2.8.0或以上,EMBOSS:TheEuropeanMolecularBiologyOpenSoftwareSuite[EMBOSS：欧洲分子生物学开放软件套件]2000Rice,P.Longden,I.和Bleasby,A.TrendsinGenetics16[遗传学趋势16],6pp276-277,http:emboss.bioinformatics.nl。对于蛋白序列，EBLOSUM62用于取代矩阵。对于核苷酸序列，使用EDNAFULL。其他可以指定。使用的任选参数是10的缺口开放罚分和0.5的缺口延伸罚分。技术人员将理解，所有这些不同的参数将产生略微不同的结果，但是当使用不同的算法时，两个序列的总体百分比同一性不会显著改变。总体同源性定义同源性或同一性是两个序列在整个比对区域包括任何缺口上相同匹配的百分比。两个比对序列之间的同源性或同一性计算如下：比对中显示两个序列中相同的氨基酸的相应位置的数目除以包括缺口的比对的总长度。这里定义的同一性可以从NEEDLE获得，并在程序的输出中标记为“IDENTITY”。最长同一性的定义两个比对序列之间的同源性或同一性计算如下：比对中显示两个序列中相同的氨基酸的相应位置的数目除以减去比对中的缺口总数后的比对的总长度。这里定义的同一性可以通过使用NOBRIEF选项从NEEDLE获得，并在程序的输出中标记为“最长同一性”。实例通用材料和方法分子和遗传技术标准的遗传和分子生物学技术是本领域已知的例如，Maniatis等人.“Molecularcloning:alaboratorymanual[分子克隆：实验室手册]”1982冷泉港实验室,冷泉港,纽约；Miller“Experimentsinmoleculargenetics[分子遗传学实验]”1972冷泉港实验室,冷泉港,纽约；SambrookandRussell“Molecularcloning:alaboratorymanual[分子克隆：实验室手册]”第3版”2001冷泉港实验室,冷泉港实验室出版社；Ausubel“Currentprotocolsinmolecularbiology[当前分子生物学方案]”1987GreenPublishingandWileyInterscience,纽约。质粒和菌株大肠杆菌：使用大肠杆菌DH5α进行克隆。产黄青霉：DS17690HarrisD.等人.,Metab.Eng.[代谢工程]20068,91-101用作产黄青霉Penicilliumchrysogenum的高水平青霉素产生菌株，DS47274HarrisD.等人.,Metab.Eng.[代谢工程]20068,91-101用作低水平的青霉素产生菌株。它们在青霉素生物合成基因簇的数量方面不同。原始菌株DS17690含有8个拷贝，而DS47274是单拷贝菌株HarrisD.等人,Metab.Eng.[代谢工程]20068,91-101。培养基：大肠杆菌：使用2xPY15gL细菌用胰蛋白胨，10gL酵母菌提取物，10gL氯化钠，pH7.0在37℃和200rpm下培养所有培养物。补充抗生素25μgmL吉欧霉素以维持pIAT质粒。产黄青霉：产黄青霉菌株在YGG培养基BartoszewskaM.等人,Appl.Environ.Microbiol.[应用与环境微生物学]201177,1413-14221.中生长。通过在25℃下在R琼脂BartoszewskaM.等人,Appl.Environ.Microbiol.[应用与环境微生物学]201177,1413-14221上生长10-11天来刺激菌丝体的孢子形成。出于遗传操作的目的，通过它们在乙酰胺作为唯一氮源的平板上生长的能力选择转化体作为共转化体。乙酰胺-琼脂包含0.04mMFeSO4、2.03mMMgSO4、51.3mMNaCl、55.5mM葡萄糖、15gl琼脂、10gl乙酰胺、5.1mMKH2PO4、4.9mMK2HPO4、和2mll微量元素溶液Na3C6H5O7149mM、FeSO489mMMgSO41.04mM、H3BO30.2mM、Na2MoO40.05mM、CuSO42.56mM、ZnSO48.76mM、CoSO42.28mM、MnSO417.99mM、CaCl210.88mM、EDTA107mM。为了产生次生代谢物，使用青霉素产生性培养基PPMNijlandJ.G.等人,Appl.Environ.Microbiol.[应用与环境微生物学]201076,7109-7115+0.05％苯氧基乙酸。实施例1MbtH样蛋白表达构建体的合成设计和克隆为了在产黄青霉中表达MbtH样蛋白，设计了包含产黄青霉IPNS启动子和产黄青霉AT终止子的表达盒。包含来自侧翼为NotINdeI位点SEQIDNo8的产黄青霉的异青霉素N合成酶IPNS，pcbC-基因的启动子盒启动子pcbC、带有cat-ccdB盒的DNA片段氯霉素抗性和大肠杆菌的毒性基因和来自侧翼为NsilNotl-位点SEQIDNo9的产黄青霉的酰基辅酶A:异青霉素N酰基转移酶AT，pcbDE基因的转录终止子盒的载体pIAT被用于克隆作为启动子和终止子之间的NdeINsil片段的MbtH编码基因。选择了五种不同的MbtH样蛋白，一种来自替考拉宁生物合成簇注释为tcp13SEQIDNO:1，GenBank:AJ605139基因组DNA；翻译：CAE53354.1并称为TCP13，一种来自通过搜索Veg簇中的同源MbtH样序列鉴定的Veg生物合成簇SEQIDNO:3，GenBank:EU874252，nt33826-34035，在veg9和veg10之间，称为VEG8，一种来自互补的生物合成簇注释为假设的蛋白SEQIDNO:6，GenBank:AF386507基因组DNA；翻译：AAK81828.1并称为COM，一种来自通过在Teg簇中搜索同源MbtH样序列鉴定的Teg生物合成簇SEQIDNO:4，GenBank:EU874253，nt32949-33158，在teg8和teg9之间，称为TEG，以及一种来自钙依赖性抗生素生物合成簇SCO0489SEQIDNO:7来自天蓝色链霉菌Hojati等人.Chem.&Biol.[化学与生物学]20029,1175-1187，被称为CDAI。合成构建编码所选蛋白的靶基因，得到核苷酸SEQIDNO:10-14，并以gBlocks侧翼为限制性位点Ndei和Nail在IDT集成DNA技术公司IntegratedDNAtechnology,克拉尔维尔，爱荷华州，美国订购，用于IPNS启动子和AT终止子之间的最终克隆。编码VEG8的基因用作野生型序列，而编码TCP13、COM、CDAI、BPS和TEG的基因经密码子优化以在产黄青霉中表达。将具有在产黄青霉中过表达MbtH样蛋白的表达构建体通过将取自gBlocks的NdeINsiI片段克隆到表达载体pIAT的NdeINsiI位点构建的最终质粒命名为pIAT-Tcp13、plAT-Veg8、pIAT-COM、pIAT-TEG和pIAT-CDAI。使用由MacrogenMacrogenEurope，阿姆斯特丹，荷兰提供的测序确认MbtH表达构建体的最终序列。实施例2MbtH样蛋白表达构建体的转化用NotI切割pIAT-MbtH质粒，并在琼脂糖凝胶上电泳。从琼脂糖凝胶上切下包含MbtH表达盒的NotI片段，纯化凝胶切割的片段并通过脱盐浓缩。如KovalchukA等人.MethodsMolBiol.[分子生物学方法]2012；835:1-16.所述，进行两种产黄青霉菌株DS17690和DS47274的原生质体形成和转化。转化作为与amdS选择标记共转化进行，所述amdS选择标记包含在构巢曲霉gpdA启动子美国专利5876988,SeltenGCM,SwinkelsBW,vanGorcomRFM.1999.Selectionmarkergenefreerecombinantstrains:methodforobtainingthemandtheuseofthesestrains[选择标记基因游离重组菌株：获得其方法和这些菌株的用途]控制下的构巢曲霉乙酰胺酶编码基因amdS，摩尔比为10:1MbtH表达构建体：amdS选择标记。该转化方法导致MbtH表达构建体和amdS选择标记在宿主生物的基因组中随机整合，并且表达盒的数量可以根据每个获得的转化体而变化。由于存在于大多数情况下的这种变化，我们已经针对每个宿主菌株MbtH表达构建体变化研究了多个独立转化体，而不仅限于一个。12天后，使用三个乙酰胺琼脂到R琼脂平板转移循环，对每个MbtHamdS共转化的若干个选择的菌落进行纯化。在纯化过程中，分别丢弃在选择性乙酰胺琼脂上表现出生长不足或在R琼脂平板上表现出过快孢子形成的菌落。为了确认MbtH整合，将来自所选菌落的菌丝块在milliQ水中匀浆，并在PCR反应设置中用作模板DNA。