CN113667650A - 一种11α-羟化酶突变体及其应用 - Google Patents
一种11α-羟化酶突变体及其应用 Download PDFInfo
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Abstract
本发明专利提供了赭曲霉甾体C11α‑羟化酶突变体I303L,DNA序列如SEQ ID NO:2所示。同C11α‑羟化酶CYP68J5相比,突变体I303L转化甾体底物孕酮显示更高的特异性,无明显副产物生成。本发明专利还提供了基于突变体I303L异源过表达的表达载体、毕赤酵母重组菌及其甾体转化工艺,为研究开发高效黄体酮C11α‑羟化生产工艺提供了宝贵材料(基因和菌种)和基础数据支撑。
Description
技术领域:
本发明属于基因工程和酶工程技术领域,具体涉及一种C11α-羟化酶突变体及其应用。
背景技术:
甾体激素类药物在临床上具有广泛的应用。该类药物主要用于治疗炎症和过敏等症状,同时也用于治疗癌症,人体激素分泌障碍以及生育控制。甾体药物的生理和药理活性取决于甾体骨架特定位点引入官能基团。对甾体结构的改造通过化学合成法或微生物转化法,或结合两种方法。全化学合成法存在着合成过程复杂、副产物不容易分离等缺点,故化学合成法难以完成的关键合成步骤,工业上利用微生物转化反应在甾体基本骨架上引入各种不同类型的官能团,尤其是羟基。重要的微生物甾体羟化反应包括在甾体骨架的C9-、C11-、C15-位上引入羟基。
11α-羟基黄体酮是合成甾体孕激素和糖皮质激素的重要中间体。由于赭曲霉(Aspergillus ochraceus)具有较强的C11α-羟基化活性和特异性,甾体工业常用于甾体的C11α-羟化,一步在孕酮的C11位引入羟基合成11α-羟基黄体酮。但赭曲霉转化甾体底物黄体酮存在明显副产物,不仅浪费了原料,同时增加了后续的分离纯化的成本。我们克隆鉴定了赭曲霉TCCC41060中的C11α-羟化酶基因 CYP68J5。
为了改善赭曲霉C11α-羟化酶CYP68J5基因对底物黄体酮的羟化特异性,利用定点突变技术对其进行分子水平上的改造,筛选获得了一个对孕酮具有高羟化特异性C11α-羟化酶突变体I303L,并构建相应的异源表达载体以及过表达重组毕赤酵母菌。发酵结果显示过表达突变体I303L的毕赤酵母重组菌对底物孕酮具有高C11α-羟化特异性和较高的羟化活性,为研究开发绿色高效孕酮C11α-羟化工艺奠定了坚实的基础。
发明内容:
针对现有甾体孕酮羟化技术的不足,本发明的第一个目标是利用分子操作技术提供一种C11α-羟化酶CYP68J5的突变体I303L。本发明的第二个目标是利用表达载体pPIC3.5K实现异源高效表达突变体I303L,构建了一个I303L重组表达质粒以及突变体I303L过表达重组毕赤酵母菌,建立了基于重组毕赤酵母菌的无副产物生成的黄体酮高效C11α-羟化反应工艺。
本发明所述的CYP68J5基因来源于赭曲霉。
本发明未定点突变的氨基酸序列如SEQ ID NO:1所示。
本发明经定点突变的编码基因DNA序列如SEQ ID NO:2所示。
利用本发明所述的基因片段CYP68J5m303所构建的表达载体、重组载体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CYP68J5m303编码的甾体化合物羟化酶可用于但不限于毕赤酵母等宿主细胞内表达。
有益效果:
高效C11α-羟化酶突变体I303L的获得以及重组毕赤酵母黄体酮C11α-羟化工程菌的构建为研究开发高效黄体酮C11α-羟化生产工艺提供了宝贵材料(基因和菌种)和基础数据支撑。
