CN115772507A - 细胞色素p450酶在合成灵芝三萜中的应用 - Google Patents
细胞色素p450酶在合成灵芝三萜中的应用 Download PDFInfo
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- CN115772507A CN115772507A CN202111050263.XA CN202111050263A CN115772507A CN 115772507 A CN115772507 A CN 115772507A CN 202111050263 A CN202111050263 A CN 202111050263A CN 115772507 A CN115772507 A CN 115772507A
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Abstract
一种细胞色素P450酶在合成灵芝三萜中的应用，通过P450酶作为催化剂，反应生成灵芝三萜类化合物，实现其在酿酒酵母中的异源生物合成，即从灵芝基因组中挖掘到两个参与三萜合成的P450基因GL19325和GL15605，并利用合成生物学技术手段，构建酿酒酵母工程菌株，实现相应三萜类化合物的异源生物合成。
Description
技术领域
本发明涉及的是一种生物工程领域的技术，具体是一种细胞色素P450酶在合成灵芝三萜中的应用。
背景技术
由于灵芝生长周期较长，不易培养，灵芝中代谢产物丰富且含量相对较低，缺少成熟全面的灵芝真菌细胞基因操作手段等因素，研究人员对灵芝三萜类化合物生物合成途径的认知较为匮乏且进展缓慢。
发明内容
本发明针对现有技术存在的上述不足，提出一种细胞色素P450酶在合成灵芝三萜中的应用，从灵芝基因组中挖掘到两个参与三萜合成的P450基因GL19325和GL15605，并利用合成生物学技术手段，构建酿酒酵母工程菌株，实现相应三萜类化合物的异源生物合成。
本发明是通过以下技术方案实现的：
本发明涉及一种细胞色素P450酶在合成灵芝三萜中的应用，通过P450基因作为催化剂，反应生成与天然化合物结构完全一致的灵芝三萜类化合物，并实现其在酿酒酵母中的异源生物合成。
所述的应用包括以下任意一种：采用P450酶GL19325催化2,3-环氧角鲨烯形成一种人工线性三萜化合物ST-3，或采用P450酶GL15605催化3-羟基-羊毛甾-8,24-二烯-26-酸(Ganoderic acid HLDOA)形成与天然化合物结构完全一致的灵芝酸Y(Ganoderic acidY)。
所述的P450基因GL19325的核苷酸序列如Seq ID No.1所示，相应酶的氨基酸序列如Seq ID No.2所示；所述的P450基因GL15605的核苷酸序列如Seq ID No.3所示，相应酶的氨基酸序列如Seq ID No.4所示。
所述的应用，具体为：将P450基因GL19325或GL15605分别克隆到酿酒酵母表达质粒中，即以灵芝cDNA为模板，通过PCR扩增得到各个P450编码区序列片段，通过同源重组的方法将表达载体pRS426、酵母HXT7p启动子、P450编码区序列片段、酵母FBA1t终止子和含有截短启动子Ura3(tP-Ura3)的KanMX基因表达框重组连接，得到一系列重组表达质粒pRS426HF-P450s-G418r(s代指不同的P450基因)；然后将这些质粒分别转入不同的重组改造后的酿酒酵母中进行异源表达，即在基因工程改造后的BY4742菌株YL-T3的基础上，向YL-T3菌株中引入表达质粒pRS425-iGLCPR-Hygr形成的菌株YL-T3-iGLCPR或在YL-T3的基础上引入酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr获得的高产Ganoderic acidHLDOA的酿酒酵母菌株YL-T3-CYP5150L8-iGLCPR。
所述的酿酒酵母表达质粒pRS425-iGLCPR-Hygr通过(Lan,X.,et al.,Efficientbiosynthesis of antitumor ganoderic acid HLDOA using a dual tunable systemfor optimizing the expression of CYP5150L8 and a Ganoderma P450reductase.Biotechnol Bioeng,2019.116(12):p.3301-3311.)构建获得，其中GL19526基因也就是iGLCPR的表达产物是一个细胞色素P450还原酶(CPR)。
所述的酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr是指：在酵母表达商业载体pRS425的基础上，通过同源重组的方法将pRS425、TEF1p启动子、GL19526基因(iGLCPR)、PGK1t终止子、含有截短启动子Ura3(tP-Ura3)的Hygromycin B基因表达框和CYP5150L8表达框重组连接，得到重组表达质粒pRS425-CYP5150L8-iGLCPR-Hygr或在已经构建好的质粒pRS425-iGLCPR-Hygr的基础上引入CYP5150L8表达框。
所述的基因工程改造后的BY4742菌株YL-T3，参考文献(Dai,Z.,et al.,Producing aglycons of ginsenosides in bakers'yeast.Sci Rep,2014.4:p.3698.)获得，其中：BY4742菌株为本领域技术人员常用的一种商品化酵母宿主。在此基础上过表达了Lanosterol生物合成途径上游的几个基因从而提高Lanosterol的合成量。
技术效果
与现有技术相比，本发明采用双质粒抗生素调控表达系统，构建新的酿酒酵母底盘细胞，使用优化后的抗生素浓度，提高不同底盘中三萜前体lanosterol或Ganodericacid HLDOA的产量，然后通过将待筛选基因在抗生素调控酿酒酵母质粒上进行表达，从而验证筛选基因的功能，解析催化产物三萜的生物合成途径的同时，并实现了这些三萜在酿酒酵母中的异源生物合成。
附图说明
图1为本发明表达质粒pRS425-iGLCPR-Hygr的示意图；
图2为本发明表达质粒pRS425-CYP5150L8-iGLCPR-Hygr的示意图；
图3为本发明表达质粒pRS426HF-GL19325-G418r的示意图；
图4为本发明表达质粒pRS426HF-GL15605-G418r的示意图；
图5为酿酒酵母YL-T3-iGLCPR-GL19325菌株和对照菌株YL-T3-iGLCPR-control发酵产物HPLC谱图及新峰MS离子图；
图6为酿酒酵母YL-T3-CYP5150L8-iGLCPR-GL15605菌株和对照菌株YL-T3-CYP5150L8-iGLCPR-control发酵产物HPLC谱图及新峰MS离子图；
图7～图13依次为GL19325催化产物ST-3的1H-NMR、13C-NMR谱图、DEPT135谱图、COSY谱图、HSQC谱图、HMBC谱图以及形成示意图；
图14～图20依次为GL15605催化产物Ganoderic acid Y的1H-NMR、13C-NMR谱图、DEPT135谱图、COSY谱图、HSQC谱图、HMBC谱图以及形成示意图。
具体实施方式
实施例1
重组改造后酿酒酵母菌株YL-T3-iGLCPR的构建，即向YL-T3菌株中引入表达质粒pRS425-iGLCPR-Hygr从而形成菌株YL-T3-iGLCPR，可用于基于lanosterol进行P450筛选，具体包括：
