CN114854727A - 生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用 - Google Patents
生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用 Download PDFInfo
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- CN114854727A CN114854727A CN202210466358.8A CN202210466358A CN114854727A CN 114854727 A CN114854727 A CN 114854727A CN 202210466358 A CN202210466358 A CN 202210466358A CN 114854727 A CN114854727 A CN 114854727A
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Abstract
本发明公开了一种生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用。本发明所述的酶专一性合成protosta‑13(17),24‑dien‑3β‑ol，克服了缺少高效、专一合成这类原萜烷的酶的问题，属国际首例报道合成植物原萜烷的酶，并且明确其727位置关键苏氨酸为其催化活性的必须氨基酸。本发明同时也明确可以通过突变其他类型C‑B‑C四环三萜环化酶如环阿屯醇合酶、葫芦二烯醇合成酶、羊毛甾醇合酶、帕克醇合酶，相应位置的苯丙氨酸突变为苏氨酸或天冬酰胺，均能产生原萜烷三萜。本发明具有普遍意义，可以适用于上述各类型环化酶。
Description
技术领域
本发明涉及生物技术领域，尤其涉及生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用。
背景技术
中药泽泻(Alisma orientale(Sam.)Juzep.)，2020版《中国药典》收录的品种是泽泻科植物东方泽泻，习称“建泽泻”的干燥块茎，是一种常用的利水渗湿药。其性寒，味甘、淡，归肾、膀胱经；具有利水渗湿，泄热，化浊降脂之功效。常用于小便不利，水肿胀满，痰饮眩晕，热淋涩痛和高脂血症。
泽泻作为经典的利水渗湿药，其利尿活性研究较多，有研究表明泽泻总三萜可显著增加盐水负荷大鼠的尿量及Na+，K+，Cl－的排泄，总三萜中的原萜烷型四环三萜类成分泽泻醇A、24-乙酰泽泻醇 A、泽泻醇B、23-乙酰泽泻醇B、23-乙酰泽泻醇C的组合化合物也具有同样的作用，证明原萜烷型四环三萜类成分为主要利尿成分。研究发现，24-乙酰泽泻醇A、23-乙酰泽泻醇B(0.64mg·kg－1)有良好的降血脂作用，能显著减少高脂饮食大鼠血清TC，TG和LDL-C水平，升高HDL-C浓度，促进肝脏分泌apo B，在体内外可剂量依赖性地降低HMG-CoA还原酶活性。一些原萜烷型四环三萜类成分如泽泻醇A、24-乙酰泽泻醇A、23-乙酰泽泻醇B、23-乙酰泽泻醇C等还能增加细胞对葡萄糖的摄入以达到降血糖的效果。值得一提的是，泽泻醇B、23-乙酰泽泻醇B的抗癌活性研究较广泛，不仅可时间及剂量依赖性抑制多种癌细胞株的增殖，还可逆转耐药细胞株对抗癌药物如长春花碱和紫杉醇的敏感性，是潜在的逆转耐药细胞反转物质。
三萜骨架都是由一类2，3-氧化角鲨烯环化酶(OSCs)环化产生的，在胞质中发生的甲戊二羟酸 (Mevalonic Acid，MVA)是植物甾醇、倍半萜和三萜类化合物的主要生物合成途径。其以2，3-氧化角鲨烯为底物，在复杂多样的环化途径支配下，形成了三萜类化合物复杂多样的结构，其中少数为链状、单环、双环和三环三萜。植物三萜OSCs催化的生物合成过程，可根据ABC环骨架的构象 (船式或椅式)，主要分为椅-船-椅(Chair-Boat-Chair，C-B-C)和椅-椅-椅(Chair-Chair-Chair，C-C-C) 两大反应途径。C-C-C反应途径需要经过中间态达玛烯基阳离子(Dammarenyl cation)，而C-B-C反应途径则经过原甾醇阳离子(Protosterol cation)中间体。常见的C-B-C型三萜环化酶有：环阿屯醇合成酶(cycloartenol synthase，CAS)、葫芦二烯醇合成酶(cucurbitadienol synthase，CBQ)、羊毛甾醇合酶(lanosterol synthase，LAS)、帕克醇合酶(Parkeol synthase，PKS)、和原萜烷合酶(protostane synthase，PDS)。