说明书附图:
图1黄体酮的C11α-羟基化反应
图2琼脂糖凝胶电泳验证条带
其中:M为DNA Marker,1为基因片段;
图3重组毕赤酵母质粒构建图
图4酶切重组毕赤酵母质粒
其中:M为DNA Marker,1-7:分别是挑选的单菌落大肠杆菌提质粒双酶切验证;
图5浓度为4.0的G418YPD平板上筛选高拷贝转化子
图6TLC硅胶板分析11α-羟基化产物
1:底物黄体酮;2:野生GS115毕赤酵母;
3:野生GS115-AOH毕赤酵母;4:经突变的GS115-CYP68J5m303毕赤酵母;
图7不同发酵时间对其底物黄体酮的转化效果
具体实施方式:
下面通过具体的实施方案叙述本发明方法。除非特别说明,本发明所用的技术手段均为本领域技术人员所公知的方法。另外,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:突变体I303L的构建
1.定点突变技术扩增目的片段:
以质粒pYES2-AOH作为模板,利用定点突变技术进行聚合酶链式反应,引物由天津金唯智生物科技有限公司进行合成。
AOH-CYP68J5m303-F:CTCTCCCTAGTTGCTATCCACACCACG
AOH-CYP68J5m303-R:AGCAACTAGGGAGAGCGTGACCTGCTT
(1)PCR反应体系:
(2)PCR反应条件:
2.Dpn I酶消化后纯化过程:
Dpn I酶具有识别甲基化的gatc碱基,模版质粒一般是从大肠杆菌中提取的带有天然甲基化修饰的质粒,故在DpnI酶作用下,定点识别并迅速消化掉模板质粒,而PCR扩增新突变质粒没有甲基化修饰,不会被DpnI酶切掉,能够有效避免未突变的模板存在,步骤如下;
1μL DpnI酶
5μL DpnI酶Buffer
44μL 纯化PCR片段
放置到37℃恒温培养箱内酶切3h。后进行回收PCR扩增片段需要使用小量DNA纯化试剂盒进行操作,方法如下:
(1)用移液枪将PCR产物加入含有250μL含有结合液的EP管中,轻柔混匀,再将混合产物溶液用移液枪转移至DNA吸附柱中,使DNA分子沉淀到吸附柱膜上2分钟,放入离心机13500r/min高速离心1min,扔弃吸附柱下层废液。
(2)移液枪吸取600μL的85%乙醇漂洗液进行漂洗,静置2min,放入离心机13500r/min高速离心1min,扔弃吸附柱下层废液。
(3)加入700μL的75%乙醇漂洗液进行第二次漂洗,使DNA分子沉淀到吸附柱膜上2分钟,放入离心机13500r/min高速离心1min,扔弃吸附柱下层废液。
(4)再次放入离心机13500r/min高速离心2min,取上层吸附柱开盖放置到无菌的EP管中静置风干10min。
(5)加入超纯水30-45μL,使DNA分子与超纯水有效结合到吸附柱膜上2分钟,放入离心机13500r/min高速离心2min,经琼脂糖凝胶电泳验证条带大小,见图2所示。
3.化转到酿酒酵母及其发酵:
(1)从甘油管中活化酿酒酵母菌株到YPD平板中,三区划线,分离双倍体酵母中单菌落的菌株,30℃培养箱培养2-3天,之后挑取一个大小均匀的单菌落转接在50ml的YPD液体培养基试管扩大培养,摇床温度为30℃、转速设定在 200r/min繁殖培养12h左右;
(2)从试管中吸取混匀的1ml的菌液再转接到50mLYPD液体培养基中二次培养,期间规定初始保持OD值为0.4左右防止细胞过老不新鲜,温度设定30℃,转速设定200r/min的摇床继续培养2-4h,期间不定时测量OD防止长过影响化转效率,OD值涨到1.3-1.5左右即可;
