CN117327669A - 一种细胞色素p450酶及其在催化合成蒽环类化合物中的应用 - Google Patents
一种细胞色素p450酶及其在催化合成蒽环类化合物中的应用 Download PDFInfo
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Abstract
本发明提供一种细胞色素P450酶DoxA(CYP129亚家族),其氨基酸序列为SEQ ID NO:1。本发明还提供所述的P450酶DoxA的一种用途,是催化蒽环类化合物13‑脱氧柔红霉素合成13‑二氢柔红霉素或13‑二氢柔红霉素衍生物;所述的方法,其中所述的450酶DoxA利用NAD(P)H催化13‑脱氧柔红霉素。本发明提供了一种能够直接利用NAD(P)H的细胞色素P450酶DoxA,能够利用NAD(P)H一步合成13‑二氢柔红霉素,无需电子传递蛋白辅助,简化了反应系统,降低了反应成本,增强了反应的可控性和稳定性;电子传递过程更加直接高效,提高反应的催化效率。
Description
技术领域
本发明属于酶工程、合成生物学和生物技术领域,具体涉及一种细胞色素P450酶DoxA(CYP129亚家族)及其在利用NAD(P)H合成蒽环类化合物的应用。
背景技术
P450酶作为万能生物催化剂,其反应过程一般需要电子传递蛋白(铁氧还蛋白还原酶FdR和铁氧还蛋白Fdx)的辅助,将来自辅因子NAD(P)H的电子传递到亚铁原卟啉(heme)中心,进而激活分子氧以催化反应。尚未在原核生物界发现能够直接利用NAD(P)H的P450蛋白。
蒽环类化合物13-脱氧柔红霉素(DOD)和13-二氢柔红霉素(DHD)分别是合成阿霉素(DXR)和柔红霉素(DNR)的重要前体和关键中间体。目前,关于13-二氢柔红霉素的生物法产生过程的研究主要集中在链霉菌的生物转化,尚未有通过酶法催化13-脱氧柔红霉素转化为13-二氢柔红霉素的报道。
发明内容
本发明提供一类细胞色素P450酶DoxA(CYP129亚家族),该蛋白直接利用NAD(P)H实现电子的获取和传递,催化13-脱氧柔红霉素合成13-二氢柔红霉素,为阿霉素的合成提供了新的路径。
本发明发现了细胞色素P450酶DoxA可以直接利用NAD(P)H催化13-脱氧柔红霉素氧化生成13-二氢柔红霉素。
本发明首先提供一种天蓝淡红链霉菌(Streptomyces coeruleorubidus)来源的P450酶DoxA,包含有:
1)氨基酸序列为SEQ ID NO:1的蛋白酶,
VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALAREVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLRRIHAPAFNPRRLAERTDRIAAIAGRLLTELADTSGRSGKPAELIGGFAYHFPLLVICELLGVPVTDPAMAREAVSVLKALGLGGPQSGGGDGTDPAGGVPDTSALESLLLEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIFAGHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLWRFAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDDPHAFHPDRPSWRRLTFGDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWSRKGAQTARLTELPVWLR(SEQ ID NO:1);
2)在序列为SEQ ID NO:1的蛋白上取代、缺失、添加一个或几个氨基所获得的,其具有1)中所述蛋白酶功效的衍生蛋白酶;
本发明还提供一种编码所述的细胞色素P450酶DoxA的基因doxA,其一种具体的核苷酸序列如下:
gtggccgtcgacccgttcgcgtgtcccatgatgaccatgcagcgcaagcccgaggtgcacgacgccttccgggaggcgggcccggtcgtcgaggtgaacgcccccgcgggcggacccgcctgggtcatcaccgatgacgccctcgcccgcgaggtgctggccgatccccggttcgtgaaggaccccgacctcgcccccgccgcctggcggggggtggacgacggtctcgacatccccgttccggagctgcgtccgttcacgctcatcgccgtggacggcgaggcccaccggcgcctgcgccgcatccacgcacccgcgttcaacccgcgccggctggccgagcggacggatcgcatcgccgccatcgccggccggctgctcaccgaactcgccgacacttccggccggtcgggcaaaccggccgagctgatcggcggtttcgcgtaccacttcccgctgttggtcatctgcgagctgctcggcgtgccggtcaccgatccggcgatggcccgcgaggccgtcagcgttctcaaggcactcggcctcggcggcccgcagagcggcgggggtgacggcacggaccctgccgggggcgtgccggacacctcggccctggagagcctgctcctcgaagccgtgcactcggcccggcggaacgacaccccgaccatgacccgcgtgctgtacgaacgcgcgcaggccgagttcggctcggtctccgacgaccagctcgtctacatgatcaccgggctcatcttcgccggccacgacaccaccggctccttcctgggcttcctgctcgcggaggtgctggcgggccgcctcgcggcggacgccgacgaggacgccgtctcccggttcgtggaggaggcgctgcgctaccacccgccggtgccctacacgttgtggaggttcgctgccacggaggtgaccatcggcggcgtccggctgccccgcggagcgccggtgctggtggacatcgagggcaccaacaccgacggccgccatcacgacgacccgcacgccttccacccggaccgtccctcgtggcggcggctcaccttcggcgacgggccgcactactgcatcggggagcagctcgcccagctggagtcgcgcacgatgatcggcgtactgcgcagcaggttccccgaggcccgactggccgtgccgtacgacgagttgcggtggtcccggaagggggcccagacggcgcggctcaccgaactgcccgtctggctgcgctga(SEQ ID NO:2)。
本发明还提供一种重组表达载体,所述的重组表达载体携带有上述的编码基因的核酸片段。
本发明再一个方面还提供一种重组工程菌株,所述的重组工程菌株携带有上述的重组表达载体。
本发明还提供所述的P450酶DoxA的一种用途,是催化13-脱氧柔红霉素合成13-二氢柔红霉素或13-二氢柔红霉素衍生物;
本发明再一个方面还提供一种催化催化13-脱氧柔红霉素合成13-二氢柔红霉素的方法,所述的方法是使用上述的P450酶DoxA进行催化;
更进一步的,所述的方法,其中所述的450酶DoxA利用NAD(P)H催化13-脱氧柔红霉素。
本发明提供了DoxA蛋白或其同源蛋白利用NAD(P)H催化产生蒽环类13-脱氧柔红霉素或13-二氢柔红霉素衍生物或类似物的方法。
更进一步的,所述的方法,其中NADPH在反应体系中的添加浓度为2-5mM。
本发明提供了一种能够直接利用NAD(P)H的细胞色素P450酶DoxA,能够利用NAD(P)H一步合成13-二氢柔红霉素,无需电子传递蛋白辅助,简化了反应系统,降低了反应成本,增强了反应的可控性和稳定性;电子传递过程更加直接高效,提高反应的催化效率。本发明的细胞色素P450酶DoxA直接利用NAD(P)H合成蒽环类化合物13-二氢柔红霉素的性质,也为抗癌药物阿霉素和柔红霉素的合成提供了一种新的合成途径。
附图说明
图1:P450酶DoxA蛋白SDS-PAGE检测图谱,
图2:P450酶DoxA利用NADPH催化13-脱氧柔红霉素产生13-二氢柔红霉素的UPLC检测图谱,
图3:P450酶DoxA利用NADH催化13-脱氧柔红霉素产生13-二氢柔红霉素的UPLC检测图谱。
具体实施方式