原萜烷型四环三萜骨架结构较特殊，目前仅在真菌烟曲霉中发现一例AfPDS基因产生两种不同构型的原萜烷三萜protosta-13(17),24-dien-3β-ol(95％)，protosta-17(20),24-dien-3β-ol(5％)。而这种骨架是合成经典抗生素夫西地酸、烟曲霉酸和头孢菌素P1骨架——缩链孢烷骨架的重要前体母核，已有研究表明该骨架可通过11步修饰生成夫西地酸和烟曲霉酸。夫西地酸作为一种典型的高效抗生素，具有其他任何一种抗生素都没有的抑菌杀菌和抗炎免疫调节的双重作用。目前已被广泛使用了近 40年，却仍对绝大多数葡萄球菌菌株保持较高的抗菌活性和很低的耐药率，因此成为抗甲氧西林耐药性金黄色葡萄球菌(MRSA)新的选择。其可通过干扰延长因子G，阻断核糖体的易位，从而阻碍细菌蛋白质的合成这种独特的抗菌机理避免了与其他抗生素的交叉耐药性，使之能有效抵抗MRSA 感染。对包括耐甲氧西林表皮葡萄球菌(MRSE)和MRSA引起的各种感染，如败血症、肺炎、脑膜炎、心内膜炎、骨与关节感染、烧伤感染、外科以及创伤感染、皮肤感染等具有良好的疗效。
然而植物中的原萜烷，如泽泻中的原萜烷主要以protosta-13(17),24-dien-3β-ol作为母核，而不是真菌中的protosta-17(20),24-dien-3β-ol。植物原萜烷主要分布在泽泻科的植物中，是泽泻科的特征性化学分类成分。目前尚未报道主要合成植物中protosta-13(17),24-dien-3β-ol的酶，从而限制了泽泻原萜烷的推广和应用。
发明内容
本发明的目的在于克服现有技术的缺点与不足，提供一种生产原萜烷三萜的三萜环化酶。
本发明的另一目的在于提供上述生产原萜烷三萜的三萜环化酶的编码序列。
本发明的另一目的在于提供上述生产原萜烷三萜的三萜环化酶的表达载体。
本发明的另一目的在于提供上述生产原萜烷三萜的三萜环化酶的应用。
本发明的目的通过下述技术方案实现：
一种生产原萜烷三萜的三萜环化酶，能够合成原萜烷型三萜，是如下酶中的至少一种：
(1)氨基酸如SEQ ID No:2所示的AoPDS酶，由760个氨基酸残基组成，其特征在于编码第 727个氨基酸为苏氨酸(Thr，T)；
(2)将环阿屯醇合酶(CAS)的氨基酸序列中序列为“FNRNCMI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(3)将葫芦二烯醇合成酶(CBQ)的氨基酸序列中序列为“FNKNCMI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(4)将羊毛甾醇合酶(LAS)的氨基酸序列中序列为“FNKNVAI”或“FNKSCAI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(5)将帕克醇合酶(PKS)的氨基酸序列中序列为“FLQTAMI”或“FNKSCAI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶。
上述的生产原萜烷三萜的三萜环化酶，是如下酶的至少一种：
(1)将泽泻(Alisma orientale(Sam.)Juzep)环阿屯醇合酶(AoCAS)第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(AoCAS-F726T/N)；
(2)将鸢尾(Iris tectorum Maxim.)环阿屯醇合酶(ItOSC3)第728位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(ItOSC3-F728T/N)；
(3)将番茄(Solanum lycopersicum)环阿屯醇合酶(SlCAS)第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(SlCAS-F726T/N)；
(4)将罗汉果(Siraitia grosvenorii)葫芦二烯醇合成酶(SgCBQ)第729位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(SgCBQ-F729T/N)；
