CN117448292A - 17β-羟基类固醇脱氢酶突变体及其在制备甾体激素中的应用 - Google Patents
17β-羟基类固醇脱氢酶突变体及其在制备甾体激素中的应用 Download PDFInfo
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- CN117448292A CN117448292A CN202311390497.8A CN202311390497A CN117448292A CN 117448292 A CN117448292 A CN 117448292A CN 202311390497 A CN202311390497 A CN 202311390497A CN 117448292 A CN117448292 A CN 117448292A
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- hydroxysteroid dehydrogenase
- beta
- dehydrogenase mutant
- hsdbn
- mutant
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Classifications
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- C12Y—ENZYMES
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- C12Y101/01051—3 (or 17)-Beta-hydroxysteroid dehydrogenase (1.1.1.51)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/19—Escherichia coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
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Abstract
本发明公开了一种17β‑羟基类固醇脱氢酶突变体及其在制备甾体激素宝丹酮中的应用。本发明将来源于Brettanomyces naardenensis的17β‑羟基类固醇脱氢酶(17β‑HSDbn)进行突变得到突变体17β‑HSDbnN222W、17β‑HSDbnG151Y、17β‑HSDbnF212I,分别于重组大肠杆菌中进行异源表达,其酶活较野生型17β‑HSDbn提高了1.7~2.4倍。本发明进一步将上述突变体应用于Mycobacterium sp.中进行异源表达,所构建的基因工程菌其宝丹酮产量均有较大提升,更适合工业应用,可降低宝丹酮的生产成本,进一步提高生产效率。
Description
技术领域
本发明属于酶工程领域,具体涉及17β-羟基类固醇脱氢酶突变体及其在制备甾体激素中的应用。
背景技术
甾体药物作为目前临床上用量仅次于抗生素的第二类药物,全球年产量已超过100余万吨,据2018年统计数据显示,全球甾体药物市场规模超过1200亿美元。常见的甾体药物中间体分为C19类与C22两类,其中以宝丹酮(BD)、雄甾-4-烯-3,17-二酮(AD)、雄甾-1,4-二烯-3,17-二酮(ADD)、9α-羟基雄甾烯-4-烯-3,17-二酮(9α-OH-AD)和睾酮(TS)为主的甾体药物中间体在医药领域市场的需求逐年上升。
宝丹酮(BD)是雄激素合成代谢类固醇和睾丸激素(TS)衍生物,可促进蛋白质合成,支持氮潴留并刺激肾脏促红细胞生成素释放。BD通常是由雄甾-4-烯-3,17-二酮(AD)或雄甾-1,4-二烯-3,17-二酮(ADD)通过化学合成制得的。然而,存在合成路线繁琐、需要添加强酸或有毒试剂、副产物多、产率低以及成本高等缺陷。与化学合成法相比,微生物转化法不仅具有合成步骤少、生产周期短、收率高、副反应少、反应条件温和、环境友好等优点,而且具有高度的立体选择性和区域选择性。目前,微生物转化法已成为甾体药物及其中间体合成路线中不可缺少的关键技术。