引物靶向每个MbtH表达盒的5'侧翼IPNS启动子SEQIDNo:15和3'AT终止子SEQIDNo:16。最后，获得3至9个含有amdS阳性并且表达MbtH的构建体的转化体MbtH表达构建体和菌株背景用于进一步表征表达构建体VEG8除外，这里仅获得一个针对菌株背景DS17690的转化体和两个针对菌株背景DS47274的转化体。表2概述了获得的转化体和为不同转化体选择的密码。表2针对产生高水平和低水平青霉素的产黄青霉菌株DS17690和DS47274，含有amdS阳性和MbtH基因的转化体的概述。实施例3用于产生次生代谢物的表达MbtH样蛋白的产黄青霉菌株的小规模发酵将表2中总结的经阳性测试的青霉菌株在100ml摇瓶中进行共5天的小规模发酵实验。作为参考菌株，采用未转化的菌株DS17690和DS47274。在体积25mL的YGG培养基中接种来自孢子作物的培养物。生长24小时后，将孢子形成的预培养物在PPM加0.25％苯氧基酸中2x30mL的总体积稀释10倍。每个菌株和实验使用两个生物学和两个技术重复。使用Innova44振荡器埃普多夫，汉堡，德国在250℃和200rpm下培养所有培养物。在预培养物转移后第2天和第5天进行取样。然后，取出1mL培养物，在40℃、14000xg下离心10分钟。随后使用PTFE注射器式滤器0.2μM，No.514-0068，VWR，拉德纳，宾夕法尼亚州，美国过滤上清液，用10mMDTT1,4-二硫苏糖醇，No.6908.2，CarlRothGmbH卡尔罗斯有限公司，卡尔斯鲁厄，德国还原并保存在-80℃直至分析。另外使用5天后培养物的剩余体积确定干重。实施例4次生代谢物的分析产黄青霉的基因组编码10种NRPS、20种聚酮合酶PKS和2种杂合NRPS-PKS基因vandenBerg等人.NatureBiotech[自然生物技术]200826,1161-1168。这些基因中的一些与特定的次生代谢物有关。选择三组次生代谢物其生物合成途径已分配给特定的NRPS分别用于分析次生代谢物产生：青霉素相关的次生代谢物NRPS:PcbAB-Pc21g21390、酪青霉毒素相关代谢物RoqA-Pc21g15480和黄青霉素相关代谢物NRPS:ChyA-Pc21g12630。根据保留时间和精确质量，使用可用标准通过LCMS分析鉴定峰。总共26种代谢物与这三种簇相关SaloO.V.,BMCGenomics[BMC基因组学]2015,16:937。表3通过应用的LCMS方法概述了可检测的参考化合物。显示了所应用的LC程序的生物合成簇、化合物名称、单同位素离子化质量MZ[H]+、分子组成和保留时间。在培养样品中，26种代谢物中的23种足够丰富，从而允许与野生型菌株进行比较。可以按以下方式将23种相关代谢物分配到三个簇中；用于青霉素生物合成的3种代谢物，用于黄青霉素生物合成的10种代谢物和用于酪青霉毒素生物合成的10种代谢物。通过测量峰面积，使干重归一化并最终计算相对于野生型代谢物丰度的比率来进一步评估代谢物水平。在表4-9中总结了在表达MbtH的转化体中鉴定的所有代谢物和中间体及其与未转化的野生型菌株相比的相对丰度的完整列表，其中表4、表5和表6分别显示了在培养2和5天后研究的在MbtH样蛋白存在下产生高水平青霉素的菌株DS17690中青霉素、酪青霉毒素和黄青霉素簇中次生代谢物的相对产生率，而表7-9显示了在产生低水平青霉素的菌株DS47274中情况。对在相同培养时间点和相同菌株背景下测量的相应簇的每种代谢物，计算所有表达MbtH的转化体的相对生产力的平均值以使观察到的趋势可视化。代谢物被分类为a增加平均值高于1.1生产率提高10％；b减少平均值低于0.9生产率降低10％以及未改变平均值x为1.1x0.9生产率+-10％。最后，确定每个生物合成簇、菌株和培养日的分类为增加、减少或未改变的代谢物总数，并总结在表1中。将从青霉素发酵获得的5μL的每种培养物上清液样品进行LCMS分析。每个样品运行两次技术重复。使用LCMSOrbitrap机器赛默飞世尔科技ThermoScientific与RP-C18柱Shimadzu日本岛津公司垫片组XR-ODS2.2；3.0x75mm组合以正离子模式进行分析。使用MiliQ水A，乙腈B和2％甲酸D运行梯度程序；0min，A90％、B5％、C5％；4min，A90％、B5％、C5％；13min，A0％、B95％、C5％；16min，A0％、B95％、C5％；16min、A90％、B5％、C5％；21min，A90％、B5％、C5％，流速0.3mlmin-1。表3-9的图例表3：应用LC程序测量的次生代谢物及其相应的生物合成途径概述。表4：在培养2天和5天后，在所示MbtH样蛋白存在下，菌株DS17690中的青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS17690不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表5a：在培养2天后，在MbtH样蛋白存在下，菌株DS17690中的黄青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS17690不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表5b：在培养5天后，在MbtH样蛋白存在下，菌株DS17690中的黄青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS17690不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表6a：在培养2天后，在MbtH样蛋白存在下，菌株DS17690中的酪青霉毒素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS17690不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表6b：在培养5天后，在MbtH样蛋白存在下，菌株DS17690中的酪青霉毒素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS17690不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表7：在培养2天和5天后，在MbtH样蛋白存在下，菌株DS47274中的青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS47274不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表8a：在培养2天后，在MbtH样蛋白存在下，菌株DS47274中的黄青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS47274不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表8b：在培养5天后，在MbtH样蛋白存在下，菌株DS47274中的黄青霉素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS47274不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表9a：在培养2天后，在MbtH样蛋白存在下，菌株DS47274中的酪青霉毒素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS47274不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表9b：在培养5天后，在MbtH样蛋白存在下，菌株DS47274中的酪青霉毒素簇中次生代谢物的相对生产率。将生产率与未经修饰的菌株DS47274不存在MbtH样蛋白进行比较，其中所有值均设定为1.0。表3表4表5a表5b表6a表6b表7表8a表8b：表9a表9b：序列表DSMSinochemPharmaceuticalsNetherlandsB.V.