(5)将黄瓜(Cucumis sativus)葫芦二烯醇合成酶(CsCBQ)第755位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(CsCBQ-F755T/N)；
(6)将西葫芦(Cucurbita pepo)葫芦二烯醇合成酶(CpCBQ)第734位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(CpCBQ-F734T/N)；
(7)将酿酒酵母(Saccharomyces cerevisiae)羊毛甾醇合酶(ERG7)第691位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(ERG7-F691T/N)；
(8)将人(Homo sapiens)羊毛甾醇合酶(HsLAS)第696位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(HsLAS-F696T/N)；
(9)将灵芝(Ganoderma lucidum)羊毛甾醇合酶(GlLAS)第692位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(GlLAS-F692T/N)；
(10)将水稻(Oryza sativa)帕克醇合酶(OsOSC7)第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶(OsOSC7-F726T/N)。
将上述生产原萜烷三萜的三萜环化酶的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有合成原萜烷型三萜功能的酶；
所述的取代和/或缺失和/或添加具体为1至10个氨基酸残基的取代和/或缺失和/或添加。
所述的酶为人工合成或先合成其编码基因，再进行生物表达得到。
所述的AoPDS酶的编码基因如SEQ ID No:1所示。
上述生产原萜烷三萜的三萜环化酶的编码基因，
1)核苷酸序列依据密码子编码规则得到；
2)在严格条件下与1)限定的核苷酸序列杂交且编码所述蛋白的DNA分子；
3)与1)或2)限定的核苷酸序列具有90％以上的同源性，并且对齐后的727位置为特征性的苏氨酸，且编码相同功能蛋白质分子的DNA分子。
上述生产原萜烷三萜的三萜环化酶的编码基因，通过将序列表中的自5’端第1至末位碱基所示的 DNA序列中缺失一个或者几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/ 或在其5’端和/或3’端连上纯化标签得到。
本发明还保护用于扩增所述基因或所述基因中的任一DNA片段的引物对。
一种重组表达载体，含有上述生产原萜烷三萜的三萜环化酶的编码基因。
所述的重组表达载体为使用现有的表达载体构建。
所述的重组表达载体在构建时，在其转录起始核苷酸前加上任何一种增强型启动子或组成型启动子；此外，还使用增强子，包括翻译增强子或转录增强子，这些增强子区域是ATG起始密码子或邻接区域起始密码子，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译；所述翻译控制信号和起始密码子的来源是天然的或是合成的；翻译起始区域为来自转录起始区域或结构基因。
所述的重组表达载体为pESC-URA。
一种转基因细胞系，通过将所述生产原萜烷三萜的三萜环化酶的编码基因导入细胞得到，具体为将所述重组表达载体导入细胞得到，优选为将重组表达载体导入酿酒酵母细胞得到。
含有所述编码基因构建的重组表达载体、表达盒、转基因细胞系、转基因植株或重组菌均属于本发明的保护范围。
上述三萜环化酶突变体的构建方法，使用环阿屯醇合酶(CAS)、葫芦二烯醇合成酶(CBQ)、羊毛甾醇合酶(LAS)、帕克醇合酶(LAS)(具体为：AoCAS、ItOSC3、SlCAS，SgCBQ、CsCBQ，CpCBQ， ERG7、HsLAS，GsLAS，AjLAS，OsOSC7)构建突变表达载体时，将其相对于AoPDS的727位苏氨酸的苯丙氨酸(Phe，F)(分别是AoCAS-F726、ItOSC3-F728、SlCAS-F726，SgCBQ-F728、 CsCBQ-F755，CpCBQ-F734，ERG7-F699、HsLAS-F696，GlLAS-F692，OsOSC7-F726)替换为苏氨酸(Thr，T)，天冬酰胺(Asn，N)。
