CN1267332A - 生产新的聚酮化合物的方法 - Google Patents
生产新的聚酮化合物的方法 Download PDFInfo
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- CN1267332A CN1267332A CN98808367A CN98808367A CN1267332A CN 1267332 A CN1267332 A CN 1267332A CN 98808367 A CN98808367 A CN 98808367A CN 98808367 A CN98808367 A CN 98808367A CN 1267332 A CN1267332 A CN 1267332A
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- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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Abstract
本发明描述了修饰的PKS基因簇,它在宿主细胞或无细胞提取物中的有效系统内产生新聚酮化合物。新的聚酮化合物通过通式(1)或(2)二酮化合物的进入而产生的,其中A部分活化二酮,R1和R2至少其一不是通常由修饰PKS簇加工的二酮中的天然取代基。聚酮化合物还可以经糖基化而产生抗生素。
Description
受联邦政府资助的研究所做发明的权利声明
本发明受美国国家健康协会的政府资助(GM22172和CA66736-01)。政府对本发明具有当然的权利。
技术领域
本发明涉及合成新的聚酮化合物的方法,所述方法使用不能利用天然第一模件(module)起始物单元的修饰模件聚酮化合物合成酶(PKS)。
背景技术
模件聚酮化合物合成酶的典型结构是脱氧赤糖酸酐(deoxyerythronolide)B合成酶(DEBS)的结构,这种酶产生β-deoxyerythronolide B(6-dEB),它是广谱抗生素红霉素的前体大环内酯。DEBS由3条大多肽组成,每条多肽具有约10个不同的活性位点。图1显示了由3个基因eryAI,eryAII和eryAIII编码的3个DEBS模件的特征。
已经提出了多种对PKS进行基因操作来产生新的聚酮化合物的方法。曾通过模件缺失产生新的聚酮化合物(Kao,C.M.等,J.Am.Chem.Soc.(1995)117:9105-9106;Kao,C.M.等,J.Am.Chem.Soc.(1996)118:9184-9185)。据报道,还原结构域内的失能诱变(Donadio,S.等,Science(1991)252:675-679;Donadio,S.等,Proc.Natl.Acad.Sci.USA(1993)90:7119-7123;Bedford,D.等,Chem Biol.(1996)3:827-831),通过酰基转移酶结构域的取代来改变起始或终止单元的特异性(Oliynyk,M.等,Chem.Biol.(1996)3:833-839;Kuhstoss,S.等,Gene(1996)183:231-236),在已有模件中引入新的催化活性即增加功能的诱变(McDaniel,R.等,J.Am.Chem.Soc.(1997)即将出版)也能够产生新的聚酮化合物。其中,有些报道指出,聚酮化合物路径中的下游酶能加工非天然中间体。但是,这些生产新聚酮化合物的方法都有缺点:需要克隆和DNA测序方面的投资,所用的系统局限于已经能够对其进行基因取代的生物生产者,引物和延伸单元只能来自可代谢CoA硫酯,只有少数辅助催化功能可被利用。
