CN117402840A - 一种高效制备11α-羟基孕酮的重组雷斯青霉菌及其应用 - Google Patents
一种高效制备11α-羟基孕酮的重组雷斯青霉菌及其应用 Download PDFInfo
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Abstract
本发明提供了一种高效制备11α‑羟基孕酮的重组雷斯青霉菌及其应用,属于基因工程和酶工程技术领域;本发明运用农杆菌介导转化的技术手段,将编码赭曲霉的甾体C11α‑羟化酶突变体CYP68J5mL491F的基因定点整合取代雷斯青霉甾体C15α‑羟化酶基因,获得的重组雷斯青霉菌能够高效转化甾体底物孕酮生成11α‑羟基孕酮。突变体CYP68J5mL491F氨基酸序列如SEQ ID NO:3所示。底物孕酮的投料量为15g/L,转化72h,产物11α‑羟基孕酮得率达到92%。本发明专利为研究开发高效C11α‑羟化孕酮生产C11α‑羟基孕酮工艺提供了宝贵的工业菌种和技术方法。
Description
技术领域:
本发明应用基因工程和酶工程技术,利用基因敲除、酶分子改造、农杆菌介导转化法等技术手段,构建一种过表达甾体C11α-羟化酶CYP68J5突变体重组雷斯青霉菌及其在高效制备C11α-孕酮中的应用。
背景技术:
甾体化合物是一类以环戊烷多氢菲为母核的化合物,甾体激素类药物的生理药理活性取决于骨架特定位点引入官能基团。因化学合成法的合成过程较为复杂,工业上常用微生物转化法合成引入关键官能团的甾体化合物。
在生产甾体激素类药物过程中,11α-羟基化反应占据重要地位,是微生物转化反应的关键技术之一。通过微生物转化可以在甾体母核的碳11位上引入羟基,生成药理活性更强的产物。
孕酮(PRG)又名***,是一种甾体激素类可以进行生育控制的一线临床药物。而11α-羟基孕酮(11α-OH-PRG)是合成糖皮质激素和甾体孕激素的重要医药中间体。相较于化学合成法,微生物催化底物孕酮仅需一步反应就可直接转化成C11α-羟基孕酮,巧妙地解决了皮质激素合成过程中的一个关键性化学合成难题。工业上主要利用赭曲霉(Aspergillusochraceus)生产11α-羟基孕酮,孕酮的投料量可以达到20g/L。但因赭曲霉在发酵过程中会产生大量的砖红色素,严重影响产物的分离提纯;另因赭曲霉会产生次级代谢产物赭曲霉毒素(OTA),不易发生降解,对肝脏和肾脏有强烈的毒性,严重制约了赭曲霉甾制药和食品等相关领域的应用。另外,赭曲霉对不同甾体底物的转化特异性有着十分明显的差异,并且转化效率较低,在很大程度上会限制它的应用范围
农杆菌介导转化法(ATMT)作为近几年来广泛应用于丝状真菌的一种遗传操作技术,在真菌的遗传转化中具有转化效率高、遗传稳定性好等诸多优点。农杆菌介导转化法是利用根癌农杆菌Ti质粒上的T-DNA区域在诱导剂乙酰丁香酮(AS)的作用下将内源的T-DNA替换成外源基因来完成遗传转化,得到表达外源基因的雷斯青霉重组菌。
本发明以雷斯青霉(Penicillium raistrickii ATCC 10490)作为出发菌株,构建了一种用于生产11α-羟基孕酮的雷斯青霉重组菌。首先对实验室前期从赭曲霉(Aspergillus ochraceus TCC41060)中鉴定的11α-甾体羟化酶CYP68J5进行定点突变,经酿酒酵母转化孕酮获得羟化活性强且特异性显著提高的突变体L491F,但存在投料量低的问题,需要更换宿主菌。鉴于实验室前期已验证了15α-羟化酶PRH在雷斯青霉中的转化活性,且雷斯青霉是甾体15羟转化的工业生产菌种,其特性适合在工业中应用。因此,利用雷斯青霉自身同源重组定点整合效率较高的优势,农杆菌(Agrobacterium tumefaciensAGL-1)介导转化的遗传转化特点,将改造好的高特异性11α-羟化酶突变体CYP68J5mL491F取代雷斯青霉的15α-羟化酶PRH,提高孕酮转化的投料量,实现C11α-羟化酶基因的高效异源表达,获得的C11α-羟化酶突变体雷斯青霉重组菌可以用于高效制备医药中间体11α-羟基孕酮。