(3)将无菌的50mL离心管进行降温零度处理5min,保持细胞活力,随即将菌液倒入,设置高速离心机转速4500r/min高速离心8min,丢弃YPD液体培养液;
(4)将10xTE溶液稀释到1xTE溶液,进行降温零度处理5min,将其溶液进行重悬挂壁菌体,设置高速离心机转速4500r/min高速离心8min,丢弃上层1xTE 液体溶液;
(5)用配置好的1×LiAc/0.5xTE试剂进行降温零度处理5min,随即用溶液吹吸均匀菌体沉淀,EP管中降温零度处理5min将菌体分别装入其内;
(6)将100μL酿酒酵母感受态细胞25℃静置4min;
(7)将10μL鲑鱼精DNA加入到酿酒酵母感受态细胞中是为了减少被酿酒酵母细胞进行降解,故再将6μL重组质粒加入其中,吹吸混匀;
(8)移液枪吸取700μL提前配置好的酵母缓冲混合溶液垂直管口加入到g步骤中,吹吸混匀;
(9)将混合液放置到30℃恒温培养箱,培养30min,此静置过程去融化SC 培养基;
(10)加入88μLDMSO,吹吸混匀后放到42℃恒温水浴锅进行静置7min;
(11)将热击后的EP管进行13000r/min高速离心10s,丢弃上层液体溶液,约留下100μL涂布在带有Amp抗性的SC平板中培养2-3天,直至长出转化子。
(12)从SC平板挑取转化子密集划线到含有Amp抗性的YPD平板中培养2-3 天;
(13)YPD平板挑取适量新鲜的菌体到3ml的YPD液体培养基中发酵24h;
(14)秤取0.025g甾体底物黄体酮溶于甲醇试剂中,放在75℃恒温水浴锅静置融化5min,期间定期摇晃混匀液体;
(15)分别投入24μL黄体酮-甲醇混合液,再加入120μL半乳糖(浓度2%) 进行72h、30℃、200r/min摇床发酵培养;
(16)混匀发酵液吸取800μL菌体发酵液,补加300μL乙酸乙酯,进行剧烈混匀摇晃,从而进行TLC定性分析。通过薄层色谱硅胶板进行初步鉴定目的基因的表达效果,重组pYES2-CYP68J5m303酿酒酵母经半乳糖诱导发酵,其无明显副产物。
5.重组pYES2-CYP68J5m303酿酒酵母菌株测序:根据TLC结果,将重组酿酒酵母菌株进行测序。测序后发现,由原来的异亮氨酸(ATA)突变成亮氨酸 (TTA),测序结果表明经过定点突变技术实现定点突变,且其余碱基并未发生突变,符合实验预期想法。
实施例2:构建pPIC 3.5K-CYP68J5m303重组质粒
1.目的片段CYP68J5m303的PCR扩增
根据开放阅读框设计带有EcoRI、SnaBI的酶切位点的上下游引物,将提好的质粒pYES2-CYP68J5m303作为模板,对其进行PCR扩增,引物由天津金唯智生物科技有限公司进行合成。
AOH3.5K-F:GGtacgtaATGCCCTTCTTCACTGGG
AOH3.5K-R:CGgaattcCTACACAGTTAAACTCGCC
(1)PCR反应体系:
(2)PCR反应条件:
设定预变性温度95℃反应5min,变性温度94℃反应45s,再设置53℃的退火温度反应45s,72℃的延伸温度反应2min,完成三个循环后重新进行变性温度为95℃反应45s,60℃的退火温度反应45s,72℃的延伸温度反应2min,一共经历27个循环后,72℃温度反应10min结束,琼脂糖凝胶电泳检测方法检测带有粘性末端的目的片段条带大小。
2.PCR产物的纯化和酶切
经DpnI酶消化模板,利用小量DNA纯化试剂盒进行回收PCR扩增片段,后再经SnaBI酶、EcoRI分别酶切纯化目的片段。