所述的转基因细胞系可通过将所述基因(AoPDS)及突变体基因(AoCAS-F726T/N、ItOSC3-F728T/N、SlCAS-F726T/N，SgCBQ-F728T/N、CsCBQ-F755T/N，CpCBQ-F734T/N， ERG7-F699T/N、HsLAS-F696T/N，GlLAS-F692T/N，AjLAS-F691T/N，OsOSC7-F726T/N)导入酵母菌，再由酵母菌表达得到产物。编码AoPDS、AoCAS-F726T、ItOSC3-F728T、SlCAS-F726T， SgCBQ-F728T、CsCPQ-F755T，CpCPQ-F734T，ERG7-F699T、HsLAS-F696T，GlLAS-F692T， AjLAS-F691T，OsOSC7-F726T的酶可产生protosta-13(17),24-dien-3β-ol；而编码AoCAS-F726N、ItOSC3-F728N、SlCAS-F726N，SgCBQ-F728N、CsCBQ-F755N，CpCBQ-F734N，ERG7-F699N、HsLAS-F696N，GlLAS-F692N，OsOSC7-F726N的酶可产生protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol。重组所用到的生物包括原核或真核宿主(例如：细菌、酵母、高等植物、昆虫和哺乳动物细胞)。
所述的原萜烷三萜为含有四环三萜骨架的三萜类化合物，优选为ABC环骨架的构象为C-B-C(椅 -船-椅)或C-C-C(椅-椅-椅)的三萜化合物，更优选为protosta-13(17),24-dien-3β-ol、 protosta-17(20),24-dien-3β-ol中的至少一种。
一种生产原萜烷三萜protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的方法，构建和培养包括环阿屯醇合酶(CAS)、葫芦二烯醇合成酶(CBQ)、羊毛甾醇合酶(LAS)、帕克醇合酶(LAS) 在内的突变体转基因细胞或突变体转基因植株，得到protosta-13(17),24-dien-3β-ol和 protosta-17(20),24-dien-3β-ol。
本发明相对于现有技术具有如下的优点及效果：
本发明提供了一种生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用，能够表达专一性产生原萜烷型三萜protosta-13(17),24-dien-3β-ol及其合成酶，属国际首例。本发明所述植物来源的 AoPDS酶专一性合成protosta-13(17),24-dien-3β-ol，克服了缺少高效、专一合成这类原萜烷的酶的问题，AoPDS酶属国际首例报道合成植物原萜烷的酶，并且明确其727位置关键苏氨酸为其催化活性的必须氨基酸。本发明同时也明确可以通过突变其他类型C-B-C四环三萜环化酶如环阿屯醇合酶 (CAS)、葫芦二烯醇合成酶(CBQ)、羊毛甾醇合酶(LAS)、帕克醇合酶，相应位置的苯丙氨酸突变为苏氨酸或天冬酰胺，均能产生原萜烷三萜。本发明具有普遍意义，可以适用于上述各类型环化酶。
本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法包含自然存在的AoPDS蛋白产生，也包含人工改造环阿屯醇合酶类(CAS)、葫芦二烯醇合成酶类(CBQ)、羊毛甾醇合酶类(LAS)、帕克醇合酶类(PKS)产生。拓展了上述两类化合物的生物合成来源，属首次发现。
以下的实施便于更好地理解本发明，但不限定本发明。
附图说明
图1是pESC-URA-AoPDS表达载体的物理图谱图。
图2是已报道的环阿屯醇合酶类(CAS)、葫芦二烯醇合成酶类(CBQ)、羊毛甾醇合酶类(LAS)、帕克醇合酶类(PKS)蛋白序列在本发明所述氨基酸残基位置的保守性对比图。
图3是基因AoPDS(a)、AoCAS-F726T/N(b)、ItOSC3-F728T/N(c)、SlCAS-F726T/N(d)， SgCBQ-F728T/N(e)、CsCBQ-F755T/N(f)，CpCBQ-F734T/N(g)，ERG7-F699T/N(h)、HsLAS-F696T/N(i)， GlLAS-F692T/N(j)，OsOSC7-F726T/N(k)转入酵母转化细胞表达得到的产物的GC谱图。