解决高浓度下菌株的转化是应用于工业生产上一个比较关键的因素。研究表明表面活性剂的憎水内核胶团对憎水性物质具有增溶作用,广泛用于疏水性底物助溶剂,而甾体物质大多都是疏水物质,所以加入合适的表面活性剂可以增加甾体底物溶解度。吐温80是一种应用非常广泛一种表面活性剂,被广泛用于增加甾体底物的溶解度。
发明内容:
本发明构建了高效制备C11α-羟基孕酮的11α-羟化酶突变体CYP68J5mL491F;提供了一种工业高效转化甾体孕酮生产11α-羟基孕酮的新型雷斯青霉重组菌,并优化了发酵工艺。
本发明技术路线如下:以实验室前期构建的重组酿酒酵母Sc-CYP68J5为基础,对该11α-羟化酶基因CYP68J5进行定点突变,获得了羟化特异性显著提高的突变体CYP68J5mL491F。突变体的表达受雷斯青霉15α-羟化酶基因PRH的ORF上游800bp序列启动子的驱动,以15α-羟化酶基因PRH基因的左右臂设计同源臂,通过同源重组技术构建了一个敲除载体质粒pPZP-ΔPRH-11αmL491F。将该重组质粒电转化至农杆菌AGL-1的感受态细胞中,得到正确的转化子。最终利用农杆菌共培养技术手段,使上述11α-羟化酶突变体CYP68J5mL491F取代野生雷斯青霉的15α-羟化酶PRH,获得了一种高效制备医药中间体11α-羟基孕酮的C11α-羟化酶突变体雷斯青霉重组菌。
本发明所述的CYP68J5基因来源于赭曲霉(Aspergillus ochraceus TCCC41060);
本发明未定点突变的氨基酸序列如SEQ ID NO:1所示,其相应核苷酸序列如SEQID NO:2所示。
本发明中突变体CYP68J5mL491F氨基酸序列如SEQ ID NO:3所示,其相应核苷酸序列如SEQ ID NO:4所示。以上序列属于本发明的保护范围。
利用本发明所述的编码C11α-羟化酶的基因片段CYP68J5所构建的重组表达载体、重组表达质粒或宿主细胞也属于本发明的保护范围,所使用的扩增引物序列、针对基因片段CYP68J5定点突变所涉及氨基酸位点也属于本发明的保护范围。
本发明所述的经定点突变技术获得的突变基因CYP68J5mL491F编码的甾体化合物羟化酶包括但不限于酿酒酵母、雷斯青霉等宿主细胞内表达。
有益效果:
11α-羟化酶突变体CYP68J5mL491F高效异源表达,甾体底物孕酮的投料量为15g/L,转化72h,产物11α-羟基孕酮得率为92%;获得的C11α-羟化酶突变体CYP68J5mL491F雷斯青霉重组菌可以用于高效制备医药中间体11α-羟基孕酮。
附图说明:
图1为C11α-羟化酶CYP68J5转化孕酮(PRG)生成11α-羟基孕酮(11α-OH-PRG)的C11α-羟基化反应;
图2.酿酒酵母重组表达质粒pYES2-11αmL491F示意图;
图3.重组酿酒酵母Sc-11αmL491F转化PRG合成11α-OH-PRG的TLC分析;
图4.重组酿酒酵母Sc-11αmL491F转化PRG合成11α-OH-PRG的HPLC分析;
图5.C11α-羟化酶突变体L491F表达盒示意图,其表达受雷斯青霉15α-羟化酶基因PRH的ORF上游800bp序列启动子的驱动;