本发明提供一种天蓝淡红链霉菌(Streptomyces coeruleorubidus)来源的P450酶DoxA,包含有:
1)氨基酸序列为SEQ ID NO:1的蛋白酶;
2)在序列为SEQ ID NO:1的蛋白上取代、缺失、添加一个或几个氨基所获得的,其具有1)中所述蛋白酶功效的衍生蛋白酶;
所述的衍生蛋白酶,就是与氨基酸序列为SEQ ID NO:1的蛋白酶具有不少于90%的同源性,最佳是99%的同源性(即与SEQ ID NO:1的氨基酸相比差不多于5个氨基酸的差异)。
本发明还提供编码上述的P450酶DoxA的编码基因,其中一种具体的核酸序列为SEQ ID NO:2;但还可以根据宿主的不同来对编码基因进行优化。
本发明还提供一种重组表达载体,用于在工程菌的宿主中重组表达上述的P450酶DoxA,所述的重组表达载体可以是蛋白重组表达任一表达载体,例如pET28b载体。
本发明再一个方面还提供一种重组工程菌株,所述的重组工程菌株携带有上述的重组表达载体。
本发明还提供所述的P450酶DoxA的一种用途,是催化13-脱氧柔红霉素合成13-二氢柔红霉素或13-二氢柔红霉素衍生物;
本发明再一个方面还提供一种催化催化13-脱氧柔红霉素合成13-二氢柔红霉素的方法,所述的方法是使用上述的P450酶DoxA进行催化;
更进一步的,所述的方法,其中所述的P450酶DoxA利用NAD(P)H催化13-脱氧柔红霉素。
本发明提供了DoxA蛋白或其同源蛋白利用NAD(P)H催化产生13-脱氧柔红霉素或13-二氢柔红霉素衍生物或类似物的方法。
下面结合实施例和附图对本发明进行详细的描述。
实施例1:P450 DoxA蛋白的重组表达
由华大生物科技有限公司对天蓝淡红链霉菌(S.coeruleorubidus)中的doxA基因进行密码子优化(其氨基酸序列为SEQ ID NO:1,编码基因的核苷酸序列为SEQ ID NO:2),克隆到pET28b载体,转化大肠杆菌菌株BL21(DE3)。将能够高效表达的DoxA蛋白的大肠杆菌菌株划线接种至LB(含50μg/mL Kan)平板,37℃培养24h。挑取单克隆于50mL LB(含50μg/mLKan)培养基,37℃、220rpm摇床培养过夜。按1%接种量接种至含500mL TB或LB(含50μg/mLKan)培养基的2L三角瓶中,37℃、200rpm培养至OD600介于0.8~1,加入终浓度200μM的IPTG、500μM的5-氨基乙酰丙酸(5-ALA)和500μM的维生素B1(VB1),随后,在18℃、150rpm条件下培养18-20h诱导蛋白表达。
离心收集菌体,加入30-40mL Lysis buffer(50mM NaH2PO4,300mM NaCl,10mM咪唑,10%甘油,pH 8.0)涡旋振荡重悬菌体,超声破碎细胞后10000rpm,4℃离心60min,上清液与Ni-NTA 4℃孵育60min。然后,使用Wash buffer(50mM NaH2PO4,300mM NaCl,20mM咪唑,10%甘油,pH 8.0)对杂蛋白进行洗脱;用Elution buffer(50mM NaH2PO4,300mM NaCl,250mM咪唑,10%甘油,pH 8.0)对目标蛋白进行洗脱,使用50KD超滤管浓缩蛋白溶液;SDS-PAGE检测获得可溶性DoxA蛋白(图1)。
实施例2:DoxA直接催化13-脱氧柔红霉素氧化生成13-二氢柔红霉素
以100μM 13-脱氧柔红霉素为底物,加入2μM DoxA,反应缓冲液为50mM NaH2PO4,300mM NaCl,pH 7.5,总体积100μL,煮沸的DoxA作为对照,30℃反应2h,向反应液中加入2倍体积甲醇终止反应,14,000×g离心10min,取上清进行UPLC检测。检测程序:流动相A为含0.1%甲酸的水,流动相B为含0.1%甲酸的乙腈,洗脱梯度为0-0.5min,20%B;0.5-5min,20-90% B;5-5.1min,90-100% B;5.1-5.6min,100% B;5.6-5.7min,100-20% B;5.7-6.5min,20% B;流速0.4mL/min,检测波长为470nm。
结果发现在未外源添加辅因子的条件下,产生目标产物13-二氢柔红霉素(保留时间为3.1min),其产率约12%(图2ii),说明DoxA可以利用自身结合的NAD(P)H实现13-二氢柔红霉素合成。