图4是本发明所述生物合成方法产生的两种原萜烷三萜的MS谱图，其中1为protosta-13(17),24-dien-3β-ol；3为protosta-17(20),24-dien-3β-ol。
图5是protosta-13(17),24-dien-3β-ol、protosta-17(20),24-dien-3β-ol、环阿屯醇(cycloartenol)、葫芦二烯醇(cucurbitadienol)、羊毛甾醇(lanosterol)、帕克醇(parkeol)的结构图。
图6是本发明所述生物合成方法产生两种原萜烷三萜的示意图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下面实施方案中若未注明具体试验条件，则通常按照常规试验条件或按照试剂公司所建议的试验条件。所使用的的材料、试剂等，若无特殊说明，均为从商业途径得到的试剂和材料。
实施例1.重组酵母表达载体pESC-URA-AoPDS的构建
从东方泽泻(Alisma orientale(Sam.)Juzep.)植物(购自福建建瓯市)中克隆AoPDS基因(序列见SEQ ID No:1)，引物如下。该引物包括该候选基因的一段序列，以及pESC-URA载体上的一段同源臂序列，小写字母表示载体同源序列，大写字母表示基因的序列。
5’-端引物：pESC-URA-AoPDS-F
5’-cgtcaaggagaaaaaaccccggatccATGTGGAGGCTGAAGATTGCGGACG-3’
3’-端引物：pESC-URA-AoPDS-R
3’-acttctgttccatgtcgacgcccgggctaTTTGAGGGACTGTAAAACCCTG-5’
以总RNA反转录的cDNA为模板，用上述两个引物对分别进行PCR扩增，具体步骤如下：
1、提取东方泽泻总RNA
收集100mg洗净的东方泽泻叶片材料，立即置于液氮中研磨成粉，应用北京华越洋生物科技有限公司的快速通用植物RNA提取试剂盒3.0(产品编号：0416-50)并按试剂盒附带操作说明书执行操作，得到东方泽泻叶片总RNA。
2、反转录获得总的cDNA
取5μg步骤1中提取的东方泽泻叶片总RNA放入无RNase的PCR管中，应用Takara公司的 PrimeScriptTM1st Strand cDNA Synthesis Kit试剂盒(货号：6210A)并按试剂盒说明书进行操作，依次加入各体系，加入量与说明书一致，得到反转录产物第一链cDNA。
3、泽泻AoPDS基因的克隆与纯化
应用Vazyme公司的Phanta Max Super-Fidelity DNA Polymerase试剂盒(货号：P505-d3)，取2μL 步骤2所得反转录产物，在5’-端引物和3’-端引物的引导下进行PCR。PCR反应体系为：25μL 2×Phanta Max Buffer、1μL dNTP Mix(10mM)、1μL 5’-端引物(10μM)、1μL 3’-端引物(10μM)、2μL模板DNA、1μL Phanta Max Super-Fidelity DNA Polymerase、加19μL ddH2O至50μL体系后吹打混匀； PCR反应条件为：95℃3分钟；95℃15秒，57℃15秒，72℃1分20秒，33个循环；72℃5分钟。反应结束后用水平凝胶电泳检测。结果表明，克隆产物长度约为2.3kbp，与泽泻AoPDS基因大小相同，得到泽泻AoPDS基因。使用PCR产物回收试剂盒(北京天根)纯化该片段得到纯化后的泽泻 AoPDS基因。
4、空载体pESC-URA质粒的线性化
用限制性内切酶BamHⅠ，SmaⅠ(赛默飞公司)对pESC-URA进行双酶切。酶切体系为：2μL Buffer、 0.5μL BamHⅠ、0.5μL Sma I、250ng pESC-URA、ddH2O补至10μL后吹打混匀；反应条件为：37℃， 45分钟。85℃，5分钟失活，得到线性化空载体pESC-URA双酶切产物。
5、构建重组酵母表达载体pESC-URA-AoPDS
1)应用诺唯赞公司的