图6.重组表达质粒pPZP-ΔPRH-11αmL491F示意图;
图7.重组雷斯青霉转化子菌落;
图8.重组雷斯青霉Pr-ΔPRH-11αmL491F转化PRG合成11α-PRG的TLC分析;
图9.重组雷斯青霉P.r-ΔPRH-11αmL491F在15g/L底物PRG投料量下的11α-OH-PRG得率。
具体实施方式:
下面通过具体的实施方案叙述本发明方法。下述实施例中的实验方法,如无特殊说明,均为常规方法。实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1.C11α-羟化酶突变体L491F的构建与功能评价
1.1突变体L491F的构建
以质粒pYES2-CYP68J5作为模板,分别以L491F-F/R为引物进行聚合酶链式反应,得到定点突变目标载体片段L491F。引物由北京华大基因公司进行合成。
引物名称 引物碱基序列
L491F-F ACTTATTTTGCGGATCCCAATACCAGGA
L491F-R ATCCGCAAAATAAAGTCATCCCGATGTTGAG
PCR反应体系(25μl)
PCR反应条件
取2μL扩增产物经琼脂糖凝胶电泳验证条带大小。PCR产物经Dpn I酶消化3h(70℃灭酶活20min)后,利用小量DNA纯化试剂盒进行回收。将回收产物化转入JM109大肠感受态,挑取转化子于LB试管中,于37℃摇床200r摇8-12h后提取质粒,进行酶切验证。
L491F定点突变重组质粒经Spe I酶切,获得0.5kb和6.8kb大小的两条条带,琼脂糖凝胶电泳图结果显示得到的片段大小与理论结果一致,即得到突变体质粒pYES2-11αmL491F,图2为表达载体构建示意图。
1.2重组酿酒酵母Sc-11αmL491F转化PRG产11α-OH-PRG的功能评价
将上述得到的重组质粒pYES2-11αmL491F通过醋酸锂转化法转入酿酒酵母,利用尿嘧啶营养缺陷的SC培养基进行初步筛选转化子,经YPD培养基再生后,进行甾体底物PRG转化发酵实验。
底物PRG的投料量为1g/L,重组酿酒酵母突变体Sc-11αmL491F进行PRG甾体转化实验,在30℃摇床振荡培养72h后取样,乙酸乙酯萃取上清液,薄层层析对产物进行定性分析,经紫外分析仪对色谱进行分析发现,突变体11αmL491F同突变之前相比副产物明显减少,即羟化特异性有些许提高。转化结果如图3所示
萃取上述突变体发酵转化产物取100μL上清液烘干,加200μL超声混合均匀的流动相液体,混合均匀后制备液相样品,进行HPLC高效液相色谱检测,进行HPLC检测,检测结果如图4所示。
液相条件:流动相A:流动相B=乙腈:水=80%:20%;温度:25℃;流速:0.8mL/min;进样量:10μL;检测波长:254m;进样时间:10min;
由液相结果分析可知:8.092min左右为底物PRG的出峰时间,4.297min左右为产物11α-OH-PRG的出峰时间。重组酿酒酵母突变体CYP68J5mL491F与基因野生型相比,副产物峰明显减少,产物11α-OH-PRG的得率提高了20.96%。
实施例2.重组质粒pPZP-ΔPRH-11αmL491F的构建
2.1载体骨架pPZp-ΔPRH-的获得
根据实验室前期已构建的重组质粒pPZP-ΔPRH-CYP68J5设计用于反向PCR除CYP68J5部分的骨架的引物进行PCR扩增,引物由北京华大基因公司进行合成。
引物名称 引物碱基序列
反-F GAATTCCAGCACACTGGCGGCCGTTAC
反-R GTCGCAGAGAACTCATCCATTATCATTG
PCR反应25μL体系:
PCR反应条件:
PCR产物经0.8%琼脂糖凝胶电泳验证正确
2.2***目的片段11αmL491F的获得
从已构建好的重组质粒pYES2-11αmL491F上扩增目的片段11αmL491F,正、反向PCR引物5’端引入20bp的线性化载体骨架pPZP-ΔPRH末端同源序列,使得***片段PCR产物5’和3’末端分别带有与线性化载体两末端对应的完全一致的序列。引物由北京华大基因公司进行合成。
引物名称 引物碱基序列
HR-F CCGCCAGTGTGCTGGAATTcATGCCCTTCTTCACTGGGC
HR-R TGGATGAGTTCTCTGCGACCTACACAGTTAAACTCGC
PCR反应条件:
PCR产物经0.9%琼脂糖凝胶电泳验证正确
2.3重组质粒pPZP-ΔPRH-11αmL491F的构建
重组反应体系(推荐冰上配制,各组分使用前需混匀)
体外重组反应:将配制好的重组反应体系置于50℃金属浴上反应20min。冰上冷却5min。
重组产物转化并验证:将重组产物化转入JM109大肠感受态,挑取转化子于LB试管中,于37℃摇床200r摇8-12h后提取质粒,经Xba I酶切验证后确有2.8kb和9.7kb的两条带,证明成功构建重组质粒pPZP-ΔPRH-11αmLA91F,如图6所示。
实施例3.C11α-羟化酶突变体CYP68J5mL491F重组雷斯青霉的构建及功能评价
3.1农杆菌感受态细胞的制备
取农杆菌甘油管三区划线,挑取农杆菌单菌落,接种于5mL LB液体培养基,于28℃,200r/min振荡培养约12h;
以2%的接种量将上述菌液接种于50mL灭过菌的LB液体培养基中,28℃,200r/min振荡培养4-6h,至OD600值为0.8;
将培养好的菌液冰浴30min(适当震荡,以确保菌液均匀冷却),然后转移至预冷的灭过菌的50mL离心管中,4℃,5000r/min离心10min,弃上清;
用10mL预冷的无菌水重新悬浮菌体,4℃,5000r/min离心10min;
用20mL预冷的10%甘油重悬菌体,4℃,5000r/min离心10min;
用1mL的10%甘油重悬菌体,按100μL/管的量分装于1.5mLEP管中,于-80℃保存备用。
3.2雷斯青霉孢子悬液制备和培养
孢子悬液的制备:用接种环将雷斯青霉ATCC 10490的孢子划线接种于PDA试管斜面上,28℃培养3-5天。待青色孢子成熟后,用预先灭过菌的ddH2O洗涤,用灭过菌的玻璃珠将其打散(震荡约15-30min),用血球计数板计数,收集浓度为107个/mL的孢子悬液,接种于装有50mL PDA液体培养基的250mL摇瓶中,于28℃、200r/min的摇床中培养约24h。
3.3潮霉素B对雷斯青霉最小抑制浓度的确定
配制PDA固体培养基,用微波炉彻底融化后凉至50-60℃,使潮霉素B终浓度分别为0μg/mL,100μg/mL,150μg/mL,200μg/mL,250μg/mL,300μg/mL。摇晃均匀后倒制平板,凝固后每个平板分四个区域滴10-15μL提前收集的雷斯青霉孢子悬液(107个/mL)
潮霉素B对雷斯青霉的生长有较大的影响作用,在一定的范围内,雷斯青霉的致死率随着潮霉素B浓度的增加而增大。当浓度为100μg/mL时,菌体生长缓慢;当浓度为150μg/mL时,菌体生长受到明显抑制;当浓度为300μg/mL时,雷斯青霉完全不能生长,所以最终确定潮霉素B对雷斯青霉的最小抑制浓度为300μg/mL
3.4.重组质粒电击转化至根癌农杆菌AGL-1
将电转杯用无水乙醇洗三次,再用ddH2O清洗三次,晾干后冰浴;
取2μL的重组质粒pPZP-ΔPRH-11αmL491F,加入提前于冰上融化的农杆菌感受态细胞中;
