CN106282080A - 一种新金色分枝杆菌来源的甾酮c27‑单加氧酶及其应用 - Google Patents
一种新金色分枝杆菌来源的甾酮c27‑单加氧酶及其应用 Download PDFInfo
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Abstract
本发明公开了一种新金色分枝杆菌来源的甾酮C27‑单加氧酶及其应用,属于基因工程和酶工程技术领域。本发明通过基因敲除及加强表达的方法,在新金色分枝杆菌中筛选到甾醇边链降解过程中关键酶SMO的三个同工酶。将其分别在高产雄甾二烯二酮(ADD)的新金色分枝杆菌中加强表达,ADD产量得到明显提高,其中SMO2的效果最明显。通过过量表达SMO2,ADD最终产量从5.2g/L提高到7.3g/L。本发明为微生物发酵法提高ADD产量的工业化提供了有益的指导。
Description
技术领域
本发明涉及一种新金色分枝杆菌来源的甾酮C27-单加氧酶及其应用,属于基因工程和酶工程技术领域。
背景技术
甾体激素类药物因其多种生理功能,对机体起着非常重要的调节作用,故在临床上具有广泛的应用。甾体激素类药物被广泛用于抗肿瘤、抗炎症、抗菌、抗病毒、抗激素和抗过敏等。此外,各类性激素类药物是治疗性器官退化和妇科疾病的主要药物,是口服避孕药的主要成分。同时甾体激素类药物还可以作为抗肥胖药物的活性成分,用于预防冠心病以及作为HIV整合酶的抑制剂,预防HIV的传染和用于艾滋病的治疗等。雄甾-4-烯-3,17-二酮(AD)和雄甾-1,4-二烯-3,17-二酮(ADD)作为甾体激素类药物的重要中间体,几乎可以合成所有的甾体激素类药物,在甾体的工业化生产中占有重要的地位。
目前制备AD和ADD主要有两种途径:一是从野生中草药黄姜、穿地龙等植物中提取薯蓣皂苷元,然后进行化学合成。但该途径存在生产成本高、过程复杂、环境污染严重等问题;二是以自然界中丰富存在的动植物甾醇为原料,通过微生物发酵技术对甾醇侧链进行选择性降解而得到目的产物。微生物法具有成本低、对环境温和等优点,越来越受到人们的重视。世界先进国家目前进行甾体药物制备,大多以动植物甾醇为起始原料进行微生物转化,得到甾体药物中间体后,再进一步制备。
众多学者对微生物转化甾醇合成AD/ADD进行了大量的研究,其中分枝杆菌是生产AD/ADD的优良菌株之一。已有报道微生物转化甾醇合成AD/ADD的代谢途径需要很多的酶,但是具体的是那些酶参与还没有完全研究明白。目前研究的进展是仅仅鉴定和证明了甾醇转化的第一步酶胆固醇氧化酶和最后一步酶3-甾酮-Δ1-脱氢酶。其他途径中的酶还未鉴定和应用。
因此,有必要对分枝杆菌转化甾醇合成AD/ADD过程中的关键酶及其功能鉴定进行深入研究,以期在此基础上开发可促进分枝杆菌转化甾醇合成AD/ADD的方法。
发明内容
为了解决上述问题,本发明通过基因敲除及回补的方法,首次在新金色分枝杆菌中筛选到甾醇边链降解过程中关键酶甾酮C27-单加氧酶(SMO)的三个同工酶(SMO1,SMO2,SMO3)。分别将这三个同工酶在高产雄甾-1,4-二烯-3,17-二酮(ADD)的新金色分枝杆菌中加强表达,ADD产量都得到明显提高,其中SMO2的加强表达对提高ADD的产量效果更明显。新金色分枝杆菌中甾酮C27-单加氧酶的功能鉴定和应用均为首次报道,且重组菌对提高ADD的产量较明显。加强表达SMO2基因的菌株的ADD最终产量从5.2g/L提高到7.3g/L。
本发明的第一个目的是提供一种甾酮C27-单加氧酶,所述甾酮C27-单加氧酶的氨基酸序列如SEQ NO.4、SEQ NO.5或SEQ NO.6所示。
本发明的第二个目的一种ADD产量提高的新金色分枝杆菌重组菌,所述重组菌是过表达了甾酮C27-单加氧酶基因的新金色分枝杆菌;所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
在本发明的一种实施方式中,所述甾酮C27-单加氧酶基因Smo1的氨基酸序列如SEQ NO.4所示,核苷酸序列如SEQ NO.1所示。
在本发明的一种实施方式中,所述甾酮C27-单加氧酶基因Smo2的氨基酸序列如SEQ NO.5所示,核苷酸序列如SEQ NO.2所示。
在本发明的一种实施方式中,所述甾酮C27-单加氧酶基因Smo3的氨基酸序列如SEQ NO.6所示,核苷酸序列如SEQ NO.3所示。
