CN113684191A - 梨头霉甾体11β-羟化酶CYP5311B2突变体构建及其应用 - Google Patents
梨头霉甾体11β-羟化酶CYP5311B2突变体构建及其应用 Download PDFInfo
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Abstract
本发明涉及利用定向进化和定点饱和突变以及利用酿酒酵母表达平台筛选高甾体羟化特异性的C11β‑羟化酶CYP5311B2突变体,其编码核苷酸序列如SEQ ID NO:1,SEQ ID NO:3所示;羟化酶突变体氨基酸序列如SEQ ID NO:2,SEQ ID NO:4所示。本发明还涉及CYP5311B2突变体异源表达的重组载体,过表达重组基因工程菌以及基于表达突变体的重组工程菌的高效甾体C11β‑羟化工艺。
Description
本发明属于基因工程和酶工程技术领域,具体涉及C11β-羟化酶突变体及其应用。
背景技术
甾体激素药物氢化可的松是一种具有显著抗炎、抗毒、抗休克等疗效的重要糖皮质激素药物,同时也是合成系列高效糖皮质激素药物的关键中间体。在甾体激素药物的生产中,微生物转化技术已成为许多甾体化合物或其中间体合成路线的关键技术。
目前基于丝状真菌蓝色梨头霉生物直接羟化底物11-脱氧可的松合成氢化可的松的生产工艺具有投料浓低(2-3g/L),副产物高(25-30%)等缺点,严重制约了氢化可的松生产效率。另一方面蓝色梨头霉遗传背景不清晰,进行菌种改造比较困难。
参与微生物甾体化合物羟化反应的羟基化酶属于P450酶系,我们克隆鉴定了蓝色梨头霉中编码甾体C11β-羟化酶的基因CYP5311B2(简称为CYP-7)并构建了重组表达载体和重组酵母菌。
同丝状真菌蓝色梨头霉一样,表达丝状真菌蓝色梨头霉甾体C11β-羟化酶的基因CYP-7重组酵母菌转化底物11-脱氧可的松形成多种副产物,为了改进C11β-羟化反应特异性,本发明提供的表达CYP-7突变体Z4(321Q、322 I、323 I、326 A/327 L)和Z8(V309I)的重组酿酒酵母工程菌转化底物11-脱氧可的松都显示明显改善的C11β-羟化特异性。基于以上突变体重组酵母的11-脱氧可的松转化工艺形成显著减少的副产物。
发明内容:
本发明涉及利用定向进化和定点饱和突变以及利用酿酒酵母表达平台筛选高甾体羟化特异性的C11β-羟化酶CYP-7突变体Z4和Z8,本发明还涉及CYP-7突变体异源表达的重组载体,过表达重组基因工程菌以及基于表达突变体的重组工程菌的高效甾体C11β-羟化工艺。
本发明所述含有基因CYP-7的表达盒、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。
含有本发明所述11β-羟化酶基因CYP-7 DNA全长或部分片段的定点突变和饱和突变引物序列也属于本发明的保护范围。
本发明所述C11β-羟化酶CYP-7突变体的氨基酸序列如SEQ ID NO:2和SEQ ID NO:4所示,其相应的DNA序列分别如SEQ ID NO:1和SEQ ID NO:3所示属于本发明的保护范围。
本发明所述C11β-羟化酶CYP-7突变体,可以用于工业转化脱氧可的松生产氢化可的松。
本发明所述C11β-羟化酶CYP-7突变体,转化甾体脱氧可的松合成氢化可的松时副产物明显减少。
本发明所述C11β-羟化酶基因CYP-7定点突变和饱和突变所涉及的特定位置氨基酸也属于本发明的保护范围。
本发明所述C11β-羟化酶CYP-7突变体重组表达载体或重组酿酒酵母宿主菌表达C11β-羟化酶CYP-7突变体的应用也属于本发明的保护范围。
本发明所述一种高效制备氢化可的松的方法,所述的CYP-7重组酿酒酵母突变体,发酵转化底物脱氧可的松并从发酵液萃取产物氢化可的松。
本发明所述C11β-羟化酶CYP-7突变体,可应用于过表达重组基因工程菌以及基于表达突变体的重组工程菌的高效甾体C11β-羟化工艺。
有益效果:
本发明实例考察了C11β-羟化酶基因CYP-7特定位点氨基酸对功能的影响,及重组酵母突变株的脱氧可的松的投料量。甾体转化实验表明两株酵母重组菌突变体对脱氧可的松投料量显著高于现有生产菌株蓝色梨头霉As3.65,本发明的研究成果具有重要的商业应用价值。
附图说明:
图1:脱氧可的松的11β-羟基化生物催化反应。
图2A:重组表达质粒pYES2-CYP-7的构建示意图。
图2B:启动子Pshn产物验证电泳图;M:Marker;CYP-7:11β-羟化酶基因。
图2C:重组表达质粒pYES2-CYP-7酶切验证图;M:10kb DNA ladder;1:酶切产物。
图3:CYP-7定点突变目标基因的获得;M:10kb DNA ladder;1、2、3:依次表示Z4(321Q、322 I、323 I、326 A/327 L)、Z8(V309I)、Z12(F147V/I)。