1.1)构建如图1所示的酿酒酵母表达质粒pRS425-iGLCPR-Hygr。
1.2)将构建好的质粒pRS425-iGLCPR-Hygr通过醋酸锂法(Gietz,R.D.andR.H.Schiestl,High-efficiency yeast transformation using the LiAc/SS carrierDNA/PEG method.Nat Protoc,2007.2(1):p.31-4.)转化到酿酒酵母细胞YL-T3中，然后将转化后的菌液涂布在SC-His-Leu(SC-HL)固体培养基(yeast nitrogen base withoutamino acids(YNB)，6.7g/L；葡萄糖，20g/L；yeast synthetic drop-out media(SD)Y2001，1.39g/L；tryptophan，76mg/L；Uracil，76mg/L；琼脂粉，2％)上。30℃培养至转化子出现，从而得到经过改造后的酿酒酵母YL-T3-CYP5150L8-iGLCPR菌株。
实施例2
重组改造后酿酒酵母菌株YL-T3-CYP5150L8-iGLCPR的构建，即将酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr转入酿酒酵母YL-T3中，形成经过重组改造后的酿酒酵母YL-T3-CYP5150L8-iGLCPR，具体包括：
2.1)酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr的构建。
2.1.1)在pRS425-iGLCPR-Hygr的基础上进行改造。pRS425-iGLCPR-Hygr质粒参考(Lan,X.,et al.,Efficient biosynthesis of antitumor ganoderic acid HLDOA usinga dual tunable system for optimizing the expression of CYP5150L8 and aGanoderma P450 reductase.Biotechnol Bioeng,2019.116(12):p.3301-3311.)构建获得。首先使用Pmel酶消化质粒pRS425-iGLCPR-Hygr得到线性化质粒载体片段。
2.1.2)然后使用引物对HF-CYP5150L8-F和HF-CYP5150L8-R，以pRS426-HXT7p-CYP5150L8-FBA1t为模板扩增出含同源臂的CYP5150L8表达框(Lan,X.,et al.,Efficientbiosynthesis of antitumor ganoderic acid HLDOA using a dual tunable systemfor optimizing the expression of CYP5150L8 and a Ganoderma P450reductase.Biotechnol Bioeng,2019.116(12):p.3301-3311.)
引物具体序列如序列表1所示：
表1：扩增含同源臂的CYP5150L8的表达框的引物序列表
| 引物名称 | 序列号 | 序列(5'to 3') |
| HF-CYP5150L8-F | Seq ID No.5 | ggcaaaggaataatctcgagtcatgtaattagttatgtca |
| HF-CYP5150L8-R | Seq ID No.6 | cgagcggtctaaggcggtttacttctcgtaggaacaattt |
F和R分别表示正向和反向引物。
2.1.3)然后将线性化的pRS425-iGLCPR-Hygr载体片段和含同源臂的CYP5150L8表达框片段进行同源重组连接，具体包括：
2.1.3.1)连接体系：线性化的pRS425-iGLCPR-Hygr质粒0.03pmol、扩增得到的含同源臂的CYP5150L8表达框片段0.06pmol、CE II Buffer 4μL、Exnase II 2μL、无菌水补至20μL。混匀，37℃反应连接30min，冰浴5min。
2.1.3.2)取出-80℃冻存的50μL DH5α感受态细胞，冰上放置，至完全解冻。约需5min。
2.1.3.3)将连接产物转移到50μL DH5α感受态细胞中，混匀，避免产生气泡，冰上放置20min，42℃热激60S，然后冰上放置2min，加入900μL LB培养基，37℃孵育60min。随后涂含100μg/mL的Amp抗性的LB平板，37℃培养箱倒置过夜培养。
2.1.3.4)等平板长出单菌落后，挑选单克隆转接含100μg/mL Amp抗性的3mL液体LB培养基，37℃220rpm过夜培养。
2.1.3.5)等菌液长至稳定期后，提取质粒，使用测序引物进行PCR验证，然后挑选可能对的质粒进行测序，比对测序结果，从而可以得到正确的重组质粒pRS425-CYP5150L8-iGLCPR-Hygr。质粒示意图如图2所示。
所使用的测序引物如表2所示：
表2：验证重组质粒pRS425-CYP5150L8-iGLCPR-Hygr正确性的测序引物序列表
| 引物名称 | 序列号 | 序列(5'to 3') |
| HF-CYP5150L8-CX-F | Seq ID No.7 | atttcgatgatgcagcttgg |
| HF-CYP5150L8-CX-R | Seq ID No.8 | acatcaaaatccacattctc |
2.2)将上述得到的测序正确的重组质粒pRS425-CYP5150L8-iGLCPR-Hygr通过醋酸锂法转化到酿酒酵母细胞YL-T3中，将转化后的酵母涂布在SC-HL固体培养基上。30℃培养至转化子出现，从而得到经过改造后的酿酒酵母YL-T3-CYP5150L8-iGLCPR菌株。
实施例3
构建表达P450基因的酿酒酵母转化菌株及对照菌株，具体包括：
3.1)以灵芝cDNA为模板，通过PCR扩增得到各个P450编码区序列片段；
3.2)通过(Lan,X.,et al.,Efficient biosynthesis of antitumor ganodericacid HLDOA using a dual tunable system for optimizing the expression ofCYP5150L8 and a Ganoderma P450 reductase.Biotechnol Bioeng,2019.116(12):p.3301-3311.)构建得到的pRS426HF-G418r质粒的基础上，通过同源重组的方法将表达载体pRS426、P450编码区序列片段、酵母HXT7p启动子、酵母FBA1t终止子和含有截短启动子Ura3(tP-Ura3)的KanMX基因表达框重组连接，得到一系列重组表达质粒pRS426HF-P450s-G418r(s表示不同的P450基因)；
3.3)将各个重组质粒分别转化到酿酒酵母细胞YL-T3-CYP5150L8-iGLCPR中，从而得到一系列表达不同候选P450基因的酿酒酵母菌株YL-T3-CYP5150L8-iGLCPR-P450s(s表示不同的P450基因)。
所述的用于从灵芝cDNA中扩增获得灵芝P450基因GL19325和GL15605编码区序列片段的引物如序列表3所示：
表3：扩增灵芝P450基因编码区序列片段所用引物序列表
| 引物名称 | 序列号 | 序列(5'to 3') |