II One Step Cloning Kit试剂盒(货号：C112-01)使用同源重组法将步骤3中基因片段与步骤4中线性化载体相连。连接体系为：1μL步骤4中得到的线性化 pESC-URA空载体、1μL步骤3中得到的纯化后的泽泻AoPDS基因、2μL5×CEⅡBuffer、1μL Exnase Ⅱ、加5μL ddH2O补充至10μL体系，吹打混匀，短暂离心；37℃温育30分钟，得到连接产物。
2)使用热击转化法，将连接产物转化至Trans5αChemically Competent Cell大肠杆菌化学感受态细胞(购自全式金，货号：CD201-01)，在含有氨苄霉素(浓度为100μg/mL)的LB固体培养基上根据pESC-URA载体上的氨苄霉素抗性标记筛选阳性克隆，得到阳性克隆。
3)将阳性克隆进行PCR鉴定，引物如下：
GAL1-F 5’-AGTACGGATTAGAAGCCGCCG-3’
GAL1-R 5’-GGGTTTTTTCTCCTTGACGTTA-3’
结果表明，AoPDS基因插入了pESC-URA，且方向正确，将该重组质粒命名为pESC-URA-AoPDS。测序结果表明该序列由2280个碱基组成，其开放阅读框(ORF)为序列表中SEQID No:1所示的自 5′末端第1-2280位脱氧核糖核苷酸，编码得到氨基酸序列为序列表SEQID No:2所示的蛋白质。将 SEQ ID No:1所示的DNA序列命名为AoPDS，将SEQ ID No:2所示的氨基酸序列命名为AoPDS，将在pESC-URA中插入SEQ ID No:1所述的核苷酸序列得到的重组载体命名为pESC-URA-AoPDS，如图1所示。
实施例2.pESC-URA-AoPDS重组酿酒酵母工程菌细胞系的转化与表达
1、醋酸锂法转化酵母菌CEN.PK2-1D
取少量CEN.PK2-1D甘油菌(购自北京科瑞思搏公司，货号：ST11663)划线接种于YPD固体培养基上30℃培养至形成单克隆菌落。挑取一个单克隆菌落于2mL YPD培养基中30℃，200rpm培养至浑浊；按1％比例接种于5mL YPD培养基中(每100mL YPD培养基由蛋白胨2g、酵母提取物 1g，2g葡萄糖溶解于100mL ddH2O中灭菌获得)培养至OD600为1.0左右，加入1mL无菌水吹打悬浮后离心弃上清；加入灭菌的200μL 100mmol/L醋酸锂溶液吹打悬浮菌体，13000rpm离心15秒后弃上清，制备成CEN.PK2-1D酵母感受态细胞；加入44μL无菌水轻柔悬浮菌体，再分别加入240μl 无菌50％聚乙二醇溶液(PEG3350)，36μl无菌1M醋酸锂溶液(LiAc)，20μl ssDNA(鲑鱼精DNA，购自索莱宝)，取实施例1步骤5中获得的重组质粒pESC-URA-AoPDS 500ng吹打混匀，30℃静置30 分钟后42℃水浴锅中热激25分钟；5000rpm，5分钟离心收集菌体，弃去上清后加入1mL YPD培养基，30℃培养2h；5000rpm，3分钟离心收集菌体，用等体积无菌水洗涤一次。涂布于SD-URA氨基酸缺陷型平板(由凝固后的SD酵母生长液体培养基获得，其中每100mL的SD酵母生长液体培养基由0.68g YNB、100×缺URA的氨基酸母液1mL，ddH2O定容至90mL组成、余量ddH2O溶解2g 葡萄糖，分别灭菌后混合获得。液体培养基中加入2g琼脂粉，加热溶解摇匀后铺于平板上冷却获得氨基酸缺陷型平板)进行筛选；30℃培养2～3天后，获得pESC-URA-AoPDS重组酿酒酵母工程菌细胞系。
2、将阳性克隆用基因特异性引物对进行PCR鉴定
对挑取的阳性克隆使用0.3M NaOH溶液裂解，裂解后混合液作为PCR模板进行扩增。PCR条件同实施例1步骤3，引物使用实施例1中的pESC-URA-AoPDS-F和pESC-URA-AoPDS-R。结果表明， pESC-URA-AoPDS成功的转入了表达载体酵母，获得pESC-URA-AoPDS酵母表达细胞系。
3、pESC-URA-AoPDS重组酿酒酵母工程菌细胞系的表达
挑步骤1中的阳性克隆子至SD-URA氨基酸缺陷型平板划线培养2～3天；将已活化的菌落挑取至50mL SG诱导URA氨基酸缺陷型培养基(含0.34g YNB、100×缺URA的氨基酸母液0.5mL、0.9 g半乳糖和0.1g葡萄糖)中，30℃，200rpm培养6天，得到含有表达产物的重组酿酒酵母菌液。
4、pESC-URA-AoPDS重组酿酒酵母工程菌细胞系表达产物的提取