特别是DEBS系统,已知它接受非天然引物单元,例如乙酰和丁酰CoA(Wiesmann,KEH等,Chem.Biol.(1995)2:583-589;Pieper,R.等,J.Am.Chem.Soc.(1995)117:11373-11374)以及它们相应二酮化合物(diketides)的N-乙酰半胱胺(NAC)硫酯(Pieper,R.等,Nature(1995)378:263-266)。但是,现在发现,即使这些非天然底物可用,来自天然起始单元的竞争使得率急遽降低。即使起始单元不是特意提供的,它们也能够通过DEBS系统所用的甲基丙二酰基延伸单元脱羧产生(Piper,R.等,Biochemistry(1996)35:2054-2060;Pieper,R.等,Biochemistry(1997)36:1846-1851)。
所以,提供一种不能利用天然起始单元的突变模件聚酮化合物合成系统将是十分有利的。这样的系统可通过提供合适的二酮化合物,例如NAC硫酯或其他合适的硫酯而诱导产生新的聚酮化合物。目前已经进行过消除天然材料竞争的突变(Daum,S.J.等,Ann.Rev.Microbiol.(1979)33:241-265)。已经用生产阿佛菌素的微生物的随机突变株合成了新的阿佛菌素衍生物(Dutton,C.J.等,Tetrahedron Letters(1994)35:327-330;Dutton,C.J.等,J.Antibiot.(1991)44:357-365)。但是,这一方法不是普遍适用,因为诱变和底物摄入的效率都不高。
所以,需要一种更有效的系统,通过对竞争性生成天然产物的抑制来制备新的聚酮化合物的。
本发明内容
本发明涉及用修饰后的模件聚酮化合物合成酶系统来制备新的聚酮化合物的方法,所述的修饰使得系统无法利用其天然起始材料。这样,就可以越过起始模件,提供多种合适的二酮底物来合成新的聚酮化合物。
所以,一方面,本发明涉及制备新聚酮化合物的方法,该方法包括在底物能被模件PKS转化为聚酮化合物产物的条件下,向具有至少两个模件的模件PKS提供硫酯二酮底物,所述的PKS经修饰而无法利用天然起始单元。另一方面,本发明涉及修饰模件PKS,它不能用天然起始底物来提供最初的两个碳单元,还涉及经修饰而含有这种失能PKS的细胞。本发明还涉及用于生产修饰PKS的重组材料和由这种系统产生的新聚酮化合物。
附图简述
图1代表DEBS模件PKS。
图2A-2C显示修饰DEBS结构的产物,结构中模件1的酮合成酶(KS)被灭活。
图3显示将6-dEB衍生物加工成红霉素-D衍生物。
本发明的实施方式
本发明提供了不能加载天然起始材料的模件PKS系统及其相应的基因。在特别优选的实施例中,模件1的酮合成酶(KS)失活,从而消除了来自天然起始单元的竞争。类似的令PKS失能的其它方法涉及使模件1的酰基转移酶(AT)或酰基运载蛋白(ACP)功能失活。
本发明的PKS必须包含至少2个模件,但也可以包含更多的模件,实际上,还可以相当于完整的合成酶系统。虽然用DEBS PKS系统来说明本发明,其实各种模件PKS均可使用,例如产生阿佛菌素和雷帕霉素等的模件PKS。可通过已知的定点诱变引入合适的突变。
也可以使用其它微生物,例如酵母和细菌。当使用正常情况下不产生聚酮化合物的宿主细胞时,例如细菌、酵母甚至哺乳动物或昆虫细胞,可能需要改变宿主细胞以获得PKS系统的翻译后加工。具体地说,为了具有功能,必须进行ACP活性的磷酸泛酰巯基乙胺化(phosphopantetheinylated)。无磷酸ACP向其活性形式的这种转化由统称为全ACP合成酶或PTT酶的酶完成。这些酶在脂肪酸合成路径中的功能形式似乎不能在PKS簇内提供全ACP功能。所以,如果是在非链霉菌的全细胞内合成聚酮化合物,必须在合成系统中加入一种合适的合成酶。如果在无细胞系统中进行合成,所用的PKS酶必须在有全ACP合成酶存在的条件下合成。
这样就可以在合适的宿主细胞内合成聚酮化合物,例如链霉菌宿主,特别是经修饰而缺失了自身PKS的链霉菌,或者经修饰可根据需要产生合适的PTT酶的其它细胞。通过重组产生相关的PKS蛋白并使得它们分泌或裂解含有这些蛋白的细胞,可用无细胞系统合成聚酮化合物。典型的无细胞系统包括催化合成聚酮化合物所需的合适的功能性PKS,NADPH和合适的缓冲液和底物。为了生产新的聚酮化合物,延伸单元的硫酯和二酮化合物的硫酯一同使用。