实施例3:DoxA利用2mM NADPH催化13-脱氧柔红霉素氧化生成13-二氢柔红霉素
在含100μM 13-脱氧柔红霉素和2μM DoxA的体系中,增加2mM NADPH,30℃反应2h,向反应液中加入2倍体积甲醇终止反应。以含水(0.1%甲酸)为流动相A,乙腈为流动相B(0.1%甲酸),洗脱梯度为0-0.5min,20% B;0.5-5min,20-90% B;5-5.1min,90-100% B;5.1-5.6min,100% B;5.6-5.7min,100-20% B;5.7-6.5min,20% B;流速0.4mL/min,检测波长为470nm。
结果发现在含有2mM NADPH条件下,产物13-二氢柔红霉素产率提高至约64%(图2iii);进一步证明P450酶DoxA直接利用NADPH催化13-脱氧柔红霉素氧化生成13-二氢柔红霉素。
实施例4:DoxA利用5mM NADPH催化13-脱氧柔红霉素氧化生成13-二氢柔红霉素
在含100μM 13-脱氧柔红霉素和2μM DoxA的体系,增加5mM NADPH,30℃反应2h,向反应液中加入2倍体积甲醇终止反应。以含水(0.1%甲酸)为流动相A,乙腈为流动相B(0.1%甲酸),洗脱梯度为0-0.5min,20% B;0.5-5min,20-90% B;5-5.1min,90-100% B;5.1-5.6min,100% B;5.6-5.7min,100-20% B;5.7-6.5min,20% B;流速0.4mL/min,检测波长为470nm。检测结果证实,在含有5mM NADPH的反应条件下,反应产物13-二氢柔红霉素的产率提高至约71%(图2iv)。
实施例5:DoxA利用NADH催化13-脱氧柔红霉素生成13-二氢柔红霉素
向含100μM 13-脱氧柔红霉素和2μM DoxA的反应体系中加入5mM NADH,30℃反应2h,加入两倍体积的甲醇混匀终止反应。以含水(0.1%甲酸)为流动相A,乙腈为流动相B(0.1%甲酸),洗脱梯度为0-0.5min,20% B;0.5-5min,20-90% B;5-5.1min,90-100% B;5.1-5.6min,100% B;5.6-5.7min,100-20% B;5.7-6.5min,20% B;流速0.4mL/min,检测波长为470nm。检测结果发现13-二氢柔红霉素产率约75%(图3)。证明P450酶DoxA能够直接利用NADH催化13-脱氧柔红霉素氧化产生13-二氢柔红霉素
综上,本发明提供了一类细胞色素P450酶DoxA,能够直接利用NAD(P)H催化13-脱氧柔红霉素合成阿霉素的重要中间体13-二氢柔红霉素。该酶催化系统简单高效,为工业生产柔红霉素和阿霉素提供了新思路;丰富了自然界P450酶的宝库,为P450酶学机制研究提供了重要素材。
Claims (10)
1.一种P450酶DoxA,其特征在于,所述的P450酶DoxA包含有:
1)氨基酸序列为SEQ ID NO:1的蛋白酶,
2)在序列为SEQ ID NO:1的蛋白上取代、缺失、添加一个或几个氨基所获得的,其具有1)中所述蛋白酶功效的衍生蛋白酶。
2.一种基因,其特征在于,所述的基因编码权利要求1所述的P450酶DoxA。
3.如权利要求2所述的基因,其特征在于,所述的基因的核苷酸序列为SEQ ID NO:2。
4.一种重组表达载体,其特征在于,所述的重组表达载体携带有权利要求2或3所述的基因的核酸片段。
5.一种重组工程菌株,其特征在于,所述的重组工程菌株中携带有权利要求4所述的重组表达载体。
6.如权利要求1所述的重组工程菌株,其特征在于,所述的重组工程菌株为大肠杆菌工程菌株。
7.权利要求1所述的P450酶DoxA在催化13-脱氧柔红霉素合成13-二氢柔红霉素或13-二氢柔红霉素衍生物中的应用。
8.一种催化13-脱氧柔红霉素合成13-二氢柔红霉素或其衍生物的方法,其特征在于,所述的方法是使用权利要求1所述的P450酶DoxA进行催化。
9.如权利要求8所述的方法,其特征在于,所述的方法,在反应体系中添加有NAD(P)H。
10.如权利要求9所述的方法,其特征在于,所述的方法,其中NAD(P)H在反应体系中的添加浓度不低于1mM。
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