| GL19325-F | Seq ID No.9 | TAATTTTAATCAAAAAGTTTATGGCGCAACTCGCAGTGCT |
| GL19325-R | Seq ID No.10 | ATTAATTTGAATTAACGTTTTCAGCGAGGTACGAGGCTGA |
| GL15605-F | Seq ID No.11 | TAATTTTAATCAAAAAGTTTATGGAGGATGCACAGACTCT |
| GL15605-R | Seq ID No.12 | ATTAATTTGAATTAACGTTTTCAGGCCGTCACTCTGCGCT |
所述的重组质粒pRS426HF-P450s-G418r，具体通过以下方式得到：
i)通过(Lan,X.,et al.,Efficient biosynthesis of antitumor ganodericacid HLDOA using a dual tunable system for optimizing the expression ofCYP5150L8 and a Ganoderma P450 reductase.Biotechnol Bioeng,2019.116(12):p.3301-3311.)构建Smal线性化pRS426HF-G418r质粒。
ii)将线性化的pRS426HF-G418r质粒与扩增得到的P450基因编码区序列片段通过重组酶连接，具体步骤如下：
a)连接体系：线性化的pRS426HF-G418r质粒0.03pmol、扩增得到的P450基因编码区序列片段0.06pmol、CE II Buffer 4μL、Exnase II 2μL、无菌水补至20μL。轻轻混匀，避免产生气泡，然后37℃反应连接30min，冰浴5min。
b)～d)同实施例2中的步骤2.1.3.2～2.1.3.4。
e)等菌液长至稳定期后，提取质粒，使用测序引物进行PCR验证，然后挑选可能对的质粒进行测序，比对测序结果，从而可以得到正确的重组质粒pRS426HF-P450s-G418r。构建好的pRS426HF-GL19325-G418r质粒如图3所示；pRS426HF-GL15605-G418r质粒如图4所示。
所使用的测序引物如表4所示：
表4：验证重组质粒pRS426HF-P450s-G418r正确性的测序引物序列表
将上述得到的测序正确的重组质粒pRS426HF-P450s-G418r通过醋酸锂法转化到YL-T3-iGLCPR或YL-T3-CYP5150L8-iGLCPR中，将转化后的酵母涂布在SC-His-Leu-Ura(SC-HLU)固体培养基上(yeast nitrogen base without amino acids(YNB)，6.7g/L；葡萄糖，20g/L；yeast synthetic drop-out media(SD)Y2001，1.39g/L；tryptophan，76mg/L；琼脂粉，2％)进行培养。30℃培养至转化子出现，从而得到过表达不同P450基因的酿酒酵母菌株，如YL-T3-iGLCPR-GL19325和YL-T3-CYP5150L8-iGLCPR-GL15605。
YL-T3-iGLCPR-GL19325的对照菌株为将pRS426HF-G418r质粒转入YL-T3-iGLCPR后得到的菌株，即YL-T3-iGLCPR-control。
YL-T3-CYP5150L8-iGLCPR-GL15605的对照菌株为将pRS426HF-G418r质粒转入YL-T3-CYP5150L8-iGLCPR后得到的菌株，即YL-T3-CYP5150L8-iGLCPR-control。
实施例4
候选P450基因功能鉴定，即将候选P450基因对应的表达质粒pRS426HF-P450s-G418r转入YL-T3-iGLCPR或YL-T3-CYP5150L8-iGLCPR中，然后将获得的转化菌株以及对照菌株进行发酵，通过比较发酵后代谢产物的差异，从而初步筛选可能和灵芝三萜类化合物相关的P450基因，同时也对相应的P450基因的功能进行初步的鉴定，具体包括：
4.1)将构建好的各个包含不同P450基因的酵母转化菌株转化子以及对照菌株分别液体转接到SC-His-Leu-Ura(SC-HLU)液体培养基中(yeast nitrogen base withoutamino acids(YNB)，6.7g/L；葡萄糖，20g/L；yeast synthetic drop-out media(SD)Y2001，1.39g/L；tryptophan，76mg/L)，30℃220rpm条件下培养至菌体长到稳定期；
4.2)然后将培养好的菌液以3％的比例再次液体转接SC-HLU液体培养基，30℃220rpm条件下培养至菌体达到对数期，种子菌液制备完成；
4.3)然后将种子液以3％的比例接种到YPD24培养基(酵母粉10g/L，牛肉蛋白胨20g/L，葡萄糖20g/L，甘油40g/L)中，然后30℃220rpm条件下发酵培养5天。
4.4)发酵培养5天后，取出发酵后的菌液，离心去除上清，细胞沉淀用甲醇萃取，上清过0.22μm针头过滤器后即可得到重组菌株发酵后粗提物，之后就可进行HPLC检测分析发酵产物。
4.5)通过观察发酵产物的HPLC图谱中是否有新峰出现，初步判断是否有和灵芝三萜生物合成相关的P450基因。
实施例5
酿酒酵母转化株发酵产物HPLC检测，具体包括：
5.1)发酵产物的HPLC分析方法：
5.1.1)仪器：安捷伦1260 Infinity II HPLC分析系统，DAD(Diode arraydetector)检测器。
5.1.2)色谱柱：Kinetex Biphenyl分析柱(2.6μm,150mm×4.6mm,Phenomenex,Torrance,CA)。
5.1.3)柱温：30℃；流速：0.5mL/min；进样量：20μL，检测波长214nm。
5.1.4)流动相：A相：超纯水，B相：甲醇(含0.1％乙酸)。
5.1.5)梯度洗脱程序：
5.1.5.1)基于lanosterol进行筛选时，洗脱程序为：0-30min，85％-100％B相；30-35min，100％B相；35-36min，100％-85％B相；36-45min，85％B相。
5.1.5.2)基于Ganoderic acid HLDOA进行筛选时，洗脱程序为：0-30min，80％-100％B相；30-35min，100％B相；35-36min，100％-80％B相；36-45min，80％B相。
5.2)通过和空质粒对照菌株发酵产物HPLC峰图对比，观察发现YL-T3-iGLCPR-GL19325和YL-T3-CYP5150L8-iGLCPR-GL15605两个菌和对照菌相比都有显著不同的峰(如图5和图6所示)，初步判断这些峰所对应的产物可能是灵芝三萜类化合物。之后再进行LC-MS测定进一步确认新峰对应的分子量是否和灵芝三萜类化合物相关。
实施例6
发酵产物LC-MS检测，具体包括：
6.1)仪器：超高效液相色谱仪-超高分辨质谱连用系统。
6.2)色谱柱：Kinetex Biphenyl分析柱(2.6μm,150mm×4.6mm,Phenomenex,Torrance,CA)。
6.3)柱温：30℃；流速：0.5mL/min；进样量：20μL，检测波长214nm。
6.4)流动相：A相：超纯水，B相：甲醇(含0.1％乙酸)。
6.5)梯度洗脱程序：
6.5.1)基于lanosterol进行筛选时，洗脱程序为：0-30min，85％-100％B相；30-35min，100％B相；35-36min，100％-85％B相；36-45min，85％B相。