图4A:Z4(321Q、322 I、323 I、326 A/327 L)基因重组菌突变体功能验证;1:酿酒酵母(野生型);2:CYP-7重组酿酒酵母;3:Z4(321Q、322 I、323 I、326 A/327 L)基因重组菌突变体。
图4B:Z8(V309I)基因重组菌突变体甾体底物功能验证;1:氢化可的松;2:CYP-7重组酿酒酵母;3:Z8(V309I)基因重组菌突变体。
图4C:Z12(F147V/I)基因重组菌突变体功能验证;1:CYP-7重组酿酒酵母;2、3:Z12(F147V/I)基因重组菌突变体。
图5:CYP-7(325A、329T/330T)饱和突变目标基因的获得;M:10kb DNA ladder;1:CYP-7(325A);2:CYP-7(329T/330T)
图6A:CYP-7(325A)饱和突变重组菌突变体甾体底物功能筛选;1:酿酒酵母(野生型);2:CYP-7重组酿酒酵母;3~8:CYP-7(325A)基因重组菌突变体。
图6B:CYP-7(329T/330T)饱和突变重组酵母菌突变体甾体底物功能筛选;1:酿酒酵母(野生型);2:CYP-7重组酿酒酵母;3:氢化可的松;4:Z6(T329L/T330V)重组酵母突变体;5:Z10(T329L/T330I)重组酵母突变体。
具体实施方式:
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中(的实验方法,如无特殊说明,均为常规方法。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。
涉及菌株:酿酒酵母(Saccharomyces cerevisiae INVS1),蓝色梨头霉(Absidiacoerulea)As3.65,天津科技大学微生物菌种保藏中心。
所用的酶及试剂:
限制性内切酶XbaI、BamHI、Solution I连接试剂盒及Pyrobest DNA聚合酶购买自Takara公司。甲醇、乙腈、乙酸乙酯、石油醚及二氯甲烷购买自天津化学试剂六厂。其他常规试剂为进口分装或国产分析纯。引物的合成及序列的测定由北京华大公司完成。
所用培养基:
YPD培养基(g/L):酵母粉1g,蛋白胨2g,D-葡萄糖2g,加蒸馏水并定容至100mL,加入琼脂1.5g,115℃灭菌20min。
SC筛选培养基(g/L):YNB 6.7g,葡萄糖20g,腺嘌呤0.1g,精氨酸0.1g,半胱氨酸0.1g,亮氨酸0.1g,赖氨酸0.1g,苏氨酸0.1g,色氨酸0.1g,异亮氨酸0.05g,甲硫氨酸0.05g,,脯氨酸0.05g,丝氨酸0.05g,酪氨酸0.05g,苯丙氨酸0.05g,天冬氨酸0.05g,缬氨酸0.05g,组氨酸0.05g,琼脂20g,溶解于800mL去离子水中,并加水定容至1L,115℃灭菌20min。
LB培养基:酵母粉0.5g,蛋白胨1g,氯化钠1g,加水溶解并定容至100mL,调节pH至7.0,加入1.5g琼脂粉,121℃灭菌20min。
诱导产酶培养基(g/L):酵母粉1g,蛋白胨2g,D-半乳糖2g,加蒸馏水并定容至100mL,115℃灭菌20min。
实施例1 pYES2-CYP-7重组表达质粒的构建
本实施例中所用表达质粒为pYES2。
构建过程所涉及引物如下:
CYP-7-BamHI-F:GGATCCATGCTTACAGAGTACATTCATC
CYP-7-XbaI-R:TCTAGATTACTTTCTTGGCACAATCTTG
CYP-7-yan-F:CAAACCATGGTGTCATCCA
CYP-7-yan-R:GTTGTCCGTCACTTTTTGG
用引物(CYP-7-BamHI-F/CYP-7-XbaI-R),以蓝色梨头霉基因组DNA为模板通过PCR扩增得到CYP-7。通过酶切/酶联构建重组表达质粒pYES2-CYP-7。
构建的表达质粒如图2A所示,对重组质粒进行酶切验证分析。载体pYES2的大小为5.8kb,CYP-7片段大小1.5kb,如图2B所示。分别经BamHI和XbaI/BamHI酶切,琼脂糖凝胶电泳检测(图2C)显示7.4kb和5.8kb/1.6kb,结果证实重组表达质粒构建成功,将其命名为pYES2-CYP-7。
实施例2 pYES2-CYP-7重组酿酒酵母的构建和功能验证
将制备好的重组质粒(pYES2-CYP-7)通过醋酸锂转化法化转入酿酒酵母中,经组氨酸营养缺陷型筛选得到的转化子接种于YPD固体培养基中,30℃培养2~3天。
通过甾体转化对重组酵母进行功能验证。