3.连接后化转大肠感受态
带有相同的SnaBI、EcoRI酶酶切的PCR纯化片段和pPIC3.5K片段进行体外连接。
放置到16℃培养箱过夜连接,随后将混合产物化转到大肠感受态中,涂布于含Ampr(100μg/mL)的LB的固体平板中,37℃培养箱静置培养12h,得到转化子。
4.验证转化子
平板长出单菌落后挑七个大小均匀的单菌落密集划线,提质粒后用EcoRI 酶和XhoI酶双酶切得到6.0kb和4.5kb的双条带(见图4),故得到转化子。
实施例3:构建毕赤酵母特异性提高的羟化酶表达重组菌
1.重组质粒DNA的线性化
使用线性化载体可以更加有利于获得阳性重组毕赤酵母 pPIC3.5K-CYP68J5m303转化子,故使用限制性内切酶NcoI酶切 pPIC3.5K-CYP68J5m303重组质粒,50μL酶切体系如下所示:
放置到37℃培养箱消化3h,再经过小量DNA纯化试剂盒纯化片段,消除 Nco I酶Buffer等离子存在的干扰。
2.线性化质粒电转至野生毕赤酵母
(1)将实验室储存的毕赤酵母GS115菌种划三区活化,30℃培养箱培养 48h,挑取一个大小适中的单菌落接种于50ml的YPD液体培养基中放大繁殖培养 12h。
(2)从YPD液体培养基摇匀后吸取500-800μL转接到50ml的YPD液体培养基培养7-8h,直至OD600=1.35。
(3)将50mL的菌液混合均匀分别平分到两个无菌的离心管中,经高速离心机适当转速短暂离心菌体。
(4)离心后,倒掉YPD液体培养基,将无菌的超纯水各加入12mL,经高速离心机适当转速短暂离心菌体。
(5)第二次水洗,每只各加入6mL无菌的超纯水,吹吸均匀后两管合一管,经高速离心机适当转速短暂离心菌体。
(6)用12ml的1mol/L山梨醇吹吸均匀,经高速离心机适当转速短暂离心菌体。
(7)离心后加入200μL1mol/L山梨醇吹吸混匀,分装80μL在降温零度处理5min的EP管中,分别加入经线性化处理的目的片段20μL,移液枪轻微混匀后置于零度冰盒冰浴10分钟左右。
(8)将其转至到电转杯中,降温零度处理10min,设定合适的电转参数。
(9)随后加入900μL的山梨醇溶液进行维持细胞通透性,移液枪吹吸混匀液体后转至无菌的离心管中,经高速离心机适当转速短暂离心菌体。
(10)将上清部分倒掉,约留100μL的菌体涂布在MD培养基上,30℃培养箱培养2到3天直至长出白色单菌落。
3.阳性转化子的鉴定及高拷贝转化子的筛选
(1)挑取MD培养基上的单菌落,依次挑在不同浓度的G418 YPD平板上 (浓度大小分别是0.5、2.0、4.0mg/ml),30℃培养箱培养2到3天,直至长出匀称的单菌落,对比2.0、4.0mg/ml的G418 YPD平板,筛选出在两种不同浓度均能生长均匀的单菌落即为高拷贝转化子(见图5)。
(2)提取基因组,后作为模板进行PCR反应。以质粒pPIC3.5K的毕赤酵母GS115作为对照,确定阳性转化子。
实施例4重组毕赤酵母pPIC3.5K-CYP68J5m303底物转化分析
(1)将重组毕赤酵母pPIC3.5K-CYP68J5m303进行YPD平板活化,划三区放置在培养箱温度为30℃培养2-3天,使之长出均匀具有新鲜活力的单菌落。
(2)挑取新鲜的单菌落转接到适当的YPG液体培养基中发酵培养,随即摇床设定温度为30℃,合理的转速200r/min下,保证繁殖条件扩大培养24h。