冰浴10min后,将混合物转移至预冷的0.1cm电转杯中,***Bio-rad电转仪的电转槽中,1800V电转:
电转后,立即取出电转杯,加入800μL的LB复苏液于28℃,200r/min复苏3h。
复苏菌液梯度涂布在相应的含抗性LB平板上,28℃培养箱培养36-48h,待转化子长出。3.5根癌农杆菌阳性克隆转化子筛选验证
挑取几个长势良好的单菌落于Kana浓度为100ug/mL的LB平板上划线,于28℃培养箱培养2-3d;
用灭过菌的牙签沾取转化子,在100μL无菌的ddH2O中混匀;
农杆菌悬液在开水中煮沸2min;
将煮沸后的菌悬液作为模板,使用ES-Taq进行菌落PCR,验证克隆扩增标记基因HYG。验证引物如下:
引物名称 引物碱基序列
HYG-F GTACCTGTGCATTCTGGGTAAACG
HYG-R TGTTTATCGGCACTTTGCATCGGC
PCR反应体系:
PCR反应条件:
取2μl PCR产物用经琼脂糖凝胶电泳验证条带大小为1.5kb,即得到正确的转化子。
3.6.农杆菌介导雷斯青霉转化
将电击转化导入重组质粒的农杆菌在LB平板上三区划线,于28℃培养箱培养2-3d,同时将缺失15α羟化酶基因PRH的雷斯青霉孢子甘油管于PDA试管斜面划线,于28℃培养箱培养3-4d;
农杆菌长出单菌落后,接种于5mL LB液体培养基中培养24h。按1%的接种量接于装有50mL LB液体培养基摇瓶中,28℃摇床200rpm培养8-10h,至OD600值为0.8;
取15mL菌液离心后用5mL含有诱导剂乙酰丁香酮的IM液体培养基重悬洗涤菌体,5000rpm离心10min,弃上清;
用15mL含有诱导剂乙酰丁香酮的IM液体培养基重悬菌体,于28℃,200rpm摇床避光诱导培养5h;
农杆菌诱导期间,收集雷斯青霉孢子,调整孢子悬液为107个/mL备用;
将诱导好的农杆菌与雷斯青霉孢子悬液按体积比1∶1混合均匀,取0.5mL涂布于铺有转移膜的IM平板上,25℃共培养48h;(转移膜为孔径0.45μm的水系膜)
将转移膜揭下置于培养皿盖上,用1mL无菌水将菌丝洗下,收集孢子悬液涂布于CM平板上(含有Amp、Kan、Cefo、HygB)初筛;
3.7雷斯青霉阳性转化子的验证
待长出转化子,在其产孢前,转移至300μg/mL HygB抗性的PDA试管斜面进行复筛,于28℃培养箱培养3-5d;
待试管中转化子产孢后,用无菌水将孢子洗下并保菌,余下孢子悬液接种于YPD液体培养基,培养菌丝,提取基因组进行PCR验证。设计验证引物进行PCR扩增验证
引物名称 引物碱基序列
验m-Hyg-F ATGGCTGTCCTCACCGAATTG
验m-Hyg-R CGGTGCCATTGCTAAAATTTTTCG
PCR反应条件:
取2μL PCR产物经琼脂糖凝胶电泳验证条带大小,野生型基因组DNA不能扩增出条带,敲除突变株则扩增出5.4kb大小的条带即为正确转化子,可保菌继续之后的甾体转化实验。3.8重组雷斯青霉Pr-ΔPRH-11αmL491F的发酵转化评价
将验证正确的重组雷斯青霉Pr-ΔPRH-11αmL491F及雷斯青霉野生型菌株分别接种于50mLYPD液体培养基中,28℃培养24h。底物PRG的投料量为1g/L,28℃,200rpm摇床发酵转化72h后取样,乙酸乙酯萃取上清液,薄层层析对产物进行定性分析。结果表明雷斯青霉重组菌可以将甾体PRG转化为11α-OH-PRG。
经紫外分析仪对色谱进行分析发现,重组雷斯青霉突变株Pr-ΔPRH-11αmL491F同基因野生型菌株相比,副产物明显减少,羟化特异性显著提高。转化结果如图8所示