来源于Brettanomyces naardenensis的17β-羟基类固醇脱氢酶可以利用雄甾-1,4-二烯-3,17-二酮作为底物,催化甾体中C-17的酮基与羟基之间的相互转化,经还原反应产生17β-羟基-1,4-雄甾二烯-3-酮,即宝丹酮。但该野生型酶在多数微生物中酶活力不高,限制了该酶的商业化应用。因此急需寻找新的合适的具有高催化活性的17β-羟基类固醇脱氢酶,促进其在生物医药领域转化甾醇的应用。
发明内容
针对现有技术中野生型17β-羟基类固醇脱氢酶酶活力低的问题,本发明提供了具有高酶活力的17β-羟基类固醇脱氢酶突变体,及其在制备甾体激素宝丹酮中的应用。本发明对所述的来源于Brettanomyces naardenensis(Gen Bank ID:VEU22018.1)的17β-羟基类固醇脱氢酶进行定点突变,将获得的突变体编码基因通过表达载体于大肠杆菌中进行了异源表达。此外,本发明还将所述的突变体编码基因与过表达启动子共同连接至重组载体中,再于新金色分枝杆菌中进行异源表达,以此获得一株高产宝丹酮的基因工程菌。所述的以新金色分枝杆菌为宿主的基因工程菌可利用植物甾醇作为底物,以上述的17β-羟基类固醇脱氢酶突变体为催化剂制备宝丹酮,更适合工业应用,降低了宝丹酮的生产成本,提高了生产效率。
本发明提供了17β-羟基类固醇脱氢酶突变体,其特征在于,由氨基酸序列如SEQID NO.1所示的17β-羟基类固醇脱氢酶经下列之一的突变所得:
(1)第222位天冬酰胺突变为色氨酸;
(2)第151位甘氨酸突变为酪氨酸;
(3)第212位苯丙氨酸突变为异亮氨酸。
作为优选,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.2所示。所述的17β-羟基类固醇脱氢酶突变体是由氨基酸序列如SEQ ID NO.1所示的17β-羟基类固醇脱氢酶的第222位天冬酰胺突变为色氨酸所得,命名为17βHSDbnN222W。
作为优选,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.3所示。所述的17β-羟基类固醇脱氢酶突变体是由氨基酸序列如SEQ ID NO.1所示的17β-羟基类固醇脱氢酶的第151位甘氨酸突变为酪氨酸所得,命名为17βHSDbnG151Y。
作为优选,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.4所示。所述的17β-羟基类固醇脱氢酶突变体是由氨基酸序列如SEQ ID NO.1所示的17β-羟基类固醇脱氢酶的第212位苯丙氨酸突变为异亮氨酸所得,命名为17βHSDbnF212I。
本发明还提供了编码上述的17β-羟基类固醇脱氢酶突变体的基因。
作为优选,所述17β-羟基类固醇脱氢酶突变体17βHSDbnN222W编码基因的核苷酸序列如SEQ ID NO.5所示。
作为优选,所述17β-羟基类固醇脱氢酶突变体17βHSDbnG151Y编码基因的核苷酸序列如SEQ ID NO.6所示。
作为优选,所述17β-羟基类固醇脱氢酶突变体17βHSDbnF212I编码基因的核苷酸序列如SEQ ID NO.7所示。
本发明还提供了含有17β-羟基类固醇脱氢酶突变体编码基因的重组载体。所述重组载体指可在宿主细胞中表达的控制序列可操作地连接的多核苷酸,在本发明中优选载体pET28a(+)或pMV306作为重组载体的骨架。
本发明还提供了含有17β-羟基类固醇脱氢酶突变体编码基因的基因工程菌。所述基因工程菌的宿主菌优为大肠杆菌或快速生长型分枝杆菌。
作为优选,所述快速生长型分枝杆菌选自包括耻垢分枝杆菌、偶发分枝杆菌、微黄分枝杆菌、新金分枝杆菌中的至少一种。进一步优选为新金色分枝杆菌Mycobacteriumsp.NRRL B-3805。新金色分枝杆菌能够降解植物甾醇生产多种甾体激素药物及中间体,同时具有较好的代谢可塑性,是一种理想的甾体激素类药物的生物合成平台。微生物转化甾醇分解代谢途径一般包括:首先通过胆固醇氧化酶(ChOx)或3β-羟基类固醇氧化酶(3β-HSD)将胆固醇转化为4-胆甾烯-3-酮;侧链经催化形成末端羧基;C-27末端酰化后,胆固醇侧链被末端CoA硫代酯化激活,然后C-27的羧酰基CoA进入β-氧化循环途径降解甾醇的侧链,获得雄甾-4-烯-3,17-二酮(AD),并进一步得到雄甾-1,4-二烯-3,17-二酮(ADD)。