6.5.2)基于Ganoderic acid HLDOA进行筛选时，洗脱程序为：0-30min，80％-100％B相；30-35min，100％B相；35-36min，100％-80％B相；36-45min，80％B相。
6.6)扫描分子量：100-1000。
通过和对照菌发酵产物HPLC，MS谱图，以及峰对应离子的比较(如图5和图6所示)，从而基本确定是否有新的灵芝三萜类产物生成。之后再进一步进行分离纯化以及结构鉴定。
实施例7
YL-T3-iGLCPR-GL19325发酵产物中新产物的分离纯化及鉴定，具体包括：
7.1)发酵产物中新产物的分离纯化
7.1.1)取-80℃冻存的酵母菌株YL-T3-iGLCPR-GL19325在SC-HLU固体平板上划线，然后30℃培养以活化菌体。
7.1.2)当单克隆长好后，挑取单克隆，转接至SC-HLU液体培养基中，30℃ 220rpm培养至菌液长到对数期。
7.1.3)菌液长好后，以3％的比例转接SC-HLU液体培养基中，然后30℃ 220rpm培养至菌体达到对数期。此时发酵种子制备完成。
7.1.4)接种发酵。将培养好种子液以3％的比例接种10LYPD24培养基中。30℃220rpm发酵5天。
7.1.5)发酵结束后，以1:1的比例向发酵液中加入乙酸乙酯，封膜后放置于30℃250rpm摇床上震荡萃取，后收集上清乙酸乙酯层。下层再用乙酸乙酯重复萃取一次。然后合并两次萃取后的乙酸乙酯，使用旋转蒸发仪旋蒸至基本蒸干。然后吸出，残余物使用甲醇溶出，合并溶出物后，体积约为20mL。
7.1.6)过正相硅胶柱纯化。方法如下：
使用34mm(柱内径)*500mm(有效柱长)层析柱；
洗脱程序为：石油醚，200mL；石油醚：乙酸乙酯＝8:1，200mL；石油醚：乙酸乙酯＝2:1，200mL；石油醚：乙酸乙酯＝1:1，600mL；石油醚：乙酸乙酯＝1:2，600mL；石油醚：乙酸乙酯＝1:4，400mL；甲醇，600mL。
洗脱过程中，分段收集，用0.22μm有机针头过滤器过滤后，用HPLC检测分析。
HPLC检测方法同实施例5中的5.1。洗脱程序同实施例5中的5.1.5.1。
检测后，将含有新产物收集液全部合并，旋蒸至蒸干，然后用少量甲醇溶出，取上清进行后续处理。
7.1.7)使用制备液相进行制备纯化。
浓缩后的粗品通过制备液相进一步进行制备纯化，方法如下：
7.1.7.1)仪器：安捷伦1260系列制备液相色谱仪。DAD检测器，检测波长214nm；
7.1.7.2)色谱柱：YMC-Pack ODS-A,20x250mm,5um,12nm；
7.1.7.3)流速：10mL/min；进样量：800μL；
7.1.7.4)流动相：A相：超纯水，B相：甲醇(含0.1％甲酸)；
7.1.7.5)梯度洗脱程序为：0-50min，85％-100％B相；50-60min，100％B相；60-60.5min，100％-85％B相；60.5-70min，85％B相。
在新产物出峰时间段，分段收集直接取少量进行HPLC检测。HPLC检测方法同实施例5中的5.1。洗脱程序同实施例5中的5.1.5.1。根据HPLC谱图中峰面积，估算新产物的纯度，将纯度高的收集液合并，后旋蒸蒸干，用HPLC级甲醇溶出于离心管中(加入前需称量离心管的重量)。真空挥干成粉末后再称重，确认纯品重量。
7.2)新化合物结构鉴定。
当确定分离纯化后得到的化合物是感兴趣的物质。可以进一步进行NMR(核磁共振波谱法)检测以确定新产物的结构。经过对一维碳谱、一维氢谱、COSY、HSQC、HMBC的谱图进行细致解析，能够最终确定分离到的化合物的结构，从而确定新化合物是否是新的灵芝三萜类化合物。
从YL-T3-iGLCPR-GL19325发酵产物中，获得并鉴定出一个线性三萜化合物ST-3。一维碳谱和氢谱数据如表5所示。1H-NMR、13C-NMR、DEPT、COSY、HSQC、HMBC谱图详见图7至图12。图13为GL19325催化产物ST-3形成的示意图。
表7 ST-3的1H-NMR、13C-NMR数据表
| C position | 13C NMR(ppm) | 1H NMR(δppm,J,N) |
| 1，24 | 18.75 | 1.26(s,3H) |
| 2，23 | 58.36 | - |
| 3，22 | 64.23 | 2.70(t,J＝6.3Hz,1H) |
| 4，21 | 27.47 | (1.56m,1.66m,2H) |
| 5，20 | 36.31 | (2.09m,2.15m,2H) |
| 6,19 | 134.01 | - |
| 7,18 | 124.93 | 5.16(m,1H) |
| 8,17 | 26.67 | (2.00m,2.09m,2H) |
| 9,16 | 39.68 | (2.00m,2.09m,2H) |
| 10,15 | 135.02 | - |
| 11,14 | 124.36 | 5.16(m,1H) |
| 12,13 | 28.27 | 2.01(d,J＝3.3Hz,2H) |
| 25，30 | 24.91 | 1.30(s,3H) |
| 26,29 | 16.05 | 1.62(s,3H) |
| 27,28 | 16.01 | 1.60(s,3H) |
实施例8
YL-T3-CYP5150L8-iGLCPR-GL15605发酵产物中新产物的分离纯化及鉴定，具体包括：
8.1)发酵产物中新产物的分离纯化
8.1.1)取-80℃冻存的酵母菌株YL-T3-CYP5150L8-iGLCPR-GL15605在SC-HLU固体平板上划线，然后30℃培养以活化菌体。
8.1.2)～8.1.5)同实施例7中的步骤7.1.2～7.1.5。
8.1.6)过正相硅胶柱纯化。方法如下：
使用34mm(柱内径)*500mm(有效柱长)层析柱；
洗脱程序为：石油醚，200mL；石油醚：乙酸乙酯＝8:1，200mL；石油醚：乙酸乙酯＝2:1，200mL；石油醚：乙酸乙酯＝1:1，600mL；石油醚：乙酸乙酯＝1:2，600mL；石油醚：乙酸乙酯＝1:4，400mL；甲醇，600mL。
洗脱过程中，分段收集，用0.22μm有机针头过滤器过滤后，用HPLC检测分析。
HPLC检测方法同实施例5中的5.1。洗脱程序同实施例5中的5.1.5.2。
检测后，将含有新产物收集液全部合并，然后旋蒸至蒸干，然后用少量甲醇溶出，取上清进行后续处理。
8.1.7)使用制备液相进行制备纯化。
浓缩后的粗品通过制备液相进一步进行制备纯化，方法如下：
8.1.7.1)～8.1.7.4)同实施例7中的步骤7.1.7.1～7.1.7.4。
8.1.7.5)梯度洗脱程序为：0-50min，80％-100％B相；50-60min，100％B相；60-60.5min，100％-80％B相；60.5-70min，80％B相。
在新产物出峰时间段，分段收集，直接取少量进行HPLC检测。HPLC检测方法同实施例5中的5.1。洗脱程序同实施例5中的5.1.5.2。根据HPLC谱图中峰面积，估算新产物的纯度，将纯度高的收集液合并，后旋蒸蒸干，用HPLC级甲醇溶出于离心管中(加入前需称量离心管的重量)。真空挥干成粉末后再称重，确认纯品重量。
8.2)新化合物结构鉴定。
当确定分离纯化后得到的化合物是感兴趣的物质。可以进一步进行NMR(核磁共振波谱法)检测以确定新产物的结构。经过对一维碳谱、一维氢谱、COSY、HSQC、HMBC的谱图进行细致解析，能够最终确定分离到的化合物的结构，从而确定新化合物是否是新的灵芝三萜类化合物。