(3)移液枪吹吸混匀YPG液体培养基,吸取750μL发酵液转接到BMGY 液体培养基中进行二级种子液的培养,摇床设定温度为30℃,合理的转速200r/min保证繁殖下,扩大培养12h左右,期间经过紫外分光光度计测定菌体细胞OD600值,直至OD600值生长至18左右,平分种子液到两个无菌的50ml离心管进行高速离心(5000r/min、5min)。
(4)弃上清,将离心后的菌体添加到BMMY诱导液体培养基进行培养发酵,此时投入底物黄体酮和甲醇进行诱导表达,记录投放底物时间(即转化时间 0h),每12h补加300μL甲醇保持液体培养基在在摇床合适的温度、适当的转速条件下扩大培养进行目的蛋白的表达。
(5)72h后取样萃取,进行产物的定性分析,自然晾干后经紫外分析仪对色谱斑点进行分析发现,重组毕赤酵母菌株转化甾体底物黄体酮时无副产物的生成(见图6)。
实施例5重组毕赤酵母pPIC3.5K-CYP68J5m303甘油批式发酵
发酵种子液摇瓶培养:甘油管接菌到YPG平板培养72h,挑取单菌落到YPG 液体培养液中培养24h,吸取5mL的一级种子液加入到YPG液体培养基中作为二级种子培养,使OD600达到12时上发酵罐接种;
发酵罐准备:以无机盐培养基为发酵培养基,接种量8%,pH 5.0;所述无机盐培养基组成为:26.7mL/L 85%磷酸,0.93g/L CaSO4,18.2g/L K2SO4,7.27 g/L MgSO4,4.13g/LKOH,40g/L甘油,微量元素0.43%;所述微量元素组成为(g/L):CuSO4·5H2O 6.0,NaI 0.08,MnSO4·H2O 3.0,NaMoO4·2H2O 0.2,H3BO3 0.02,CoCl20.5,ZnCl220.0,FeSO4·7H2O 65.0,生物素0.2,硫酸5.0mL。
菌体生长阶段:发酵温度为30℃,DO维持20%以上,当发酵培养基中甘油耗尽后,DO快速上升,随即进入补料生长阶段;
补料生长阶段:流加50%甘油(每升甘油中事先添加了12ml微量元素母液),初始流加速度为0.83mL/min,当DO低于20%时停止流加,待甘油再次耗尽, DO快速上升后,使菌体保持饥饿状态1h,然后进入诱导产酶时期。
底物黄体酮转化阶段:流加甲醇(每升甲醇中事先添加了12ml微量元素母液)。初始流加速率为0.15mL/min,当DO低于20%时停止流加,待DO快速上升后,重新开始流加,2h后流加速率提高至0.3mL/min,继续诱导2h后流加甲醇速率提高至0.454mL/min,同时流加底物黄体酮(溶于甲醇中)至终浓度 10g/L,转化96-120h(见图7),转化效率高达89%且无副产物生成。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以权利要求为准。
Claims (5)
1.C11α-羟化酶基因突变体I303L其特征在于,所述突变体的氨基酸序列是在如SEQ IDNO:1所示的序列基础上,由原来的异亮氨酸密码(ATA)突变成亮氨酸密码(TTA)。
2.权利要求1所述的C11α-羟化酶基因突变体I303L其特征在于,用于工业生产C11α-羟基黄体酮的衍生产物。
3.如权利要求2所述的用途,其特征在于,C11α-羟化酶基因突变体I303L转化甾体黄体酮时转化效率能达到89%且无明显副产物的生成。
4.如权利要求3所述的用途,其特征在于,重组表达载体或重组毕赤酵母宿主菌表达C11α-羟化酶突变体的应用。
5.一种制备C11α-羟基黄体酮的方法,其特征在于,培养权利要求4所述的重组毕赤酵母,并从发酵培养基中萃取回收C11α-羟基黄体酮产物。
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