基于上述的微生物转化甾醇分解代谢途径,本发明还提供了一种基因工程菌的构建方法,所述方法包括如下步骤:
(1)以新金色分枝杆菌Mycobacterium sp.NRRL B-3805为底盘菌,连续敲除基因kshA1、MnOpccR、SalA,得到菌株Mn B-3805△kMS;
(2)将权利要求5所述的17β-羟基类固醇脱氢酶突变体编码基因与过表达启动子以及载体连接,得到重组载体;
(3)将步骤(2)中的重组载体转化到步骤(1)所述的菌株Mn B-3805△kMS中,得到所述的基因工程菌。
本发明首先对新金色分枝杆菌Mycobacterium sp.NRRLB-3805的kshA1、MnOpccR、SalA基因进行敲除,所获得的菌株Mn B-3805△kMS可有效控制22-羟基-23,24-双降胆甾-4-烯-3-酮(4-HBC)、22-羟基-23,24-双降胆甾-1,4-二烯-3-酮(1,4-HBC)、9-羟基-4-雄烯-3,17-二酮(9-OH-AD)等副产物的合成,进一步提升底物植物甾醇的利用率。其次,本发明将所述的17β-羟基类固醇脱氢酶突变体编码基因与过表达启动子进行连接,并通过重组载体的形式导入新金色分枝杆菌,以增强突变体在新金色分枝杆菌中的异源表达。
作为优选,所述过表达启动子为L2启动子。
作为优选,所述载体为pMV306载体。
本发明还提供了所述的17β-羟基类固醇脱氢酶突变体、及其重组载体、及其基因工程菌在制备甾体激素中的应用。
作为优选,所述应用包括:将按上述构建方法构建得到的基因工程菌湿菌体于静息细胞反应体系中在25~40℃、100~300rpm条件下进行生物转化,反应时间为48~120h,制得所述的宝丹酮。
具体地,所述静息细胞反应体系包括:40~50g/L基因工程菌湿菌体,10~30g/L植物甾醇培养液,10~40g/L的葡萄糖。其中,终浓度为100g/L的植物甾醇培养液母液包括:10g植物甾醇,30g羟丙基-β-环糊精,2g吐温80,定容至100ml。
本发明的有益效果:本发明提供的17β-羟基类固醇脱氢酶突变体17β-HSDbnN222W、17β-HSDbnG151Y、17β-HSDbnF212I,于重组大肠杆菌中进行异源表达,17β-HSDbn突变体酶活较野生型17β-HSDbn分别提高2.34倍、1.79倍、1.71倍;进一步应用于新金色分枝杆菌Mycobacterium sp.中进行异源表达,构建得到的基因工程菌Mn B-3805△kMS pMV307-17β-HSDbnN222W、Mn B-3805△kMS pMV307-17β-HSDbnG151Y、Mn B-3805△kMS pMV307-17β-HSDbnF212I经过120h转化,分别产生3.54g/L、3.84g/L、3.05g/L宝丹酮,较过表达野生型17β-HSDbn产量分别提升45.08%、57.37%、25.0%,更适合工业应用,可降低宝丹酮的生产成本,进一步提高生产效率。
附图说明
图1为本发明实施例1中17β-羟基类固醇脱氢酶野生型及突变体的核酸凝胶电泳图;其中,条带1:250bp marker,条带2:pET-28a(+)-17β-HSDbn全质粒扩增产物,条带3:pET-28a(+)-17β-HSDbnN222W全质粒扩增产物,条带4:pET-28a(+)-17β-HSDbnG151Y全质粒扩增产物,条带5:pET-28a(+)-17β-HSDbnF212I全质粒扩增产物。
图2为本发明实施例2中17β-羟基类固醇脱氢酶突变体17β-HSDbnN222W、17β-HSDbnG151Y、17β-HSDbnF212I的相对酶活。
图3为本发明实施例4中新金色分枝杆菌异源过表达17β-羟基类固醇脱氢酶突变体的宝丹酮产量图。
具体实施方式