在本发明的一种实施方式中,所述新金色分枝杆菌重组菌是以新金色分枝杆菌出发菌过表达了甾酮C27-单加氧酶基因。
在本发明的一种实施方式中,所述出发菌可以是以下任意一种:Mycobacteriumneoaurum ATCC 25795、Mycobacterium neoaurum ZJUVN(CGMCC 5477)、Mycobacteriumneoaurum NwIB-01(CCTCC M 209094)、Gordonia neofelifaecis NRRL B-59395(CCTCCAB-209144)、Arthrobactersimplex(TCCC 11037)、Mycobacteriumfortuitumsubsp.fortuitum(MTCC 929)或Mycobacterium neoaurum JC-12。
其中,Mycobacterium neoaurum ATCC 25795已经在文献Yao K,Xu L-Q,Wang F-Q,Wei D-Z(2014)Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC25795 to produce9α-hydroxy-4-androstene-3,17-dione through the catabolism ofsterols.Metab Eng 24(0):181-191doi:http://dx.doi.org/10.1016/j.ymben.2014.05.005中公开了。
Mycobacterium neoaurum ZJUVN(CGMCC 5477)已经在文献Zhang X-y,Peng Y,SuZ-r,Chen Q-h,Ruan H,He G-q(2013)Optimization of biotransformation fromphytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08.Journal of Zhejiang University SCIENCE B 14(2):132-143 doi:10.1631/jzus.B1200067中公开了。
Mycobacterium neoaurum NwIB-01(CCTCC M 209094)已经在文献Wei W,Fan S-Y,Wang F-Q,Wei D-Z(2014)Accumulation of androstadiene-dione by overexpressionof heterologous3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurumNwIB-01.World J Microbiol Biotechnol 30(7):1947-1954 doi:10.1007/s11274-014-1614-3中公开了。
Gordonia neofelifaecis NRRL B-59395(CCTCC AB-209144)已经在文献Liu Y,Chen G,Ge F,Li W,Zeng L,Cao W(2011)Efficient biotransformation of cholesterolto androsta-1,4-diene-3,17-dione by a newly isolated actinomycete Gordonianeofelifaecis.World J Microbiol Biotechnol 27(4):759-765 doi:10.1007/s11274-010-0513-5中公开了。
Arthrobacter simplex(TCCC 11037)已经在文献Wang M,Zhang LT,Shen YB,MaYH,Zheng Y,Luo JM(2009)Effects ofhydroxypropyl-β-cyclodextrin on steroids 1-en-dehydrogenation biotransformation by Arthrobacter simplex TCCC 11037.J MolCatal,B Enzym 59(1–3):58-63doi:http://dx.doi.org/10.1016/j.molcatb.2008.12.017中公开了。