从YL-T3-CYP5150L8-iGLCPR-GL15605发酵产物中，获得并鉴定出一个灵芝三萜化合物Ganoderic acid Y。一维碳谱和氢谱数据如表6所示。1H-NMR、13C-NMR、DEPT、COSY、HSQC、HMBC谱图详见图14至图19。图20为GL15605催化产物Ganoderic acid Y形成的示意图。
表6 Ganoderic acid Y的1H-NMR、13C-NMR数据表
| C position | 13C NMR(ppm) | 1H NMR(δppm,J,N) |
| 1 | 27.92 | (1.32m,1.99m,2H) |
| 2 | 27.8 | (1.67m,1.72m,2H) |
| 3 | 78.98 | 3.25(dd,J＝11.6,4.3Hz,1H) |
| 4 | 38.71 | - |
| 5 | 49.11 | 1.09(dd,J＝11.4,4.3Hz,1H) |
| 6 | 23.01 | (2.07m,2.10m,2H) |
| 7 | 120.34 | 5.48(d,J＝5.1Hz,1H) |
| 8 | 142.57 | - |
| 9 | 145.95 | - |
| 10 | 37.38 | - |
| 11 | 116.2 | 5.32(d,J＝6.3Hz,1H) |
| 12 | 37.81 | (2.08m,2.21d,J＝18.0Hz,2H) |
| 13 | 43.81 | - |
| 14 | 50.32 | - |
| 15 | 31.49 | (1.39m,1.61m,2H) |
| 16 | 35.72 | (1.43m,1.99m,2H) |
| 17 | 50.86 | 1.58(m,1H) |
| 18 | 15.67 | 0.57(s,3H) |
| 19 | 22.76 | 0.98(s,3H) |
| 20 | 36.16 | 1.42(m,1H) |
| 21 | 18.31 | 0.93(d,J＝6.5Hz,3H) |
| 22 | 34.75 | (1.19m,1.56m,2H) |
| 23 | 25.92 | (2.13m,2.27m,2H) |
| 24 | 145.67 | 6.90(t,J＝7.5Hz,1H) |
| 25 | 126.48 | - |
| 26 | 172.01 | - |
| 27 | 12.04 | 1.84(s,3H) |
| 28 | 28.15 | 1.01(s,3H) |
| 29 | 15.8 | 0.88(s,3H) |
| 30 | 25.56 | 0.88(s,3H) |
实验表明，通过灵芝基因组挖掘，得到两个参与三萜类化合物生物合成的P450基因GL19325和GL15605。其中，GL19325能催化2,3-oxidosqualene形成一种人工线性三萜类化合物ST-3。GL15605能催化Ganoderic acid HLDOA形成一种四环结构灵芝三萜类化合物Ganoderic acid Y。并通过将基因在酿酒酵母中进行异源表达，实现ST-3和Ganodericacid Y的异源生物合成。
与现有技术相比，本发明采用双质粒抗生素调控表达系统，构建新的酿酒酵母底盘细胞，使用优化后的抗生素浓度，提高不同底盘中灵芝酸前体lanosterol或Ganodericacid HLDOA的产量，然后通过将待筛选基因在抗生素调控酿酒酵母质粒上进行表达，从而验证筛选基因的功能，解析催化产物三萜的生物合成途径的同时，实现了这些三萜类化合物在酿酒酵母中的异源生物合成。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
序列表
<110> 中国科学院天津工业生物技术研究所
上海交通大学
<120> 细胞色素P450酶在合成灵芝三萜中的应用
<130> fnc610e
<141> 2021-09-08
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1635
<212> DNA
<213> P450基因GL19325(Artificial Sequence)
<400> 1
atggcgcaac tcgcagtgct agtcgcgtcg ggcctagtcc tctacttcct atggctcctc 60
gtgcgaaact acgtcgtgaa gtctccattg gacaaaattc ctggccctcc gtcgggctca 120
gttctttcag gaaacatgtt tcagttgttt aaccataaca gttggggatt cgttgatgac 180
ctcattcaga cctatggccc gctctccaag tgctatggtt tctttggcac gaggatgctg 240
cacatctacg acccgaaagc gatgcacagc atctacgtca aagaccagga caactattac 300
cggggtgaga aaaatgtcag cacgatacgt ctgctccttg gtccgggtct gttgggcacg 360
tatggcggcc cccacaagaa gcaacgcaag atgctgaacc ccgtcttctc gggagcacac 420
atgcgtaatt tgacacccct gttctacgac gtcgccggta gacttcggac cgctctggag 480
tcccgagtcg aggagggtcc caaggacctc gacatcctcg catggatggg acgcaccgcc 540
ctcgagctcg tcgggcgggg tggcctaggc tactccttcg acccacttgt cgctgaatcc 600
cgggacgtgt tcacagagtc cgtcaaatca ttcgtccccg cttccaacga tattttgtgg 660
gtgagagaaa tcaccccgta cctgtcctac ttcggtccag cctggttccg ccgcttgctc 720
ctccaccttg tgcccgtccc gagcatccag cgtatgaaga aaattacgaa catcatgacc 780
aagaggacgg aggagattta ctatgccaag aaggctgcga tccagaccgg ggataaggag 840
tcgttgcacg ccctgggaga aggaaaagac atcatgagcg ttctgttgcg ggagaacgtg 900
aaggcgtctg aagaggacag gctccccgac gaggaagttc ttgctcaaat gggaactttc 960
atcttggccg gtgtcgacac tacttcgaac gccctttcgc gaatcctgca cctcctgtgc 1020
atgaaccaag acgtgcaagg caagctccgc gcggagctgc gtgacgcgca agagcagtac 1080
ggcagtgaga tcccgtacga cgagctttgc gcgctgcctt acctcgacgc cgtctgtcgt 1140
gagacgctcc gtctgtatgc cccggtgaac atcaacggca gacaagcgaa ggtcgacacg 1200
gtcattccgc tttctcagcc tgtacgctgc accgacggta ccgtcctcac ggaggtcccc 1260