以下通过特定的具体实施例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。需说明的是,在不冲突的情况下,以下实施例及实施例中的特征可以相互组合。本发明实施例中如无特殊说明所用方法均为常规方法,所用试剂均可从商业途径获得。
以下实施例中所使用的培养基:
LB液体培养基:蛋白胨10.0g/L,酵母提取物5.0g/L,Na Cl 10.0g/L。
M3培养基:磷酸二氢钾0.5g/L、磷酸氢二钾0.5g/L、磷酸氢二铵1.5g/L、酵母提取物5g/L、甘油5g/L、七水合硫酸亚铁0.005g/L、七水合硫酸锌0.002g/L、七水合硫酸镁0.2g/L及吐温80 0.5g/L。
以下实施例中所使用的引物:
表1构建重组菌表达质粒及验证引物
实施例1:重组菌株E.coli BL21(DE3)/pET-28a(+)-17βHSDbn及突变株的构建载体质粒的构建:将Brettanomyces naardenensis(Gen Bank ID:VEU22018.1)来源的17β-HSD序列(SEQ ID NO.1)根据分枝杆菌的密码子偏好进行密码子优化后,交于北京擎科生物科技有限公司进行合成。利用限制性内切酶(BamHI和XhoI)对表达质粒pET-28-28a(+)进行酶切;同时以合成的17β-HSD基因为模板,扩增带有限制性内切酶位点(BamHI和XhoI)的目的基因片段,所需引物PET-BN-F、PET-BN-R(如表1所示);目的基因片段纯化后,将目的基因片段与酶切后的载体连接,将连接产物转化到E.coli BL21(DE3)感受态细胞中,涂布于卡那霉素抗性LB平板上,37℃置培养15~18h。挑取单菌落,接种于10mL LB试管培养,提取质粒,送测至北京擎科生物科技有限公司,测序结果正确即可用于后续实验,得到载体质粒pET-28a(+)-17βHSDbn。
突变菌株的构建:以pET-28a(+)-17βHSDbn质粒为模板,分别以N222W-F/N222W-F,G151Y-F/G151Y-R、F212I-F/F212I-R为引物(如表1所示),反向PCR扩增质粒进行定点突变。跑胶验证条带大小(参见图1)。验证正确,添加DpnI 1μL,消化模板2h,80℃灭活15min。将PCR产物分别转化到E.coli BL21(DE3)感受态细胞中,涂布于卡那霉素抗性LB平板上,37℃置培养15~18h。挑取单菌落,接10mL LB试管培养,提取质粒,送测至北京擎科生物科技有限公司,测序结果正确即可用于后续实验,分别得到突变菌株E.coli BL21(DE3)/pET-28a(+)-17βHSDbnN222W、E.coli BL21(DE3)/pET-28a(+)-17βHSDbnG151Y、E.coli BL21(DE3)/pET-28a(+)-17βHSDbnF212I
实施例2:17β-羟基类固醇脱氢酶单点突变对酶活的影响
将实施例1中构建的重组菌株E.coli BL21(DE3)/pET-28a(+)-17βHSDbn、E.coliBL21(DE3)/pET-28a(+)-17βHSDbnN222W、E.coli BL21(DE3)/pET-28a(+)-17βHSDbnG151Y、E.coli BL21(DE3)/pET-28a(+)-17βHSDbnF212I分别挑取单菌落接种于10mL LB培养基中,37℃培养12h得到种子液。分别按2%接种量将种子液接种于100mL LB培养基中,37℃培养2h,待OD600达到0.6~0.8时,添加0.1mM IPTG,28℃培养12~16h。离心收集菌体,用50mM的PBS缓冲液洗涤两次。PBS缓冲液重新悬浮至细胞湿重为100g/L,加入0.5g/L底物(所述底物包括质量比为1:3的ADD与羟丙基-β-环糊精),2.5g/L的葡萄糖,2mM NADPH,在37℃下反应5min,用盐酸终止反应,加入3倍乙酸乙酯萃取,离心5min,取上清挥干,加甲醇复溶,经0.22μm滤膜过滤除杂,吸取200μL加入液相瓶的内衬管中,进行高效液相检测。对照组采用同体积的PBS缓冲液(50mM,pH 7.5)代替菌液。
相对酶活=突变体产量/出发菌株产量