Mycobacteriumfortuitum subsp.fortuitum(MTCC 929)已经在文献Gulla V,Banerjee T,Patil S(2010)Bioconversion of soysterols to androstenedione byMycobacterium fortuitum subsp.fortuitum NCIM 5239,a mutant derived from totalsterol degrader strain.J Chem Technol Biotechnol 85(8):1135-1141 doi:10.1002/jctb.2410中公开了。
Mycobacterium neoaurum JC-12已经在文献Shao ML,Zhang X,Rao ZM,Xu MJ,Yang TW,Li H,Xu ZH(2015)Enhanced production of androst-1,4-diene-3,17-dioneby Mycobacterium neoaurum JC-12 using three-stage fermentation strategy.PLoSONE 10(9):e0137658中公开了。
在本发明的一种实施方式中,所述过表达,具体是将甾酮C27-单加氧酶基因连接到质粒pMV261上得到重组质粒,然后将重组质粒转化到出发菌中。
本发明的第三个目的是提供一种通过过表达甾酮C27-单加氧酶基因提高ADD产量的方法,所述方法是利用本发明的所述的新金色分枝杆菌重组菌为生产菌株发酵生产ADD;所述重组菌过表达甾酮C27-单加氧酶基因;所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
在本发明的一种实施方式中,所述甾酮C27-单加氧酶基因的氨基酸序列如SEQNO.4、SEQ NO.5或者SEQ NO.6所示。
在本发明的一种实施方式中,所述发酵生产是以植物甾醇或/和胆固醇为底物。
在本发明的一种实施方式中,用于发酵的发酵培养基成分:胆固醇20g/L,葡萄糖20g/L,蛋白胨10g/L,牛肉膏6g/L,K2HPO43g/L,MgSO4·7H2O 0.5g/L,MnCl2·4H2O 5×10-4g/L,羟丙基-β-环糊精60g/L,pH 7.5。
在本发明的一种实施方式中,所述发酵发酵生产是在30℃,160rpm条件下发酵168h。
本发明的第四个目的是提供所述重组菌生产得到的ADD。
本发明的第五个目的是提供所述重组菌或者所述甾酮C27-单加氧酶基因在制备药物方面的应用。
在本发明的一种实施方式中,所述药物是指抗肿瘤、抗炎症、抗菌、抗病毒、抗激素或者抗过敏的药物。
在本发明的一种实施方式中,所述药物是指治疗性器官退化药物、妇科疾病药物、抗肥胖药物、预防冠心病的药物或者HIV整合酶的抑制剂等。
本发明的有益效果:
(1)本发明通过基因工程手段筛选到新金色分枝杆菌中甾酮C27-单加氧酶的3个同工酶基因,借助基因敲除和敲除基因回补的方法,鉴定了甾酮C27-单加氧酶(SMO)甾醇转化过程中的第二步酶。本发明证明了这三个同工酶在甾醇代谢过程中的功能,确定了其在甾醇转化过程中的关键作用,验证了其是分枝杆菌转化甾醇合成AD/ADD过程中的关键酶。
(2)本发明在确定关键酶的基础上,对其在甾醇转化合成ADD的过程中进行应用以期提高ADD的产量,发现SMO2对提高ADD产量效果最为明显。最终过量表达SMO2使得ADD的产量从5.2g/L提高到7.3g/L,比原始菌提高了40.4%。本发明为微生物发酵法提高ADD产量的工业化提供了有益的指导。
具体实施方式
主要试剂:植物甾醇(大豆甾醇≥95%)购自礼来生物技术(湖州)有限公司,ADD购自美国SIGMA公司。
ADD的HPLC分析:ADD在254nm紫外波长下均有特征吸收峰,所以采用HPLC法测定产物浓度。色谱条件:色谱柱:DimosoilC18(5μl,250mm×4.6mm),流动相:甲醇-水(V/V=70:30),检测器:UV Detector,检测波长:254nm,柱温:30℃,进样量:10μL,流速:1.0ml/min。
实施例1:SMO敲除菌株及相应回补菌株的构建