[中化帝斯曼制药有限公司荷兰公司]RijksuniversiteitGroningen真核NRPS催化过程中的MBTH样蛋白PCTP20131492BiSSAP1.3.5169PRT游动放线菌（Actinoplanesteichomyceticus）1MetThrAsnProPheAspAsnGluAspGlySerPheLeuValLeuVal151015AsnGlyGluGlyGlnHisSerLeuTrpProAlaPheAlaGluValPro202530AspGlyTrpThrGlyValHisGlyProAlaSerArgGlnAspCysLeu354045GlyTyrValGluGlnAsnTrpThrAspLeuArgProLysSerLeuIle505560SerGlnIleSerAsp65269PRT游动放线菌2MetThrAsnProPheAspAsnGluAspGlySerPheLeuValLeuVal151015AsnGlyGluGlyGlnHisSerLeuTrpProAlaPheAlaGluValPro202530AspGlyTrpThrGlyValHisGlyProAlaSerArgGlnAspCysLeu354045GlyTyrValGluGlnAsnTrpThrAspLeuArgProArgSerLeuVal505560GluGlnAlaAspAla65369PRT未培养的土壤细菌3MetThrAsnProPheAspAsnGluAspGlyThrPhePheValLeuVal151015AsnAspGluGlyGlnHisSerLeuTrpProThrPheAlaGluValPro202530AlaGlyTrpThrArgValHisGlyGluAlaThrArgGlnGluCysLeu354045AlaTyrValGluGluAsnTrpThrAspLeuArgProLysSerLeuIle505560GlnAlaLeuGlyAla65469PRT未培养的土壤细菌4MetThrAsnProPheAspAsnGluAspGlySerPhePheValLeuVal151015AsnAspGluGlyGlnHisSerLeuTrpProThrPheAlaGluValPro202530AlaGlyTrpValCysValTyrGlyGluAlaThrArgGlnGluCysLeu354045ThrPheValGluGluAsnTrpThrAspLeuArgProLysSerLeuIle505560GlnGluValGlyGly65569PRTAmycolatopsisbalhimycina5MetSerAsnProPheAspAsnGluAspGlySerPhePheValLeuVal151015AsnAspGluGlyGlnHisSerLeuTrpProThrPheAlaGluValPro202530AlaGlyTrpThrArgValHisGlyGluAlaGlyArgGlnGluCysLeu354045AlaTyrValGluGluAsnTrpThrAspLeuArgProLysSerLeuIle505560ArgGluAlaSerAla65673PRT淡紫灰链霉菌（Streptomyceslavendulae）6MetThrAsnProPheAspAsnGluAsnGlyThrPheLeuValLeuVal151015AsnAspGluGlyGlnHisSerLeuTrpProValPheAlaGluIlePro202530GlnGlyTrpThrThrAlaPheGlyGluAlaSerArgAlaGluCysLeu354045GluPheValGluGlnAsnTrpThrAspMetArgProLysSerLeuVal505560AlaArgMetGluGlyThrAlaThrAla6570770PRT天蓝色链霉菌（Streptomycescoelicolor）7MetSerThrAsnProPheAspAspAlaAspGlyArgPheLeuValLeu151015ValAsnAspGluGlyGlnHisSerLeuTrpProAlaPheAlaAlaVal202530ProGlyGlyTrpThrThrValPheGluGluAsnThrArgAspAlaCys354045LeuAlaTyrValGluAlaAsnTrpThrAspLeuArgProArgSerLeu505560AlaArgThrAlaAspAla65708955DNA人工序列"合成的"合成的8gcggccgcaaagcttgatatcgaattccttatactgggcctgctgcattggtctgccatt60gcagggtatatatggctgacctggccaatctccatcgagaatctgggcgactgaagcact120gcccgcagacaagatggagactttcgtctagcacggtctagggcagatccgatgccattg180gctctgtcaactgtcgactacatgtatctgcatgttgcatcgggaaatcccaccacaggg240acagccaagcggccccgcgacttggcagtgggcaaactacgcccgattctggtgccaaga300accgagaagaatgagacagacccacgttgcactctaaccggatgctatcgacttacggtg360gctgaagattcaacacgctgcaacgagagccaaggtggtccggacattttctacgtgccg420gtttaccttggaacatcgccgtcgttgagtgcacgttgccttctctctcgtggcttggct480gggcccacgagcccgattgactcgacggcgttacttgggtatctatggccccgttttctg540gcacggtaatgataagtacttactagtcttcgagcggggaagtgttgctctgcccgagca600tcaacgattggcctgatcgcaccgtctgcaaatgccacggtgcggaccgactgaaatctc660agaccaccaaagaccctccgacttcgagatacggttactaattttacactggctccagcg720gccccatccagtaagcatctgggctgcaagcgtataatgtctccaggttgtctcagcata780aacaccccgcccccgctcaggcacacaggaagagagctcaggtcgtttccattgcgtcca840tactcttcactcattgtcatcgcaggagaacttcccctgtccctttgccaagccctctct900tcgtcgttgtccacgccttcaagttttcaccattatttttctagacacccatatg9559538DNA人工序列"合成的"合成的9atgcatcttttgtatgtagcttcaaccgactccgtcttcacttcttcgcccgcactgcct60accgtttgtaccatctgactcatataaatgtctagcccctacctacactatacctaaggg120agagaagcgtagagtgattaacgtacgggcctatagtaccccgatctctagatagaacat180ttagtagagattaggatgcctaactaatttaacttgagcattgtcccgttcatattgatt240ttcagtccattatacactcttaatcgtttcccggtagaagcctgatatatacgaccatag300ggtgtggagaacagggcttcccgtctgcttggccgtacttaagctatatattctacacgg360ccaatactcaatgtgcccttagcacctaagcggcactctagggtaagtgcgggtgatata420ggtgagaagtcttaagactgaagacaggatatcacgcgttaccctgcaccgtacctacta480ccttcaatatcaactctttcaggatggacagggtcgacactagttctagagcggccgc53810219DNA人工序列"合成的"合成的10catatgacgaacccatttgacaacgaggacggatcatttttggtattggttaacggtgaa60ggtcaacacagcctgtggccggcattcgccgaggtgccggatggctggaccggcgttcat120ggtccggcgagccgccaagactgcctgggttacgtcgaacagaattggaccgatctgcgt180ccgaaaagcctgatctcccagattagcgattgaatgcat21911219DNA人工序列"合成的"合成的11catatgaccaacccgttcgacaacgaggacggcaccttcttcgtgctggtcaacgacgag60ggccagcactccctctggccgaccttcgccgaggtgcctgccggctggacccgcgtgcac120ggtgaagccacccggcaggagtgcctcgcgtatgtcgaggagaactggacggacctgcgg180ccgaagagcctcatccaggccctcggcgcctgaatgcat21912231DNA人工序列"合成的"合成的12catatgactaacccgttcgacaacgagaacggcactttccttgttcttgttaacgacgag60ggacagcactctctttggccggttttcgctgagattccgcagggctggactactgctttc120ggcgaggcttctcgtgctgagtgccttgagttcgttgagcagaactggactgacatgcgt180ccgaagtctcttgttgctcgtatggagggcactgctactgcttgaatgcat23113219DNA人工序列"合成的"合成的13catatgactaacccgttcgacaacgaggacggctctttcttcgttcttgttaacgacgag60ggccagcactctctttggccgactttcgctgaggttccggctggctgggtttgcgtttac120ggcgaggctactcgtcaggagtgccttactttcgttgaggagaactggactgaccttcgt180ccgaagtctcttattcaggaggttggcggctgaatgcat21914222DNA人工序列"合成的"合成的14catatgtctactaacccgttcgacgacgctgacggccgtttccttgttcttgttaacgac60gagggccagcactctctttggccggctttcgctgctgttccgggcggctggactactgtt120ttcgaggagaacactcgtgacgcttgccttgcttacgttgaggctaactggactgacctt180cgtccgcgttctcttgctcgtactgctgacgcttgaatgcat2221526DNA人工序列"合成的"合成的15gtctgccattgcagggtatatatggc261630DNA人工序列"合成的"合成的16cgggacaatgctcaagttaaattagttagg301744PRT人工序列合成的合成的位点2,4,6,8,11,12,13,14,15,17,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的17AsnXaaGluXaaGlnXaaSerXaaTrpProXaaXaaXaaXaaXaaPro151015XaaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35401844PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的18AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35401944PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的19AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402044PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的20AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402144PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的21AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402244PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的22AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402344PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的23AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402444PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的24AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402544PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的25AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402644PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的26AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402744PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的27AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402844PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的28AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35402944PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的29AsnXaaGluXaaGlnXaaSerXaaTrpProAlaPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403044PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的30AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403144PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的31AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403244PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的32AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403344PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的33AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403444PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的34AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403544PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的35AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403644PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的36AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403744PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的37AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403844PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的38AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35403944PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