结果如图2所示。突变体17βHSDbnN222W酶活提升至出发菌株2.34倍,突变体17βHSDbnG151Y酶活提升至发菌株1.79倍,突变体17βHSDbnF212I酶活提升至发菌株1.71倍。
实施例3:过表达17β-羟基类固醇脱氢酶及突变体基因工程菌的构建
过表达17β-羟基类固醇脱氢酶质粒的构建:利用限制性内切酶(XbaI和KspAI)对整合型质粒pMV306进行酶切,同时用引物L2-F/L2-R扩增L2启动子,307BN-F/307BN-R扩增17β-HSDbn基因片段。琼脂糖凝胶电泳检测PCR产物条带大小后,利用纯化试剂盒进行纯化备用。将目的片段L2启动子片段、17β-HSDbn基因片段与载体连接,将连接产物转化到E.coli DH5α感受态细胞中,涂布于卡那霉素抗性LB平板上,37℃置培养15~18h。从平板上挑取单菌落,用设计的验证引物:307YZ-F、307YZ-R(如表1所示)进行菌落PCR验证,用琼脂糖凝胶电泳检测PCR产物条带,将条带正确的PCR产物送测至北京擎科生物科技有限公司,测序结果正确即可接单菌落至试管,提取质粒用于后续实验,构建得到载体质粒pMV307-17βHSDbn。在此将连接有L2启动子的pMV306载体简称为pMV307载体。
突变质粒的构建:以pMV307-17βHSDbn质粒为模板,分别用N222W-F/N222W-F,G151Y-F/G151Y-R、F212I-F/F212I-R为引物(如表1所示),反向PCR扩增质粒进行定点突变。跑胶验证条带大小。验证正确,添加DpnI 1μL,消化模板2h,80℃灭活15min。将PCR产物转化到E.coli DH5α感受态细胞中,涂布于卡那霉素抗性LB平板上,37℃置培养15~18h。挑取单菌落,接种于10mL LB试管培养,提取质粒,送测至北京擎科生物科技有限公司,测序结果正确即可接单菌落至试管,提取质粒用于后续实验,得到突变质粒pMV307-17β-HSDbnN222W、pMV307-17β-HSDbnG151Y、pMV307-17β-HSDbnF212I。
新金色分枝杆菌的电转化及验证:本实施例所用宿主菌为Mycobacteriumsp.NRRL B-3805,通过宝赛生物公司购买得到,并连续敲除kshA1、MnOpccR、SalA三个基因,从而获得基因敲除菌株Mn B-3805△kMS,所用敲除方法及敲除基因序列均参考CN115747238A。首先制备Mn B-3805△kMS感受态细胞:挑取单菌落于10mL LB试管中,37℃振荡培养36h;将试管中的培养液接种于50mL LB摇瓶中,接种量2%,30℃摇床培养12-14h,待OD600为1.2-1.8时将摇瓶置于冰上静置10min,于4000rpm,4℃条件下离心10min,去上清;用蒸馏水重悬,于4000rpm,4℃条件下离心10min,去上清,洗涤两次;用2mL 15%甘油水溶液重悬,分装到EP管中,每管100μL,于-80℃保存备用。
分别将上述构建完成的质粒pMV307-17β-HSDbn、pMV307-17β-HSDbnN222W、pMV307-17β-HSDbnG151Y、pMV307-17β-HSDbnF212I加入Mn B-3805△kMS感受态细胞,置于冰上静置10min,使用电脉冲仪电击两次(电击条件:设定电压2.5kV,电击杯孔径选择2mm)。加入700μL预冷过后的LB液体培养基,垂悬菌体后,转移至无菌EP管;37℃、180rpm振荡温育4h,6000rpm离心3min,弃去上清,100μL重悬,涂布于卡那霉素抗性LB平板上,30℃倒置培养3~5d。挑取单菌落进行菌落PCR,选择条带长度正确的进行送测。将测序正确的单菌落接种于10mL LB试管中,37℃振荡培养36h后保藏备用。构建得到基因工程菌Mn B-3805△kMSpMV307-17βHSDbnN222W、Mn B-3805△kMS pMV307-17βHSDbnG151Y、Mn B-3805△kMS pMV307-17βHSDbnF212I以及对照菌株Mn B-3805△kMSpMV307-17βHSDbn。
实施例4:新金色分枝杆菌异源过表达17β-羟基类固醇脱氢酶及突变体转化性能检测
