CN1084791C - 通过扩环酶活性作用于青霉素g生产7－adca的方法 - Google Patents
通过扩环酶活性作用于青霉素g生产7－adca的方法 Download PDFInfo
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Abstract
用表达扩环酶的产黄青霉转化菌株，通过对青霉表G的酶促扩环作用制备和回收7-氨基去乙酸头孢烷酸(7-ADCA)的方法。
Description
本发明涉及制备和回收7-氨基去乙酸头孢烷酸(7-ADCA)的生物合成方法。
β一内酰胺抗生素构成最重要的一类抗生素化合物，并具有悠久的临床应用史。这类抗生素中，突出的有青霉素类和头孢菌素类。这些化合物分别由丝状真菌产黄青霉(Penicillium chrysogenum)和产黄枝顶孢(Acremonium chrysogenum)天然产生。
经过经典的菌种改进技术，使产黄青霉和产黄枝顶孢的抗生素生产水平比过去几十年大大提高了。随着对产生青霉素和头孢菌素的生物合成途径的了解增多和重组DNA技术的出现，现在有了改进生产菌株和将这些化合物体内衍生化的新工具。
β-内酰胺生物合成中涉及的大部分酶已得以鉴定，对其相应基因已进行了克隆，如见Ingolia和Queener的Med.Res.Rev.9(1989)，245-246(生物合成途径和酶)，及Aharonowitz，Cohen，和Martin的Ann.Rev.Microbiol.46(1992)，461-495(基因克隆)。
产黄青霉中青霉素生物合成的头两步是：三种氨基酸L-5-氨基-5-羧基戊酸(L-α-氨基己二酸)(A)、L-半胱氨酸(C)和L-缬氨酸(V)缩合成三肽LLD-ACV，然后该三肽环化成异青霉素N。该化合物含有典型的β-内酰胺结构。
第三步涉及通过酰基转移酶(AT)的作用以疏水侧链交换L-5-氨基-5-羧基戊酸的亲水侧链。如EP-A-0448180中所述，由AT介导的酶促交换反应发生在一种细胞器-微体中。
头孢菌素比青霉素贵得多。一个原因是一些头孢菌素(如cephalexin)是由青霉素经若干步化学转化而制得的。另一个原因是迄今只能发酵出具有D-5-氨基-5-羧基戊酰侧链的头孢菌素。在此方面迄今最重要的起始物头孢菌素C在任何pH下都非常易溶于水，这就意味着使用麻烦而又昂贵的柱技术进行费时费钱的分离工艺。这样获得的头孢菌素C须经若干化学和酶促转化才能转变为治疗用头孢菌素。
使用复杂的化学步骤使青霉素G发生扩环和衍生是目前工业上制备中间体7-ADCA的流行方法。生产7-ADCA所需的一个化学步骤5-元青霉素环结构扩环成为6-元的头孢菌素环结构(参见US4,003,894)。但这种复杂的化学步骤既昂贵又对环境有害。
所以目前急需用如发酵过程中的酶催化这类酶促反应取代化学方法。而用生物方法取代化学扩环方法的关键是头孢菌素生物合成途径中的中心酶-去乙酸头孢菌素C合成酶或扩环酶。
在一些实例中，人们发现来自细菌带棒链霉菌(Streptomycesclavuligerus)的扩环酶可在体外使青霉素环结构扩环(Baldwin等，四面体43(13)，3009，(1987))。在Cantwell等人的文章中(现代遗传学(Current Genetics)，17，213-221(1990))，描述了在产黄青霉中表达带棒链霉菌扩环酶。如该文中所示，在发酵过程中表达扩环酶并没有形成头孢菌素。又如Cantwell等人在Proc.R.Soc.Lond.B.248(1992)，283-289中所描述，只有当扩环酶和带棒链霉菌异青霉素N异构酶基因一起引入产黄青霉，才能观察到青霉素N(它的天然底物)的青霉素环结构转化为去乙酸头孢菌素C(它的天然产物)的头孢菌素环结构。已对扩环酶从生化和功能方面进行了充分表征(EP-A-0366354)，也对其相应的基因进行了表征。已描述了cefE基因的物理图谱(EP-A-0341892)和DNA序列，及cefE在产黄青霉中的转化研究。
扩环酶的另一个来源是细菌Nocardia lactamdurans(早期称为Streptomyces lactamdurans)。已描述了酶的生化特征和基因的DNA序列(分别见Cortes等，J.Gen.Microbiol.133(1987)，3165-3174及Coque等，Mol.Gen.Genet.236(1993)，453-458)。