修饰PKS簇产生的新聚酮化合物在终产物内对应于起始单元残基的取代基是不同的。而且,因为二酮中间体被提供给修饰PKS簇,所以紧靠起始单元进入的延伸单元的特征可能也不同。所以,用来制造本发明新聚酮化合物的二酮具有以下通式:其中的A活化二酮化合物,通常是巯基,例如后文的N-乙酰半胱胺硫酯,R1和R2中至少一个不是修饰PKS簇通常所加工的二酮化合物天然具有的取代基。通常,R1是取代或非取代、饱和或非饱和的烃基(1-15C),所述的烃基可以含有一到两个杂原子,特别是N、O或S,R2是取代或非取代、饱和或非饱和的烃基(1-4C)或者是OR、SR或NHR,其中R是取代或非取代、饱和或非饱和的1-4C烃基。但是,R1和R2不可以都是甲基,而且,如果R2是甲基,R1不能是乙基。
典型的取代基包括卤素,OR3,SR3,NR2,-OOR3,-NHOCR3,R3CO-,R3COO-和R3CONH-,其中每个R3各自是H或(4-4C)低级烷基。
本发明还涉及具有通式(1)或(2)所示的二酮化合物的聚酮化合物,还涉及它们的糖基化形式。
以下实施例用来说明而不是限定本发明。
制剂A
起始材料
如WO95/08548所述构建天蓝色链霉菌CH999,通过基因工程处理去除天然PKS基因簇。该专利公开中还记述了用来在CH999中表达PKS基因的穿梭质粒pRM5。该专利公开中还记载了含有完整DEBS模件系统的质粒pCK7。
实施例1
DEBS1+2+TE的制备
称为DEBS1+2+TE的修饰DEBS PKS系统只含有模件1和2和硫酯酶(TE)活性,对其进行定点诱变,即以丙氨酸取代签名序列cys-ser-ser-ser-leu中的活性位点半胱氨酸残基,由此灭活模件1KS。所得的表达质粒pKAO179编码一个2-模件PKS,它在合成天然产物的标准条件下,即丙酰基-CoA、甲基丙二酰基-CoA和NADPH存在下,没有活性。构建详情参见Kao,C.M.等,Biochemistry(1996)35:12363-12368。当有二酮化合物硫酯(2S,3R)-2-甲基-3-羟基-戊酰基-N-乙酰基半胱胺硫酯,甲基丙二酰基-CoA和NADPH存在时,将得到三酮化合物产物。
在上述条件下,当PKS孵育在无细胞系统中时,可产生三酮产物,或者,提供合适的二酮硫酯类似物使得含有修饰表达质粒的CH99活跃生长,也可以在胞内同样获得该产物。
形成天蓝色链霉菌CH999/pKAO179培养物:将孢子接种在200mL SMM培养基(5%PEG-800,0.06%MgSO4,0.2%(NH4)2SO4,25mM TES,pH7.02,25mM KH2SO4,1.6%葡萄糖,0.5%酪蛋白氨基酸,微量元素)中。培养物在30℃,325rpm振荡培养。分三次加入总共1L的(2S,3R)-2-甲基-3-羟基-戊酰基-N-乙酰基半胱胺硫酯(100mg)和4-戊炔酸(5mg):50小时后(400mL);62小时后(300mL);86小时后(300mL)。总共144小时后,离心去除培养物中的菌丝体。NaCl饱和发酵液,用乙酸乙酯(5×100mL)抽提。合并有机抽提液,用Na2SO4干燥,过滤,浓缩。经硅胶层析得到(2R,3S,4S,5R)-2,4-二甲基-3,5-二羟基-正己酸δ内酯。
实施例2
由DEBS簇制备聚酮化合物
质粒pCK7具有eryAI(DEBS1基因)的活性位点发生了突变的模件1KS结构域,该质粒还含有eryAI,eryAII(DEBS2基因)和eryAIII(DEBS3基因),它们位于actI启动子调控下。pJRJ2的表达产生适当修饰的全长PKS系统。(Kao,C.M.等,Science(1994)265:409-512)。将pJRJ2转化到CH999中,培养在R2YE培养基上。经测定没有6-dEB样产物的生成。
更具体地说,将CH999/pJRJ2于30℃培养在含0.3mg/ml丙酸钠的R2YE琼脂培养板上。3天后,在每块琼脂板上铺以1.5mL 20mM底物的9∶1水:DMSO溶液。再过4天,将琼脂培养基制成匀浆,用乙酸乙酯抽提3次。用硫酸镁干燥溶剂并浓缩。用硅胶层析(乙酸乙酯的己烷梯度溶液)纯化浓缩后的抽提液,得到产物。
但是,在系统中加入用Cane等,J.Am.Chem.Soc.(1993)115:522-526;Cane等,J.Antibiol.(1995)48:647-651所述的方法制备的底物2(天然二酮的NAC硫酯)时,见图,大量产生正常产物6-dEB。在少量如上所述培养在培养板上的培养物(300ml)中添加100mg底物2时,将产生30mg 6-dEB,得率为18%。
实施例3
新的聚酮化合物的产生
然后在生长于实施例2条件下的CH999/pJRJ2培养物中加入结构如图2所示,通式分别为3,4和5的二酮化合物。底物3和4的制备与底物2相同,只是在羟缩醛反应中分别用戊醛和苯基乙醛代替丙醛。底物5的制备参见Yue,S.等,J.Am.Chem.Soc.(1987)109:1253-1255。底物3和底物4分别产生55mg/L产物6和22mg/L产物7。底物5出人意料地产生25mg/L的16元内酯8。
实施例4
其它新聚酮化合物
将结构如图2B和2C所示的二酮化合物,即化合物9-18加入生长于实施例2条件下的CH999/pJRJ2培养物中。产物如图2B和2C所示,即化合物19-28。
实施例5
空间要求
用与实施例2相同的系统,但以图2A中通式2的三种非对映异构体代替化合物2,经测定都没有聚酮化合物的合成。类似的,用2-位的对映异构体代替化合物12,也没有测得聚酮化合物的合成。
实施例6
聚酮化合物产物的加工
DEBS的“下游”模件对非天然中间体的成功加工促使人们进行实验以确定红霉素天然合成路径中的后PKS酶是否也接受非天然底物。在天然微生物生产者saccharopolyspora erythrea中,6-dEB经历数次酶催化的转化。C6位的氧化和C3及C5位的糖基化产生红霉素D(图3中的通式9),此后的转化产生红霉素A、B和C。S.erythrea突变体(A34)(Weber,J.M.等,J.Bactiol.(1985)164:425-433)不能合成6-dEB。该菌株生长于R2YE培养板上时不生产红霉素(根据提取物抑制红霉素敏感菌蜡状芽孢杆菌生长的能力来判断)。但是,当培养基中加入6-dEB(没有抗生素活性)时,抽提物表现出强抗菌活性。
对该菌株转化6-dEB衍生物6和7样品进行了测试。部分纯化的抽提物抑制蜡状芽孢杆菌的生长,还用质谱法鉴定出抽提物的主要成分是图3中的通式10(来自衍生物6)和通式11(来自衍生物7)。
更具体地说,将纯化的6和7(5mg溶于7.5ml 50%乙醇水溶液)铺在R2YE培养板上(200ml培养基/试验),自然干燥。然后接种S.erythrea A34,任其生长。7天后,将培养基制成匀浆,用98.5∶1.5的乙酸乙酯∶三乙胺抽提3次。将每次试验的抽提液合并,用硫酸镁干燥,并浓缩。用硅胶层析(甲醇和三乙胺的氯仿梯度溶液)将抽提液部分纯化。用TLC和质谱检查部分纯化的抽提液。为了进行抗生素活性试验,将滤片浸在红霉素D,10和11,以及不加任何6-dEB类似物培养的S.erythrea A34浓缩抽提液的400μM乙醇溶液中。干燥滤片,盖在新鲜的蜡状芽孢杆菌培养板上。37℃培养12小时后,除对照抽提液之外,所有化合物都抑制细菌生长。
Claims (13)
1.一种修饰的功能性模件聚酮化合物合成酶(PKS),它包含至少两个模件,所述的PKS经修饰而不能利用所述模件PKS的天然起始单元,其中的模件1的酮合成酶(KS)催化结构域失活。
2.根据权利要求1所述的修饰PKS,它是完整的PKS。
3.一种PKS基因簇,它编码修饰PKS,所述的PKS经修饰而不能利用所述模件PKS的天然起始单元,其中模件1的酮合成酶(KS)催化结构域失活。
4.根据权利要求3所述的基因簇,它编码完整的PKS。
5.一种重组宿主细胞,它经修饰而含有权利要求3所述的基因簇,还可含有生产全-ACP合成酶的重组表达系统,全-ACP合成酶能将产生的PKS的ACP区磷酸泛酰巯基乙胺化。