gtcccccggg gcacgaacct cttgctcaac ttgcgcgggt gcaacaccaa caaggcgctg 1320
tggggagagg acgcgtggga gtggaaaccg gagcgatggc tcaagcccct gcccaaggcg 1380
gtcgaggacg cacgtatccc cgggatatac gcgaatttga tgactttcat cagcggcgga 1440
aatgcgtgca ttggtttcaa gttctcccaa ctcgaaatga agatcgtcct ctccacgctt 1500
atctccagct tccggttcgg gctctgtccg gagaagccga tcttttggaa cttcgcaggc 1560
atcgcatacc cggtcgcaga ccacgccttt tccaaacccg agatgacctt aaatgtcagc 1620
ctcgtacctc gctga 1635
<210> 2
<211> 544
<212> PRT
<213> P450酶GL19325(Artificial Sequence)
<400> 2
Met Ala Gln Leu Ala Val Leu Val Ala Ser Gly Leu Val Leu Tyr Phe
1 5 10 15
Leu Trp Leu Leu Val Arg Asn Tyr Val Val Lys Ser Pro Leu Asp Lys
20 25 30
Ile Pro Gly Pro Pro Ser Gly Ser Val Leu Ser Gly Asn Met Phe Gln
35 40 45
Leu Phe Asn His Asn Ser Trp Gly Phe Val Asp Asp Leu Ile Gln Thr
50 55 60
Tyr Gly Pro Leu Ser Lys Cys Tyr Gly Phe Phe Gly Thr Arg Met Leu
65 70 75 80
His Ile Tyr Asp Pro Lys Ala Met His Ser Ile Tyr Val Lys Asp Gln
85 90 95
Asp Asn Tyr Tyr Arg Gly Glu Lys Asn Val Ser Thr Ile Arg Leu Leu
100 105 110
Leu Gly Pro Gly Leu Leu Gly Thr Tyr Gly Gly Pro His Lys Lys Gln
115 120 125
Arg Lys Met Leu Asn Pro Val Phe Ser Gly Ala His Met Arg Asn Leu
130 135 140
Thr Pro Leu Phe Tyr Asp Val Ala Gly Arg Leu Arg Thr Ala Leu Glu
145 150 155 160
Ser Arg Val Glu Glu Gly Pro Lys Asp Leu Asp Ile Leu Ala Trp Met
165 170 175
Gly Arg Thr Ala Leu Glu Leu Val Gly Arg Gly Gly Leu Gly Tyr Ser
180 185 190
Phe Asp Pro Leu Val Ala Glu Ser Arg Asp Val Phe Thr Glu Ser Val
195 200 205
Lys Ser Phe Val Pro Ala Ser Asn Asp Ile Leu Trp Val Arg Glu Ile
210 215 220
Thr Pro Tyr Leu Ser Tyr Phe Gly Pro Ala Trp Phe Arg Arg Leu Leu
225 230 235 240
Leu His Leu Val Pro Val Pro Ser Ile Gln Arg Met Lys Lys Ile Thr
245 250 255
Asn Ile Met Thr Lys Arg Thr Glu Glu Ile Tyr Tyr Ala Lys Lys Ala
260 265 270
Ala Ile Gln Thr Gly Asp Lys Glu Ser Leu His Ala Leu Gly Glu Gly
275 280 285
Lys Asp Ile Met Ser Val Leu Leu Arg Glu Asn Val Lys Ala Ser Glu
290 295 300
Glu Asp Arg Leu Pro Asp Glu Glu Val Leu Ala Gln Met Gly Thr Phe
305 310 315 320
Ile Leu Ala Gly Val Asp Thr Thr Ser Asn Ala Leu Ser Arg Ile Leu
325 330 335
His Leu Leu Cys Met Asn Gln Asp Val Gln Gly Lys Leu Arg Ala Glu
340 345 350
Leu Arg Asp Ala Gln Glu Gln Tyr Gly Ser Glu Ile Pro Tyr Asp Glu
355 360 365
Leu Cys Ala Leu Pro Tyr Leu Asp Ala Val Cys Arg Glu Thr Leu Arg
370 375 380
Leu Tyr Ala Pro Val Asn Ile Asn Gly Arg Gln Ala Lys Val Asp Thr
385 390 395 400
Val Ile Pro Leu Ser Gln Pro Val Arg Cys Thr Asp Gly Thr Val Leu
405 410 415
Thr Glu Val Pro Val Pro Arg Gly Thr Asn Leu Leu Leu Asn Leu Arg
420 425 430
Gly Cys Asn Thr Asn Lys Ala Leu Trp Gly Glu Asp Ala Trp Glu Trp
435 440 445
Lys Pro Glu Arg Trp Leu Lys Pro Leu Pro Lys Ala Val Glu Asp Ala
450 455 460
Arg Ile Pro Gly Ile Tyr Ala Asn Leu Met Thr Phe Ile Ser Gly Gly
465 470 475 480
Asn Ala Cys Ile Gly Phe Lys Phe Ser Gln Leu Glu Met Lys Ile Val
485 490 495
Leu Ser Thr Leu Ile Ser Ser Phe Arg Phe Gly Leu Cys Pro Glu Lys
500 505 510
Pro Ile Phe Trp Asn Phe Ala Gly Ile Ala Tyr Pro Val Ala Asp His
515 520 525
Ala Phe Ser Lys Pro Glu Met Thr Leu Asn Val Ser Leu Val Pro Arg
530 535 540
<210> 3
<211> 1509
<212> DNA
<213> P450基因GL15605(Artificial Sequence)
<400> 3
atggaggatg cacagactct gctgtacacg ttggtggctg tcttcgcggc ggtgtatgtc 60