经HPLC高效液相色谱检测分析,与C11α基因野生型的11α-OH-PRG产率72%相比,重组雷斯青霉Pr-ΔPRH-11αmL491F的11α-OH-PRG产率可以达到90.7%。
实施例4.重组雷斯青霉Pr-ΔPRH-11αmL491F转化PRG产11α-OH-PRG的工艺建立
4.1底物溶剂对甾体转化的影响
考虑到底物PRG的溶解性,为提高底物PRG的转化率,研究了在丙酮、甲醇、乙醇、二甲基亚砜、乙酸乙酯等溶剂中的转化率。综合极性、PRG的转化率、11α-OH-PRG得率、经济成本及安全性角度选择甲醇作为底物溶剂。
4.2底物浓度对甾体转化的影响
研究了重组菌在较高投料浓度下的甾体转化能力,研究了重组菌株分别在1g/L、3g/L、5g/L、10g/L、15g/L、20g/L、25g/L底物浓度下72h底物的转化率。研究发现,底物浓度在15g/L时底物转化率比野生菌株较高。重组菌株在底物浓度为15g/L时,11α-OH-PRG的得率达到最大,这也说明该重组菌株对高浓度的底物有更好的适应性。
4.3菌体接种量对甾体转化的影响
收集107个/mL的重组雷斯青霉孢子悬液,按0.5%、1%、1.5%、2%、2.5%的接种量接于YPD培养基装液量为50ml的250ml摇瓶中进行发酵转化。最终选择2%的接种量。
4.4不同浓度吐温80对甾体转化的影响
吐温80即聚山梨醇酯80,是一种亲水性表面活性剂,对改善底物PRG表面润湿性有良好的效果,可以大幅度提高PRG的溶出度。为研究吐温80对甾体转化的影响,选取重组雷斯青霉Pr-ΔPRH-11αmL491F,底物PRG的投料浓度为15g/L,分别投加不同浓度的吐温80:0g/L、0.5g/L、1.0g/L、1.5g/L、2.0g/L后分析菌株对底物的转化率。吐温80浓度为1g/L时,PRG的转化率明显高于其他吐温80浓度。表面活性剂浓度的提高,对细胞会造成一定程度的伤害,并且表面活剂在水中易于起泡沫,加入量过多会增加消泡剂的用量,综合考虑在底物浓度为15g/L时,最佳转化时间为72h,最佳添加吐温80浓度为1g/L。
4.5重组雷斯青霉Pr-ΔPRH-11αmL491F在15g/L底物投料量下的甾体转化分析
根据上述条件的优化,重组雷斯青霉Pr-ΔPRH-11αmL491F建立以甲醇为底物溶剂溶解底物PRG浓度至15g/L,重组雷斯青霉孢子悬液(孢子数107个/mL)接种量为2%,吐温80浓度为1g/L的工艺条件下,重组雷斯青霉Pr-ΔPRH-11αmL491F转化底物PRG,在48h、72h和96h取样,发酵转化72h产物11α-OH-PRG的得率为92%,结果如图9所示。
上述实施例仅表达了本发明的几种实施方式,但其并非用以限定本发明。应当指出的是,任何本领域技术人员,在不脱离本发明的精神和范围内,上述各实施方式还可以做出若干改进。因此,本专利的保护范围应以权利要求书所限定的内容为准。
Claims (3)
1.C11α-羟化酶突变体L491F其特征在于,所述突变体的氨基酸序列是在序列SEQ IDNO:1的基础上进行突变;突变体L491F的491位氨基酸进行突变。
2.权利要求1所述突变体的表达受雷斯青霉15α-羟化酶基因PRH的ORF上游800bp序列启动子的驱动。
3.权利要求1所述C11α-羟化酶突变体L491F用于工业高效转化孕酮生产11α-羟基孕酮,其特征在于,C11α-羟化酶突变体L491F转化甾体底物孕酮生成11α-羟基孕酮时羟化特异性和得率提高。
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