将重组酵母培养好后,首先将甾体底物脱氧可的松通过球磨机研磨后,使其颗粒直径在10~15μm。向甾体底物脱氧可的松加入适量甲醇,适当加热使底物充分溶解,以0.05%RSA和2%半乳糖的量加入重组酵母发酵液中,摇晃均匀后放入摇床(28℃,180r/min)继续振荡培养,培养5天后取样测定转化情况。TLC检测结果如图4所示。
实施例3 CYP-7定点突变目标基因的获得
本实施例中所用重组表达质粒为pYES2-CYP-7。
实施例3所用定点突变引物如下:
CYP-7(321-327)F:ACAGTTTTAGTTTTTGCATCTATTCACACAACCAGTGAAAACGG
CYP-7(321-327)R:GTGAATAGATGCAAAAACTAAAACTGTCATCCAATCGCAACAATG
CYP-7(V309I)F:CATATTGATGTCACGGATCATTGTTGC
CYP-7(V309I)R:ATCAATATGAGCTGGGATATCGCC
CYP-7(F147V/I)F:AAGRTTGCGGGTAGTATCAAGAAGAA
CYP-7(F147V/I)R:CGCAAYCTTGTGTACAGGCAAGTCTCG
以制备好的重组质粒pYES2-CYP-7为模板,分别以设计的定点突变引物进行PCR扩增,获得长约7.4kb的产物片段,琼脂糖凝胶电泳检测,结果如图3所示。将CYP-7(321Q、322I、323 I、326 A/327 L)、CYP-7(V309I)、CYP-7(F147V/I)三种定点突变目标载体片段及重组酵母突变体均分别记为Z4、Z8和Z12。
实施例4重组酵母突变体Z4、Z8和Z12的构建和功能分析
将定点突变目标载体片段Z4、Z8和Z12分别通过醋酸锂转化法转入酿酒酵母中,通过甾体转化对重组酵母进行功能验证。
将重组酵母培养好后,首先将甾体底物脱氧可的松通过球磨机研磨后,使其颗粒直径为10~15μm。向甾体底物脱氧可的松加入适量甲醇,适当加热使底物充分溶解,以0.05%RSA和2%半乳糖的量加入重组酵母发酵液中,摇晃均匀后放入摇床(28℃,180r/min)继续振荡培养,培养5天后取样测定转化情况。TLC检测结果如图4所示。
实施例5 CYP-7(325A、329T/330T)饱和突变目标基因的获得
实施例5所用饱和突变引物如下:
SM325A-F:ATCTTTNNKGCTCTACACACAACCAGTG
SM325A-R:TAGAGCMNNAAAGATAATTTGTGTCATCCAATCG
SM330T-F:CACACANNKAGTGAAAACGGCACATTATCC
SM330T-R:TTCACTMNNTGTGTGTAGAGCTGCAAAG
以制备好的质粒pYES2-CYP-7为模板,分别以设计的饱和突变引物进行PCR扩增,获得长约7.4kb的产物片段,琼脂糖凝胶电泳检测,结果如图5所示。CYP-7(325A)、CYP-7(329T/330T)PCR扩增体系记为a和b。
实施例6饱和突变重组酵母的构建和功能筛选
将饱和突变PCR扩增纯化片段分别通过醋酸锂转化法转入酿酒酵母中,通过甾体转化及结果检测对重组酵母进行功能筛选。
将重组酵母培养好后,首先将甾体底物脱氧可的松通过球磨机研磨后,使其颗粒直径为10~15μm。向甾体底物脱氧可的松加入适量甲醇,适当加热使底物充分溶解,以0.05%RSA和2%半乳糖的量加入重组酵母发酵液中,摇晃均匀后放入摇床(28℃,180r/min)继续振荡培养,培养5天后取样测定转化情况。筛选得到几个11β-羟基化特异性影响较大的重组酵母突变体,TLC检测结果如图6所示,其余结果未展示。
虽然上述已公开了本发明较有效的实施方式,但其并非用以限定本发明,任何本领域技术人员,在不脱离本发明的精神和范围内,都可做各种的改动和修饰,因此本发明的保护范围应该以权利要求书所限定的为准。
Claims (5)
1.C11β-羟化酶CYP5311B2突变体Z4和Z8,其特征在于,所述突变体的氨基酸序列如SEQID NO:2和SEQ ID NO:4所示,其相应的DNA序列分别如SEQ ID NO:1和SEQ ID NO:3所示。
2.权利要求1所述的C11β-羟化酶CYP5311B2突变体,其特征在于,可以用于工业转化11-脱氧可的松生产氢化可的松。
3.如权利要求2所述的用途,其特征在于,C11β-羟化酶CYP5311B2突变体转化甾体脱氧可的松合成氢化可的松时副产物明显减少。
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酿酒酵母宿主菌表达C11β-羟化酶CYP5311B2突变体的应用。
5.一种高效制备氢化可的松的方法,其特征在于,利用权利要求4所述的重组酿酒酵母,发酵转化底物脱氧可的松并从发酵液萃取产物氢化可的松。
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