既然扩环酶可催化青霉素N的5-元噻唑烷环结构扩环为去乙酸头孢菌素C的6-元二氢噻嗪环结构，这种酶自然成为取代化学方法扩环的合理的候选物。但不幸的是，这种酶只作用于头孢菌素生物合成途径的青霉素N中间体，而不作用于包括青霉素G在内的由产黄青霉产生的廉价易得的青霉素。青霉素N没有商业供应，而且即使经过扩环，青霉素酰基转移酶也不能轻易地将其D-氨基己二酰侧链除去。
最近发现扩环酶可将具有特定侧链的青霉素扩环成为相应的7-ADCA衍生物。EP-A-268343描述了使用去乙酸头孢菌素C合成酶对带有3-羧苯基乙酰或己二酰侧链的青霉素进行扩环的体外方法。而且，在EP-A-0532341、EP-A-0540210、WO95/04148及WO95/04149
中，扩环酶的这一特性已得到技术开发。在这些专利文件中，由青霉素G经传统的化学转变制备7-ADCA的方法已被含有扩环酶基因的重组产黄青霉株内的特定6-氨基青霉烷酸(6-APA)衍生物的体内转化所替代。
更具体地讲，EP-A-052341描述了扩环酶结合5-羧基戊酰侧链作为原料在产黄青霉中的体内应用。5-羧基戊酰是产黄青霉中酰基转移酶的底物。这导致形成5-羧基戊酰-6-青霉烷酸，后者通过引入产黄青霉株中的扩环酶产生5-羧基戊酰-7-ADCA。最后，提到去除5-羧基戊酰侧链生成终产物7-ADCA。
在WO95/04148和WO95/4149中，指出3′-羧甲基硫代丙酸和3′3-硫代二丙酸被发现是扩环酶的底物，分别生成2-(羧乙基硫)乙酰-和3-(羧甲基硫)丙酰-7-ADCA。
但是，由于青霉素生产菌株具有很高的青霉素G合成能力且提取苯乙酰-7-ADCA的方法更有利，因此，本发明具有更多优势。而且，青霉素G的苯乙酰基侧链很容易被产生6-APA的几种微生物的青霉素G酰胺酶(如EP-A-0453047中所指出的分离酶G)催化裂解。
各种出版物都报告说扩环酶不能以青霉素G作为底物进行扩环(Baldwin&Abraham(1988)，天然产物报告(Natural Product Reports)，5(2)，p.129-145；Maeda等(1995)，酶和微生物技术(Enzyme andMicrobial Technology)，17，231-234；Crawford等(1995)，生物技术(Bio/technology)，13，p.58-61；Wu-KuangYeh等，于“青霉素应用五十年(50 years Penicillin Application)(Kleinkauf和VonDohren编)，209(1991)，特别请见表3A)。
但令人惊讶的是，现在发现用扩环酶编码基因转化的产生青霉素G的产黄青霉能够产生苯乙酰-去乙酸头孢烷酸。
本发明提供一种制备和回收7-氨基去乙酸头孢烷酸(7-ADCA)的方法，包括：
a)在真菌表达信号的转录和翻译调控下，用扩环酶基因转化产黄青霉菌株；
b)在适于产生青霉素G的培养基中发酵所述菌株并在所述培养基中加入苯乙酸或其盐或其酯，青霉素G经扩环形成苯乙酰-7-ADCA；
c)从发酵液中回收苯乙酰-7-ADCA；
d)将苯乙酰-7-ADCA脱酰基化；及
e)回收结晶的7-ADCA。
e)步优选为一过滤步骤。
优选在低于约4.5的pH下用一种与水不溶混的有机溶剂萃取发酵液滤液并在pH为4-10时用水将其反萃取，从发酵液中回收苯乙酰-7-ADCA。
另外，还提供了包含扩环酶编码DNA的重组DNA载体，其中编码扩环酶之DNA与如构巢曲霉gpdA基因和黑曲霉glcA基因等真菌基因的转录和翻译控制元件有效地连接，以及用此载体转化的宿主细胞。
本发明涉及应用产黄青霉中的功能性基因构建物体内扩张青霉素G环结构形成头孢菌素生物合成的关键中间体：7-氨基去乙酸头孢烷酸，或7-ADCA的衍生物。该衍生物具有允许进行有效溶剂萃取的化学组成，从而提供一种在经济效益上有吸引力的回收方法。
产黄青霉的转化原则上可用不同的DNA转移技术如PEG-Ca介导的原生质体摄入、电穿孔或基因枪技术，及转化子选择来完成。例如，见Van den Hondel en Punt在真菌的应用分子遗传学(Peberdy，Laten，Ogden，Bennett，编)中的“对丝状真菌的基因转移和载体发展”一文，(Cambridge University Press(1991))。已描述了显性和非显性选择标记的应用(VandenHondel，同上)。已描述了同源性(产黄青霉衍生的)和异源性(非产黄青霉衍生的)选择标记(Gouka等，生物技术杂志，20(1991)，189-200)。