6.一种制备聚酮化合物的方法,该方法包括提供权利要求1所述的修饰PKS的硫酯二酮化合物底物。
7.根据权利要求6所述的方法,它是在宿主细胞内进行的。
8.根据权利要求6所述的方法,它是在无细胞系统中进行的。
9.一种制备抗生素的方法,所述的方法包括用聚酮化合物糖基化酶处理权利要求6所产生的聚酮化合物。
10.一种新聚酮化合物,它具有图2中通式6-8的结构或图2B和2C中通式19-28的结构。
11.一种新的抗生素,它是权利要求10所述聚酮化合物的糖基化形式。
12.一种新的聚酮化合物,它由以下通式的二酮化合物通过权利要求6所述的方法得到:
其中A活化二酮;
R1和R2至少其一不是通常由修饰PKS簇加工的二酮中的天然取代基;而且,
R1是取代或非取代、饱和或非饱和的烃基(1-15C),所述的烃基可以含有一到两个杂原子,特别是N、O或S,R2是取代或非取代、饱和或非饱和的烃基(1-4C)或者是OR、SR或NHR,其中R是取代或非取代、饱和或非饱和的1-4C烃基。但是,R1和R2不可以都是甲基,而且,如果R2是甲基,R1不能是乙基。
13.一种新的抗生素,它是权利要求12所述新聚酮化合物的糖基化形式。
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1997
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1998
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- 1998-07-17 NZ NZ502360A patent/NZ502360A/en unknown
- 1998-07-17 WO PCT/US1998/014911 patent/WO1999003986A2/en not_active Application Discontinuation
- 1998-07-17 HU HU0003728A patent/HUP0003728A2/hu unknown
- 1998-07-17 CN CN98808367A patent/CN1267332A/zh active Pending
- 1998-07-17 EP EP98934641A patent/EP0996714A2/en not_active Withdrawn
- 1998-07-17 BR BR9813004-8A patent/BR9813004A/pt not_active IP Right Cessation
- 1998-07-17 CA CA002296728A patent/CA2296728A1/en not_active Abandoned
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2001
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US20010024811A1 (en) | 2001-09-27 |
IL134074A0 (en) | 2001-04-30 |
WO1999003986A2 (en) | 1999-01-28 |
AU736972B2 (en) | 2001-08-09 |
KR20010021933A (ko) | 2001-03-15 |
WO1999003986A3 (en) | 1999-04-08 |
CA2296728A1 (en) | 1999-01-28 |
NZ502360A (en) | 2001-11-30 |
HUP0003728A2 (hu) | 2001-02-28 |
EP0996714A2 (en) | 2000-05-03 |
US6261816B1 (en) | 2001-07-17 |
JP2001510038A (ja) | 2001-07-31 |
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