atccgatggc gtacgaaccc gctcaactct atcccgacgg tcggcggacc atcagcccca 120
atactctcgt atttgagcgc gataaagttc ctgctccacc ccagagagct tctcacggaa 180
gggtaccaga agttccacaa ctcggcattc aaggtcgcac ttctcgacca atggctcgtc 240
gttgtgagcg ggtcgaagct cctcgacgaa ttccggaagc gctcggacga cgagctgtcg 300
ttcatcgagg gagttgagga tgtcatccga actcgataca cgctcggtcc agagacgacc 360
catgacccgt accatgtcga catcatcaag gagaagctca tgcgcacgct gcccgcggtg 420
ctgccggacg tcattgacga gctcaactac gcggttccgg actatattcc ggcaactgaa 480
gatggctgga tctccgtgcc tgtcatgaag accatgctga acgttgtagc gcgagtaagc 540
aatcgcgtct tcgttgggct tcccctatgt cgcaaccaga agtacctcga catcgcaatt 600
gcattcacca ttgacatcgt caaagaccgc accatcatca atatctttcc cgacttcatg 660
aaaacgtatg tcgcacggat gaccactaac gtgaagtcga gcatccgccg cgccatcttc 720
cacatcaagc ctctgctcta tgaacgcaag gcaaaggttc tggagaacgg cctcggggag 780
aactggcctg ggaagccgaa cgacatgctc gaatggattc tggagcaggc gattccaaga 840
aatagcagcg accagtctat cacccagaga atcctggtgg tcaacttcgc cgccatccac 900
acctcttctc atagcatgac gcatgcactc tttgatctcg ccgccgcgcc ccagtacttg 960
cagcctctgc gcgaggagat tgagcccatc attgcggcgg agggctggac gaaggcggcg 1020
atgggcaaga tgtggaagct cgacagcttc ctgcgcgagt ctcagcggtt caacggcatc 1080
ggcctcacgt cggtcacacg caaggcaatg aaggacgtca ccctcagcaa cggaaccgtg 1140
atccccaagg gcgcgctcgt cgtcgcagct tcgtacccca cgcaccacga cgacgcgatc 1200
tacgagaacg cgaacacgtt cgaccccttc cgcttctcgc gcatgcgcga ggtcgaaggc 1260
gagggcatca agtatcaatt cgtcaacacg tccctcgact acgtctcctt cggccacggc 1320
aagcatgcat gcccgggtcg gttcttcgcg gcgaacgagc tcaaggcgat cctggcgtac 1380
atcgtggtta actacgacct gaagatcgcc ggggacggcg agcgccccgc caacctgtac 1440
ttcgctgcga gcgtcgtgcc gagcccgaag gggcagatcc tgttcaggaa gcgcagagtg 1500
acggcctga 1509
<210> 4
<211> 502
<212> PRT
<213> P450酶GL15605(Artificial Sequence)
<400> 4
Met Glu Asp Ala Gln Thr Leu Leu Tyr Thr Leu Val Ala Val Phe Ala
1 5 10 15
Ala Val Tyr Val Ile Arg Trp Arg Thr Asn Pro Leu Asn Ser Ile Pro
20 25 30
Thr Val Gly Gly Pro Ser Ala Pro Ile Leu Ser Tyr Leu Ser Ala Ile
35 40 45
Lys Phe Leu Leu His Pro Arg Glu Leu Leu Thr Glu Gly Tyr Gln Lys
50 55 60
Phe His Asn Ser Ala Phe Lys Val Ala Leu Leu Asp Gln Trp Leu Val
65 70 75 80
Val Val Ser Gly Ser Lys Leu Leu Asp Glu Phe Arg Lys Arg Ser Asp
85 90 95
Asp Glu Leu Ser Phe Ile Glu Gly Val Glu Asp Val Ile Arg Thr Arg
100 105 110
Tyr Thr Leu Gly Pro Glu Thr Thr His Asp Pro Tyr His Val Asp Ile
115 120 125
Ile Lys Glu Lys Leu Met Arg Thr Leu Pro Ala Val Leu Pro Asp Val
130 135 140
Ile Asp Glu Leu Asn Tyr Ala Val Pro Asp Tyr Ile Pro Ala Thr Glu
145 150 155 160
Asp Gly Trp Ile Ser Val Pro Val Met Lys Thr Met Leu Asn Val Val
165 170 175
Ala Arg Val Ser Asn Arg Val Phe Val Gly Leu Pro Leu Cys Arg Asn
180 185 190
Gln Lys Tyr Leu Asp Ile Ala Ile Ala Phe Thr Ile Asp Ile Val Lys
195 200 205
Asp Arg Thr Ile Ile Asn Ile Phe Pro Asp Phe Met Lys Thr Tyr Val
210 215 220
Ala Arg Met Thr Thr Asn Val Lys Ser Ser Ile Arg Arg Ala Ile Phe
225 230 235 240
His Ile Lys Pro Leu Leu Tyr Glu Arg Lys Ala Lys Val Leu Glu Asn
245 250 255
Gly Leu Gly Glu Asn Trp Pro Gly Lys Pro Asn Asp Met Leu Glu Trp
260 265 270
Ile Leu Glu Gln Ala Ile Pro Arg Asn Ser Ser Asp Gln Ser Ile Thr
275 280 285
Gln Arg Ile Leu Val Val Asn Phe Ala Ala Ile His Thr Ser Ser His
290 295 300
Ser Met Thr His Ala Leu Phe Asp Leu Ala Ala Ala Pro Gln Tyr Leu