在存在或不存在载体序列时，与非选择性DNA物理连接或不连接的不同转化子选择标记(同源或异源)在转化子选择中的应用是熟知的。
按此方法，由扩环酶介导的扩环反应被引入产黄青霉中，并在其中表达，例如在菌株PanlabsP14-B10，DS18541(保存于CBS，寄存号为455.95)中。很明显地，如果扩环反应在其突变体中进行，应对培养基条件稍做调整以得到有效生长。
另外，将cefE基因置于真菌(丝状或非丝状)基因控制元件的转录和翻译控制之下，优选来自产黄青霉Y基因(如EP-A-0549062所述)、产黄青霉IPNS基因、β微管蛋白基因、构巢状曲霉gpdA基因、或黑曲霉glcA基因。
总之，本发明描述了引入产黄青霉中的扩环酶活性如何在体内影响青霉素G环结构的扩张。
根据本发明，通过在培养基中加入苯乙酸或其盐或酯，在产黄青霉中产生β-内酰胺中间体苯乙酰基-7-ADCA。适当的盐例如钠盐或钾盐。通过简单的溶剂萃取从培养基中有效回收7-ADCA，例如以下方法：
过滤发酵液，向滤液中加入与水不混溶的有机溶剂，调节pH值以便从水层中萃取头孢菌素。pH范围必须低于4.5；优选在4-1之间，更优选在2-1之间。这样就将头孢菌素与发酵液中存在的许多其他杂质分离。优选使用小体积有机溶剂，得到头孢菌素浓溶液，以使体积流速减小。第二种可能性是在pH4或更低时，萃取全部发酵液，优选在pH4-1之间用与水不混溶的有机溶剂萃取发酵液。
可以使用不影响头孢菌素的任何溶剂。适宜的溶剂如：乙酸丁酯、乙酸乙酯、甲基异丁酮、醇如丁醇等。优选使用乙酸丁酯。
然后在pH4-10，优选在6-9下用水反萃取头孢菌素，再次大大减小终体积。在0-50摄氏度，优选室温下进行回收。
这样得到的头孢菌素水溶液用适当的酶处理，以除去苯乙酰基侧链，得到所需的7-ADCA。适当的酶例如EP-A-0453047中所描述的青霉素G酰基转移酶，也称为青霉素酰胺酶。
优选使用固定化酶以能够重复使用该酶。EP-A-0222462中广泛描述了这种颗粒的制备方法和酶的固定化方法。水溶液的pH值例如为4-9，此时头孢菌素的降解反应最小，而所希望的酶转化此时是最优化的。因此，向头孢菌素水溶液中加入酶的同时保持pH在此适当的水平，例如通过加入无机碱如氢氧化钾溶液，或应用阳离子交换树脂。当反应完成后过滤除去固定化酶。另一个可能性是在固定床或流化床柱中应用固定化酶，或在溶液中使用酶并通过膜过滤除去产物。然后，在与水不混溶的有机溶剂存在下酸化反应混合物。
调节pH值至0.1-1.5后，分离各层，调节水层的pH值为2-5。然后过滤出结晶的7-ADCA。
脱酰化还可以按本领域已知的化学方法进行，例如通过在低于10摄氏度的温度下加入五氯化磷，然后于室温或更低温度下加入异丁醇形成偕氯代亚胺侧链。
提供以下实施例用于说明，但不限制本发明。
实施例1
苯乙酰基-7-ADCA的发酵生产
使用Panlabs P14-B10产黄青霉菌株(保存于CBS，入藏号为455.95)作为扩环酶表达框构建物宿主菌株。
Crawford等人的文章(同上)描述了在产黄青霉IPNS基因转录和翻译调控信号控制下含有扩环酶基因的表达框。Crawford等人的文章(同上)还描述了转化和培养的条件。同一文章内还描述了通过测试产生己二酰基-7-ADCA的能力对表达扩环酶的转化子进行纯化和分析。
产生己二酰基-7-ADCA的转化子如产黄青霉株PC100(保存于ATCC，入藏号为74182)以2.106分生孢子/ml接种于下列组成的种子培养基中(g/l)：葡萄糖，30；Pharmamedia(棉籽粉)，10；玉米浸出物，20；(NH4)2SO4，20；CaCO3，5；KH2PO4，0.5；乳糖，10；酵母提取物，10，在消毒前，培养基pH为5.6。
将20ml种子培养基置于250ml锥形瓶中，用棉塞将瓶口塞住，在25摄氏度、220转/分下孵育。48小时后，将1ml种子培养物接种于15ml如下组成的生产培养基中(g/l)：KH2PO4，0.5；K2SO4，5；(NH4)2SO4，17.5；乳糖，140；Pharmamedia，20；CaCO3，10；猪油，10，消毒前pH为6.6。
用种子培养物接种后，在发酵液中加入已用KOH将pH调整为7的10％苯乙酸溶液0.15-0.75ml。
将生产培养基置于用乳滤纸封口的250ml锥形瓶中，在25摄氏度220转/分下培养168小时，每隔一天补充一次蒸发掉的水。