的39AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404044PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的40AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404144PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的41AsnXaaGluXaaGlnXaaSerXaaTrpProThrPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404244PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的42AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404344PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的43AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404444PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的44AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404544PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的45AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404644PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的46AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404744PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的47AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404844PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的48AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35404944PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的49AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405044PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的50AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405144PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的51AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405244PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的52AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405344PRT人工序列合成的合成的位点2,4,6,8,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的53AsnXaaGluXaaGlnXaaSerXaaTrpProValPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro3540543791PRT产黄青霉54MetThrGlnLeuLysProProAsnGlyThrThrProIleGlyPheSer151015AlaThrThrSerLeuAsnAlaSerGlySerSerSerValLysAsnGly202530ThrIleLysProSerAsnGlyIlePheLysProSerThrArgAspThr354045MetAspProCysSerGlyAsnAlaAlaAspGlySerIleArgValArg505560PheArgGlyGlyIleGluArgTrpLysGluCysValAsnGlnValPro65707580GluArgCysAspLeuSerGlyLeuThrThrAspSerThrArgTyrGln859095LeuAlaSerThrGlyPheGlyAspAlaSerAlaAlaTyrGlnGluArg100105110LeuMetThrValProValAspValHisAlaAlaLeuGlnGluLeuCys115120125LeuGluArgArgValSerValGlySerValIleAsnPheSerValHis130135140GlnMetLeuLysGlyPheGlyAsnGlyThrHisThrIleThrAlaSer145150155160LeuHisArgGluGlnAsnLeuGlnAsnSerSerProSerTrpValVal165170175SerProThrIleValThrHisGluAsnArgAspGlyTrpSerValAla180185190GlnAlaValGluSerIleGluAlaGlyArgGlySerGluLysGluSer195200205ValThrAlaIleAspSerGlySerSerLeuValLysMetGlyLeuPhe210215220AspLeuLeuValSerPheValAspAlaAspAspAlaArgIleProCys225230235240PheAspPheProLeuAlaValIleValArgGluCysAspAlaAsnLeu245250255SerLeuThrLeuArgPheSerAspCysLeuPheAsnGluGluThrIle260265270CysAsnPheThrAspAlaLeuAsnIleLeuLeuAlaGluAlaValIle275280285GlyArgValThrProValAlaAspIleGluLeuLeuSerAlaGluGln290295300LysGlnGlnLeuGluGluTrpAsnAsnThrAspGlyGluTyrProSer305310315320SerLysArgLeuHisHisLeuIleGluGluValValGluArgHisGlu325330335AspLysIleAlaValValCysAspGluArgGluLeuThrTyrGlyGlu340345350LeuAsnAlaGlnGlyAsnSerLeuAlaArgTyrLeuArgSerIleGly355360365IleLeuProGluGlnLeuValAlaLeuPheLeuAspLysSerGluLys370375380LeuIleValThrIleLeuGlyValTrpLysSerGlyAlaAlaTyrVal385390395400ProIleAspProThrTyrProAspGluArgValArgPheValLeuAsp405410415AspThrLysAlaArgAlaIleIleAlaSerAsnGlnHisValGluArg420425430LeuGlnArgGluValIleGlyAspArgAsnLeuCysIleIleArgLeu435440445GluProLeuLeuAlaSerLeuAlaGlnAspSerSerLysPheProAla450455460HisAsnLeuAspAspLeuProLeuThrSerGlnGlnLeuAlaTyrVal465470475480ThrTyrThrSerGlyThrThrGlyPheProLysGlyIlePheLysGln485490495HisThrAsnValValAsnSerIleThrAspLeuSerAlaArgTyrGly500505510ValAlaGlyGlnHisHisGluAlaIleLeuLeuPheSerAlaCysVal515520525PheGluProPheValArgGlnThrLeuMetAlaLeuValAsnGlyHis530535540LeuLeuAlaValIleAsnAspValGluLysTyrAspAlaAspThrLeu545550555560LeuProPheIleArgArgHisSerIleThrTyrLeuAsnGlyThrAla565570575SerValLeuGlnGluTyrAspPheSerAspCysProSerLeuAsnArg580585590IleIleLeuValGlyGluAsnLeuThrGluAlaArgTyrLeuAlaLeu595600605ArgGlnArgPheLysAsnArgIleLeuAsnGluTyrGlyPheThrGlu610615620SerAlaPheValThrAlaLeuLysI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lnAsn115511601165LeuLeuThrIleArgLeuGlnSerLeuGlnMetProProGlyAlaLeu117011751180IleGluLeuProGluAsnGluAsnHisAspLeuLysPheAlaSerTyr1185119011951200AlaLeuSerIleValAlaGlnGlnGluGlyThrSerLeuGlyValLys120512101215AlaIlePheAsnSerCysIleLeuGlyAlaAspArgThrGluAlaLeu122012251230LeuGluGlnPheAspThrLeuLeuGlnArgIleLeuArgGluProGly123512401245ThrLysMetLysAspLeuArgThrGlnLeuSerProGluTrpGlnGln125012551260LeuAlaAlaIleAsnLysLysSerProSerHisLeuArgCysLeuHis1265127012751280AspIleIleAsnHisPheSerIleThrGlnProAsnSerGluAlaVal128512901295CysAlaTrpAspGlySerLeuThrTyrSerGluLeuValAlaLeuAla130013051310ArgArgLeuAlaGlyLeuLeuGlnSerPheGlySerGlySerGluPro131513201325GlyAlaValIleGlyIleCysValGluArgSerLysTrpPheProVal133013351340AlaIleLeuGlyValMetMetSerGlyAlaAlaMetValLeuLeuGlu1345135013551360ProAsnPheProThrGlnArgLeuArgHisIleLeuArgAspAlaGly136513701375AlaArgThrMetIleCysSerThrValPheGlnGluLysCysAlaGly138013851390LeuValAspAspMetLeuValLeuThrHisAspIleValThrGlnAla139514001405AspTyrAspAlaTrpThrProSerAlaValSerHisHisAspProMet141014151420TyrValAlaPheThrSerGlySerThrGlyAlaProLysGlyValVal1425143014351440IleGluHisGlyMetValTyrSerMetLeuLysAlaHisLysAspIle144514501455IleGlyAlaSerIleAlaSerArgGlyLeuLeuPheAlaSerProAla146014651470PheAspIleCysLeuAlaGluIleValLeuIleLeuCysSerGlyGly147514801485CysValCysValProSerGluAlaGlnArgMetAsnSerLeuAlaLys149014951500ThrMetThrThrMetGlnValAsnMetAlaMetLeuThrProSerVal1505151015151520AlaArgThrLeuAlaProAlaAlaIleProCysLeuGlnThrLeuIle152515301535LeuGlyGlyGluAlaProSerAlaSerAspLeuGluThrTrpAlaSer154015451550ArgValLysLeuHisGlnSerTyrGlyProAlaGluCysAlaMetTyr155515601565ThrThrThrThrHisProLeuThrSerSerSerAspLeuGlyAsnVal157015751580GlySerSerGlnAsnAlaSerCysTrpIleValAspProAspAsnHis1585159015951600AspGluLeuGlnProValGlySerIleGlyGluLeuLeuIleGlyGly160516101615ProIleValGlyArgGlyTyrIleAsnArgAlaGlnGluSerAlaAla162016251630AlaPheIleCysAspProValTrpSerGluAsnPheProPheLeuGln163516401645GlyAlaArgLeuTyrLysThrGlyAspLeuAlaIleLeuAsnAlaAsp165016551660GlySerLeuAsnLeuValGlyArgLysAspThrGlnValLysLeuAsn1665167016751680GlyGlnArgIleGluLeuHisGluIleGluHisCysAlaGluArgTyr168516901695GlnHisGlyThrAlaValIleAlaGluLeuIleLysProValGlyIle170017051710GlnArgProArgLeuIleMetPheValTyrAspProAlaThrValGlu171517201725ThrThrValGlyIleAspSerThrCysHisAspHisArgGlyLeuPhe173017351740LeuProProSerArgGlnAsnGlnAlaTyrLeuGluGlyValArgAsp1745175017551760HisLeuAsnGlnHisLeuProProTyrMetIleProSerHisPheLeu176517701775SerLeuSerArgLeuProLeuSerProSerGlyLysAlaAspArgLys178017851790ThrLeuArgGlnValAlaSerLysMetAspArgGluThrLeuGluMet179518001805TyrLeuAspAsnProValAlaGluLysArgLysProThrThrGluGln181018151820GluArgPheValArgAlaSerPheAlaThrAlaLeuSerLeuAsnGlu1825183018351840GluAlaIleGlyMetAspAspSerPhePheAlaLeuGlyGlyAspSer184518501855IleThrAlaMetArgValLeuThrLeuCysArgArgArgAsnMetAla186018651870IleSerMetGlnGluPheLeuSerLysAsnThrValThrLeuPheCys187518801885LysHisValIleLeuIleGlnGlyGlnAlaValAspSerLysArgGln189018951900LysLeuLeuAspSerGlnAspLeuValArgGlyGluAspHisLeuVal1905191019151920GlnPheGlnHisLeuAspTyrGlnLeuAspMetValArgSerGlnLeu192519301935AsnLeuLeuLysSerAspSerIleGlnGluIleTyrProCysSerAsp194019451950AlaHisSerGlyValLeuGluLeuTyrThrSerAsnTyrThrSerThr195519601965AlaIlePheGluIleArgAlaThrGlySerValThrProMetGlnVal197019751980SerAsnAlaTrpSerGlnLeuValHisArgHisValAlaLeuArgThr1985199019952000ValLeuMetLysGluProLysValHisAlaAspTyrLeuHisValVal200520102015LeuAspLysGlyProAlaGlnIleLeuAlaLeuProArgSerLysAsn202020252030AlaLeuSerGluLeuLysGlyLeuGluProValLysSerTrpGlyLeu203520402045SerProProHisArgLeuIleIleGlyHisAspHisSerGlyThrLeu205020552060PheMetArgLeuGluThrGlyTyrAlaLeuIleAspAlaPheSerMet2065207020752080ThrIleLeuLeuGluGluLeuSerLeuLeuLeuGlnGlyGlnProLeu208520902095ProGluArgGlyValSerTyrArgGluTyrLeuSerAsnLeuArgSer210021052110GlnSerSerAlaGluThrLeuGlnTyrTrpThrGlnValLeuTyrGly211521202125ValTyrProSerHisLeuProArgValProValThrGlnSerProLeu213021352140ProGluProArgSerGlnSerArgCysLeuProPheAlaGlnSerLys2145215021552160ArgLeuAspSerPheTrpArgSerAsnAsnLeuThrIleThrAsnVal216521702175PheGlnLeuAlaTrpAlaLeuThrLeuAlaHisTyrThrAsnSerArg218021852190AspValCysPheGlyThrIleThrSerGlyArgAspIleProHisLeu219522002205GluValTrpAsnIleValGlySerPhePheAsnValLeuProCysArg221022152220IleAlaIleGluProThrArgThrValIleAspThrLeuArgGlnAsn2225223022352240GlnGluAspIleGlnArgArgAsnAspHisGlnTyrCysSerIlePro224522502255AspMetIleArgLysSerGlyIleArgSerLeuAspAsnAsnGlnGln226022652270LeuPheAsnThrValLeuThrValGlnAsnProPheSerIleGlnSer227522802285SerThrAlaLysAspGlySerAsnGluIleAspValLysLeuIleAsp229022952300LeuGluAspAlaThrGluTyrAspLeuCysValAlaIleLeuProSer2305231023152320ProSerHisLeuLysValGluLeuArgTyrTrpSerThrThrValSer232523302335GluGlyTyrAlaSerAspIleLeuAspArgLeuPheSerGlnLeuGlu234023452350GlnIleValHisHisAlaThrLysProAspPheValGlnValTyrGlu235523602365CysThrLysHis23705744PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的57AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405844PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的58AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35405944PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的59AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406044PRT人工序列"合成的"合成的信号2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的60AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406144PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的61AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406244PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的62AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406344PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的63AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406444PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的64AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406544PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的65AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406644PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的66AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406744PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的67AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406844PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的68AsnXaaGluXaaGlnXaaSerLeuTrpProAlaPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35406944PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的69AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407044PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的70AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407144PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的71AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407244PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的72AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407344PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的73AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407444PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的74AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407544PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的75AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407644PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的76AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407744PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的77AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407844PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的78AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35407944PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的79AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408044PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的80AsnXaaGluXaaGlnXaaSerLeuTrpProThrPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408144PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的81AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408244PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的82AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408344PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的83AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408444PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的84AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408544PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的85AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaAlaValPro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408644PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的86AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaAlaValPro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408744PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的87AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaAlaValPro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408844PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的88AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaAlaValPro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35408944PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的89AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluIlePro151015AspGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35409044PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的90AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluIlePro151015AlaGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35409144PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的91AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluIlePro151015GlyGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro35409244PRT人工序列"合成的"合成的位点2,4,6,20,21,22,23,24,25,26,27,28,29,30,31,33,34,35,36,37,38,42Xaa是未知的92AsnXaaGluXaaGlnXaaSerLeuTrpProValPheAlaGluIlePro151015GlnGlyTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeu202530XaaXaaXaaXaaXaaXaaTrpThrAspXaaArgPro3540

权利要求：1.一种改善由非核糖体肽合成酶催化的次生代谢物的产生或导致所述次生代谢物的途径中发生的前体的产生的方法，该方法包括在真核宿主中使所述非核糖体肽合成酶与MbtH样蛋白接触，其特征在于所述非核糖体肽合成酶来自真核起源并且不是杂合体。2.根据权利要求1所述的方法，其中所述真核宿主是真菌。3.根据权利要求1至2中任一项所述的方法，其中所述次生代谢物是β-内酰胺、色素或真菌毒素。4.根据权利要求3所述的方法，其中所述β-内酰胺是6-氨基青霉烷酸、7-氨基去乙酰氧基头孢烷酸、己二酰基7-氨基去乙酰氧基头孢烷酸、头孢菌素C、青霉素G或青霉素V，其中所述色素是黄青霉素或其中所述真菌毒素是酪青霉毒素。5.根据权利要求2至4中任一项所述的方法，其中所述真菌是产黄青霉。6.根据权利要求5所述的方法，其中所述真核宿主是多拷贝株。7.根据权利要求1-6中任一项所述的方法，其中所述MbtH样蛋白具有SEQIDNO:1-7或与SEQIDNO:1-7至少70％同源的序列。8.根据权利要求1至7中任一项所述的方法，其中所述MbtH样蛋白包含SEQIDNO:17的氨基酸密码。9.根据权利要求8所述的方法，其中所述MbtH样蛋白包含以下中任一项的氨基酸密码：SEQIDNO:18至SEQIDNO:53或SEQIDNO:57至SEQIDNO:92。10.包含非杂合体的真核非核糖体肽合成酶和原核MbtH的组合物。11.根据权利要求10所述的组合物，其是SEQIDNO:54和SEQIDNO:1或SEQIDNO:54和SEQIDNO:3或SEQIDNO:54和SEQIDNO:4或SEQIDNO:54和SEQIDNO:6或SEQIDNO:54和SEQIDNO:7或SEQIDNO:55和SEQIDNO:1或SEQIDNO:55和SEQIDNO:3或SEQIDNO:55和SEQIDNO:4或SEQIDNO:55和SEQIDNO:6或SEQIDNO:55和SEQIDNO:7或SEQIDNO:56和SEQIDNO:1或SEQIDNO:56和SEQIDNO:3或SEQIDNO:56和SEQIDNO:4或SEQIDNO:56和SEQIDNO:6或SEQIDNO:56和SEQIDNO:7、或与其最终90％同源的序列。12.一种真核宿主细胞，其包含非杂合体的非核糖体肽合成酶和允许表达MbtH样蛋白的多核苷酸，其特征在于所述非核糖体肽合成酶来自真核起源。13.根据权利要求12所述的宿主细胞，其中所述MbtH样蛋白具有SEQIDNO:1-7或与SEQIDNO:1-7至少70％同源的序列。14.根据权利要求12所述的宿主细胞，其中所述MbtH样蛋白包含SEQIDNO:17的氨基酸密码。15.根据权利要求12-14中任一项所述的宿主细胞，该宿主细胞是产黄青霉。

百度查询： 灿盛制药荷兰公司;格罗宁根大学 真核NRPS催化过程中的MBTH样蛋白

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