通过查询新金色分枝杆菌的全基因组信息,筛选到3个SMO同工酶。以实验室具有SMO酶活力的新金色分枝杆菌作为出发菌株,以其染色体为模板,利用PCR手段获得这3个酶的基因。通过基因敲除引物的设计,利用PCR手段获得敲除基因,连接分枝杆菌敲除质粒p2NIL,构建敲除质粒,经PCR验证构建成功后,转化新金色分枝杆菌中。以胆甾-4-烯-3-酮作为底物,检测敲除菌株对底物的降解情况。在敲除菌株的基础上,构建敲除基因回补菌株,通过设计引物,利用PCR手段扩增出完整的SMO基因,连接整合载体pMV306构建整合型质粒,经PCR验证构建成功后,转化相应的敲除菌株中,构建敲除基因回补菌株,相同条件下,以胆甾-4-烯-3-酮作为底物,检测回补菌株对底物的降解情况。检测结果表明基因敲除菌株对胆甾-4-烯-3-酮的降解能力受到阻碍,而敲除基因回补菌株在一定程度上减少了这种阻碍作用。本发明最终证明SMO这三个酶在甾醇降解过程中是关键酶。
重组菌株的具体构建方法如下:
(1)甾酮C27-单加氧酶(SMO)基因全序列的克隆
根据GENBANK网站公布的Mycobacterium neoaurum VKM Ac-1815D的全基因组序列(NC_023036)查找到3个SMO基因(Smo1,Smo2,Smo3),根据具体的基因序列设计相应的基因引物。以制备的新金色分枝杆菌染色体DNA为模板,通过PCR扩增出相应的基因全序列。
PCR反应体系:10×ExTaq Buffer 5μL,dNTP 4μL,模板DNA 1μL,上下游引物各0.5μL,ExTaq酶1μL,ddH2O补齐至总体积50μL。PCR反应条件:94℃5min,94℃30s,65℃45s,72℃90s,循环35次,72℃10min,12℃10min。
参照上海捷瑞公司胶回收试剂盒说明书回收PCR产物,胶回收产物按一定比例与pMD18-T vector过夜连接,转化E.coli JM109感受态细胞,使用氨苄青霉素抗性平板筛选重组菌,重组质粒经酶切释放出大小约为2.7kb和1.3kb的基因条带,表明重组质粒构建成功,重组质粒命名为pMD18-T-Smo1,pMD18-T-Smo2和pMD18-T-Smo3。
(2)新金色分枝杆菌基因敲除质粒的构建
设计特定引物通过PCR的方式从Smo1,Smo2和Smo3上游扩增出300bp的序列,从下游扩增出450bp的序列。将上下游扩增出来的基因序列混合作为模版,利用上游300bp序列的上游引物和450bp序列的下游引物扩增出750bp的序列。将扩增出来的序列和敲除质粒p2NIL经Kpn I和Hind III双酶切,胶回收纯化,T4DNA连接酶过夜连接两片段,过夜后将连接物热击转化E.coli JM109感受态细胞,使用卡那霉素抗性平板筛选阳性转化子。提取转化子质粒,重组质粒经酶切验证构建成功。用Pac I单酶切将质粒pGOAL19中的选择标记基因盒切下来插入到构建好的重组质粒的Pac I酶切位点中,构建敲除质粒p2N-ΔSmo1,p2N-ΔSmo2和p2N-ΔSmo3。经PCR验证,敲除质粒构建成功。
(3)新金色分枝杆菌敲除基因回补质粒的构建
质粒pMV306用于敲除基因的回补。用Xba I/Cla I双酶切,将重组质粒p261-Smo1,p261-Smo2和p261-Smo3中的相应的基因片段连同热激启动子hsp60一同酶切下来连接到相应的pMV306酶切位点上,构建敲除基因回补质粒p306-Smo1,p306-Smo2和p306-Smo3。
(4)重组质粒电转化法转化到新金色分枝杆菌
A将构建成功的重组质粒用电转化法转化新金色分枝杆菌中。转化方法采用改进的Gordhan and Parish法。
B重组菌株M.neoaurum阳性转化子的筛选;
挑取在具有相应抗生素压力平板上长出的菌落,摇瓶发酵,提取质粒进行酶切验证。
实施例2:SMO加强表达重组菌株的构建
质粒pMV261用于新金色分枝杆菌中基因的过量表达。用Sac I和Hind III双酶切pMD18-T-Smo1,用BamH I和EcoR I分别双酶切pMD18-T-Smo2和pMD18-T-Smo3,同时用相应的酶切位点酶切质粒pMV261,胶回收纯化相应的基因片段和质粒pMV261片段,T4DNA连接酶过夜连接两片段,过夜后将连接物热击转化E.coli JM109感受态细胞,使用卡那霉素抗性平板筛选阳性转化子。提取转化子质粒,重组质粒p261-Smo1,p261-Smo2和p261-Smo3经酶切验证构建成功,将构建成功的重组质粒,分别电转化到高产ADD的新金色分枝杆菌JC-12(Mycobacterium neoaurum JC-12已经在文献Shao ML,Zhang X,Rao ZM,Xu MJ,Yang TW,Li H,Xu ZH(2015)Enhanced production of androst-1,4-diene-3,17-dione byMycobacterium neoaurum JC-12using three-stage fermentation strategy.PLoSONE10(9):e0137658中公开)中,分别获得重组菌株JC-12S1,JC-12S2和JC-12S3。