生产发酵结束后，用离心或过滤方法除去菌丝体，用高效液相色谱(HPLC)分析青霉素G和苯乙酰基-7-ADCA。
实施例2
苯乙酰基-7-ADCA产物的分析
用高效液相色谱(HPLC)分析转化过的青霉菌株发酵产物。HPLC系统含有如下光谱物理组件：P1500
溶剂传送系统，AS1000注射器，UV1000可变波长检测器(设定于214nm)和ISM100整合器或类似物。固定相采用ChrompackChrompherC18柱，流动相含有的75％pH2.6磷酸缓冲液和25％乙腈。产物与苯乙酰基-7-ADCA和青霉素G标准曲线对照进行定量分析。培养液滤液经酸萃取得到氘代氯仿溶液，用600MHz核磁共振下确认苯乙酰基-7-ADCA。酸萃取液中苯乙酰基-7-ADCA的共振现象与生物合成样品相一致。
Claims (4)
1.一种制备和回收7-氨基去乙酸头孢烷酸(7-ADCA)的方法，包括：a)在真菌表达信号的转录和翻译调控下，用扩环酶基因对产黄青霉进行转化；b)将上述菌株在适于产生青霉素G的培养基中发酵，并在所述培养基中加入苯乙酸或其盐或其酯，青霉素G经扩环形成苯乙酰基-7-ADCA；c)从发酵液中回收苯乙酰基-7-ADCA；d)将苯乙酰基-7-ADCA脱酰基化；并且e)回收结晶的7-ADCA。
2.根据权利要求1的方法，其中(e)步为过滤步骤。
3.根据以上权利要求任一项的方法，其中(c)步为过滤步骤、和在pH低于4.5时，用与水不溶混的有机溶剂萃取发酵液滤液，再在pH4-10时用水将其反萃取。
4.根据权利要求1的方法，其中扩环酶基因来自带棒链霉菌(Streptomyces clavuligerus)或Nocardia lactamdurans。
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| EP0540210A1 (en) * | 1991-10-15 | 1993-05-05 | Merck & Co. Inc. | Novel bioprocesses for preparing 7-aca and 7-adac |
| WO1995004149A1 (en) * | 1993-07-30 | 1995-02-09 | Gist-Brocades B.V. | Process for the efficient production of 7-adca via 3-(carboxyethylthio)propionyl-7-adca |
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| DE69434023D1 (de) * | 1993-07-30 | 2004-10-28 | Dsm Ip Assets Bv | Verfahren zur effizienten Herstellung von 7-ADCA via 2-(carboxyethylthio)acetyl-7-ADCA und 3-(carboxymethylthio)propionyl-7-ADCA |
| US5731165A (en) * | 1995-06-02 | 1998-03-24 | Gist-Brocades, B.V. | Process for the production of 7-ADCA via expandase activity on penicillin G |
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| EP0540210A1 (en) * | 1991-10-15 | 1993-05-05 | Merck & Co. Inc. | Novel bioprocesses for preparing 7-aca and 7-adac |
| WO1995004149A1 (en) * | 1993-07-30 | 1995-02-09 | Gist-Brocades B.V. | Process for the efficient production of 7-adca via 3-(carboxyethylthio)propionyl-7-adca |
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