将实施例3中构建完成的基因工程菌以及对照菌株分别在含卡那霉素抗性的LB固体平板上划线活化,30℃培养2~3d;挑取单菌落于5mL LB试管中,30℃振荡培养36h;取4%转接到100mLM3培养基中,30℃振荡培养48h。用高速冷冻离心机收菌(4℃,6000rpm,离心10min),去除上清培养基,再用50mM pH 7.5的PBS缓冲液重悬清洗2次,最后再用适量的PBS缓冲液重悬湿菌体。
构建静息细胞反应体系(50ml):45g/L基因工程菌湿菌体,10g/L植物甾醇培养液(100g/L植物甾醇培养液母液:10g植物甾醇,30g羟丙基-β-环糊精,2g吐温80,定容至100ml),30g/L的葡萄糖。30℃,180rpm振荡培养,反应144h。
样品处理方法:500μL样品加入1.5mL乙酸乙酯萃取,振荡混匀,离心5min,取上层有机相200μL于EP管中挥发,加入800μL甲醇复溶,用0.22μm有机膜过滤,取200μL与液相瓶内衬管中待测。
高效液相色谱法(HPLC):选用Agilent1260高效液相色谱仪,紫外检测器,色谱柱为C18柱(5um,250mmx4.6 mm)型色谱柱,进样量10uL,检测波长为254nm,柱温为30℃。流动相:甲醇:水(75:25,v/v),流速1mL/min。
结果如图3所示,对照菌株Mn B-3805△kMSpMV307-17β-HSDbn转化120小时达到最高产量2.44g/L。构建得到的基因工程菌Mn B-3805△kMSpMV307-17βHSDbnN222W、Mn B-3805△kMSpMV307-17βHSDbnG151Y、Mn B-3805△kMSpMV307-17βHSDbnF212I经过120h转化,分别产生3.54g/L、3.84g/L、3.05g/L宝丹酮,较过表达野生型17β-HSDbn产量分别提升45.08%、57.37%、25.0%。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明的保护范围内。
Claims (10)
1.17β-羟基类固醇脱氢酶突变体,其特征在于,由氨基酸序列如SEQ ID NO.1所示的17β-羟基类固醇脱氢酶经下列之一的突变所得:
(1)第222位天冬酰胺突变为色氨酸;
(2)第151位甘氨酸突变为酪氨酸;
(3)第212位苯丙氨酸突变为异亮氨酸。
2.如权利要求1所述的17β-羟基类固醇脱氢酶突变体,其特征在于,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的17β-羟基类固醇脱氢酶突变体,其特征在于,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.3所示。
4.如权利要求1所述的17β-羟基类固醇脱氢酶突变体,其特征在于,所述17β-羟基类固醇脱氢酶突变体的氨基酸序列如SEQ ID NO.4所示。
5.编码权利要求1~4任一所述的17β-羟基类固醇脱氢酶突变体的基因。
6.含有权利要求5所述编码基因的重组载体。
7.含有权利要求5所述编码基因的基因工程菌。
8.如权利要求7所述的基因工程菌,其特征在于,所述基因工程菌的宿主菌为大肠杆菌或快速生长型分枝杆菌。
9.一种如权利要求7所述的基因工程菌的构建方法,其特征在于,所述方法包括如下步骤:
(1)以新金色分枝杆菌Mycobacterium sp.NRRL B-3805为底盘菌,连续敲除基因kshA1、MnOpccR、SalA,得到菌株Mn B-3805△kMS;
(2)将权利要求5所述的17β-羟基类固醇脱氢酶突变体编码基因与过表达启动子以及载体连接,得到重组载体;
(3)将步骤(2)中的重组载体转化到步骤(1)所述的菌株Mn B-3805△kMS中,得到所述的基因工程菌。
10.如权利要求1~4任一所述的17β-羟基类固醇脱氢酶突变体、或如权利要求6所述的重组载体、或如权利要求7~8任一所述的基因工程菌、或如权利要求9所述的构建方法构建得到的基因工程菌在制备甾体激素中的应用。
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