305 310 315 320
Gln Pro Leu Arg Glu Glu Ile Glu Pro Ile Ile Ala Ala Glu Gly Trp
325 330 335
Thr Lys Ala Ala Met Gly Lys Met Trp Lys Leu Asp Ser Phe Leu Arg
340 345 350
Glu Ser Gln Arg Phe Asn Gly Ile Gly Leu Thr Ser Val Thr Arg Lys
355 360 365
Ala Met Lys Asp Val Thr Leu Ser Asn Gly Thr Val Ile Pro Lys Gly
370 375 380
Ala Leu Val Val Ala Ala Ser Tyr Pro Thr His His Asp Asp Ala Ile
385 390 395 400
Tyr Glu Asn Ala Asn Thr Phe Asp Pro Phe Arg Phe Ser Arg Met Arg
405 410 415
Glu Val Glu Gly Glu Gly Ile Lys Tyr Gln Phe Val Asn Thr Ser Leu
420 425 430
Asp Tyr Val Ser Phe Gly His Gly Lys His Ala Cys Pro Gly Arg Phe
435 440 445
Phe Ala Ala Asn Glu Leu Lys Ala Ile Leu Ala Tyr Ile Val Val Asn
450 455 460
Tyr Asp Leu Lys Ile Ala Gly Asp Gly Glu Arg Pro Ala Asn Leu Tyr
465 470 475 480
Phe Ala Ala Ser Val Val Pro Ser Pro Lys Gly Gln Ile Leu Phe Arg
485 490 495
Lys Arg Arg Val Thr Ala
500
<210> 5
<211> 40
<212> DNA
<213> HF-CYP5150L8-F(Artificial Sequence)
<400> 5
ggcaaaggaa taatctcgag tcatgtaatt agttatgtca 40
<210> 6
<211> 40
<212> DNA
<213> HF-CYP5150L8-R(Artificial Sequence)
<400> 6
cgagcggtct aaggcggttt acttctcgta ggaacaattt 40
<210> 7
<211> 20
<212> DNA
<213> HF-CYP5150L8-CX-F(Artificial Sequence)
<400> 7
atttcgatga tgcagcttgg 20
<210> 8
<211> 20
<212> DNA
<213> HF-CYP5150L8-CX-R(Artificial Sequence)
<400> 8
acatcaaaat ccacattctc 20
<210> 9
<211> 40
<212> DNA
<213> GL19325-F(Artificial Sequence)
<400> 9
taattttaat caaaaagttt atggcgcaac tcgcagtgct 40
<210> 10
<211> 40
<212> DNA
<213> GL19325-R(Artificial Sequence)
<400> 10
attaatttga attaacgttt tcagcgaggt acgaggctga 40
<210> 11
<211> 40
<212> DNA
<213> GL15605-F(Artificial Sequence)
<400> 11
taattttaat caaaaagttt atggaggatg cacagactct 40
<210> 12
<211> 40
<212> DNA
<213> GL15605-R(Artificial Sequence)
<400> 12
attaatttga attaacgttt tcaggccgtc actctgcgct 40
<210> 13
<211> 20
<212> DNA
<213> P450-CX-F(Artificial Sequence)
<400> 13
gccaatactt cacaatgttc 20
<210> 14
<211> 20
<212> DNA
<213> P450-CX-R(Artificial Sequence)
<400> 14
tcattttgtc attgaccttc 20
Claims (4)
1.一种细胞色素P450酶在合成灵芝三萜中的应用，其特征在于，通过P450基因作为催化剂，反应生成与天然化合物结构完全一致的灵芝三萜类化合物，并实现其在酿酒酵母中的异源生物合成；
所述的P450基因GL19325的核苷酸序列如Seq ID No.1所示，对应酶的氨基酸序列如Seq ID No.2所示；所述的P450基因GL15605的核苷酸序列如Seq ID No.3所示，对应酶的氨基酸序列如Seq ID No.4所示。
2.根据权利要求1所述的细胞色素P450酶在合成灵芝三萜中的应用，其特征是，所述的应用包括以下任意一种：采用P450酶GL19325催化2,3-环氧角鲨烯形成一种人工线性三萜化合物ST-3，或采用P450酶GL15605催化3-羟基-羊毛甾-8,24-二烯-26-酸(Ganodericacid HLDOA)形成与天然化合物结构完全一致的灵芝酸Y(Ganoderic acid Y)。
3.根据权利要求1或2所述的细胞色素P450酶在合成灵芝三萜中的应用，其特征是，所述的应用，具体为：将P450基因GL19325或GL15605分别克隆到酿酒酵母表达质粒中，即以灵芝cDNA为模板，通过PCR扩增得到各个P450编码区序列片段，通过同源重组的方法将表达载体pRS426、酵母HXT7p启动子、P450编码区序列片段、酵母FBA1t终止子和含有截短启动子Ura3的KanMX基因表达框重组连接，得到一系列重组表达质粒pRS426HF-P450s-G418r；然后将这些质粒分别转入不同的重组改造后的酿酒酵母中进行异源表达，即在基因工程改造后的BY4742菌株YL-T3的基础上，向YL-T3菌株中引入表达质粒pRS425-iGLCPR-Hygr形成的菌株YL-T3-iGLCPR或在YL-T3的基础上引入酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr获得的酿酒酵母菌株YL-T3-CYP5150L8-iGLCPR。
4.根据权利要求3所述的细胞色素P450酶在合成灵芝三萜中的应用，其特征是，所述的酵母表达质粒pRS425-CYP5150L8-iGLCPR-Hygr是指：在酵母表达商业载体pRS425的基础上，通过同源重组的方法将pRS425、TEF1p启动子、GL19526基因、PGK1t终止子、含有截短启动子Ura3的Hygromycin B基因表达框和CYP5150L8表达框重组连接，得到重组表达质粒pRS425-CYP5150L8-iGLCPR-Hygr或在已经构建好的质粒pRS425-iGLCPR-Hygr的基础上引入CYP5150L8表达框。
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