实施例3:SMO加强表达重组菌株提高ADD的产量
将重组菌株JC-12S1,JC-12S2和JC-12S3在种子培养基上活化后,按照5%的接种量接入发酵培养基,以20g/L的胆固醇为底物,在30℃,160rpm条件下发酵168h,进行发酵转化实验。其中,种子培养基:葡萄糖10g/L,蛋白胨10g/l,牛肉膏6g/L,NaCl 10g/L,pH 7.5;发酵培养基:胆固醇20g/L,葡萄糖20g/L,蛋白胨10g/L,牛肉膏6g/L,K2HPO43g/L,MgSO4·7H2O0.5g/L,MnCl2·4H2O 5×10-4g/L,羟丙基-β-环糊精60g/L,pH 7.5。
检测ADD的产量变化。结果表明发酵转化168h,过量表达SMO2的重组菌JC-12S2的ADD的产量提高最明显,从出发菌株新金色分枝杆菌JC-12的5.2g/L提高到7.3g/L。此外,重组菌JC-12S1和JC-12S3的ADD的产量分别提高到6.5g/L和6.1g/L。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种ADD产量提高的新金色分枝杆菌重组菌,所述重组菌是过表达了甾酮C27-单加氧酶基因的新金色分枝杆菌;所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
2.根据权利要求1所述的新金色分枝杆菌重组菌,其特征在于,所述甾酮C27-单加氧酶基因Smo1、Smo2、Smo3的氨基酸序列分别如SEQ NO.4、SEQ NO.5、SEQ NO.6所示。
3.根据权利要求1所述的新金色分枝杆菌重组菌,其特征在于,所述过表达,具体是将甾酮C27-单加氧酶基因连接到质粒pMV261上得到重组质粒,然后将重组质粒转化到出发菌中。
4.一种通过过表达甾酮C27-单加氧酶基因提高ADD产量的方法,其特征在于,所述方法是将利用权利要求1~3任一所述的新金色分枝杆菌重组菌为生产菌株发酵生产ADD;所述新金色分枝杆菌重组菌过表达甾酮C27-单加氧酶基因;所述甾酮C27-单加氧酶基因是基因Smo1、Smo2、Smo3中的任意一个或者两个以上。
5.根据权利要求4所述的方法,其特征在于,所述甾酮C27-单加氧酶基因的氨基酸序列如SEQ NO.4、SEQ NO.5或者SEQ NO.6所示。
6.根据权利要求4所述的方法,其特征在于,所述发酵生产是以植物甾醇或/和胆固醇为底物进行发酵生产。
7.一种甾酮C27-单加氧酶基因,其特征在于,所述甾酮C27-单加氧酶基因的氨基酸序列如SEQ NO.4、SEQ NO.5或者SEQ NO.6所示。
8.权利要求1~3任一所述的新金色分枝杆菌重组菌或者权利要求7所述的甾酮C27-单加氧酶基因在制备药物方面的应用。
9.根据权利要求8所述的应用,其特征在于,所述药物是指抗肿瘤、抗炎症、抗菌、抗病毒、抗激素或者抗过敏的药物。
10.根据权利要求8所述的应用,其特征在于,所述药物是指治疗性器官退化药物、妇科疾病药物、抗肥胖药物、预防冠心病的药物或者HIV整合酶的抑制剂。
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CN111454871A (zh) * | 2020-03-03 | 2020-07-28 | 天津大学 | 一种高产雄烯二酮的重组分枝杆菌及构建方法及应用 |
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US20190153404A1 (en) | 2019-05-23 |
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