取步骤3中得到的含有表达产物的全部重组酿酒酵母菌液4000rpm，10min收集菌体于试管；在菌体中加入10mL碱裂解液(50％乙醇作为溶剂配制的20％氢氧化钾乙醇混合液)，涡旋振荡1min，使菌体完全重悬与裂解液混合；将试管置于沸水中沸腾5min后室温静置冷却；加入等体积正己烷，振荡2min，吸取有机层(上层)至西林瓶；重复向水层(下层)中加入等体积正己烷，振荡后取有机层，合并两次的萃取液；过夜挥干后，即可获得pESC-URA-AoPDS重组酿酒酵母的全部表达产物。
5、pESC-URA-AoPDS重组酿酒酵母工程菌细胞系表达产物的检测
用1mL乙酸乙酯将步骤4中样品转移至1.5mL离心管，高速离心15min后取50μL至内插管放入离心浓缩仪中挥干；转至进样瓶后加入25μL四甲基硅烷TMS和25μL无水吡啶，85℃，700rpm，衍生化20分钟，衍生化结束后的样品用GC-MS平台进行分析，结果如图3a所示。由结果可知， pESC-URA-AoPDS重组酿酒酵母工程菌细胞系产生专一性的protosta-13(17),24-dien-3β-ol，且产量较高。这说明本发明所述的生产原萜烷三萜的三萜环化酶AoPDS是具备高效性和专一性的产生植物原萜烷型三萜protosta-13(17),24-dien-3β-ol的酶。
实施例3.东方泽泻(Alisma orientale(Sam.)Juzep)环阿屯醇合酶(AoCAS)突变体 (AoCAS-F726T/N)重组酿酒酵母工程菌细胞系的构建与表达
一、pESC-URA-AoCAS-F726T/N突变体构建
突变重组载体构建方法采用全质粒克隆方法。根据公开的AoCAS基因(ID:ANG59904)，由商业公司合成核苷酸序列并构建pESC-URA-AoCAS重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-AoCAS-F726T/N，筛选阳性重组子测序验证后扩繁提取供酵母转化的稳定重组质粒，具体步骤如下：
1、选择已经将泽泻环阿屯醇基因(AoCAS，ID：KU856664.1)构建到表达载体上的pESC-URA-AoCAS质粒(具体构建步骤参照实施例1)，突变其氨基酸序列的726位置的苯丙氨酸为苏氨酸或天冬酰胺。
设计突变726位置的苯丙氨酸为苏氨酸(T)全质粒突变引物如下：
5’-端引物：pESC-URA-AoCAS-F726T-F
5’-GAAATAATGGGGGTAACCAACAGAAATTGCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-AoCAS-F726T-R
3’-CATGCAATTTCTGTTGGTTACCCCCATTATTTC-5’
设计突变726位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-AoCAS-F726N-F
5’-GAAATAATGGGGGTAAACAACAGAAATTGCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-AoCAS-F726N-R
3’-CATGCAATTTCTGTTGTTTACCCCCATTATTTC-5’
应用Vazyme公司的Phanta Max Super-Fidelity DNA Polymerase试剂盒(货号：P505-d3)并按试剂盒说明书进行操作：在突变726位置的苯丙氨酸为苏氨酸(T)或天冬酰胺(N)的各自5’-端引物和3’-端引物的引导下，进行PCR。PCR反应体系为：25μL 2×PhantaMax Buffer、1μL dNTP Mix(10 mM)、2μL 5’-端引物(10μM)、2μL 3’-端引物(10μM)、2μL模板质粒稀释液、1μL Phanta Max Super-Fidelity DNA Polymerase、加17μL ddH2O至50μL体系，并轻弹混匀；PCR反应条件为：95℃ 3分钟；95℃15秒，57℃30秒，72℃4分钟，此过程设置20个循环；最后72℃10分钟。反应结束后用1％琼脂糖凝胶检测。使用DpnI(赛默飞公司)在37℃下消化亲代甲基化DNA 1小时，85℃失活。获得pESC-URA-AoCAS-F726T/N重组载体突变产物。
2、使用热击转化法及筛选突变成功的重组子，操作同实施例1，获得构建成功的突变体质粒。
二、pESC-URA-AoCAS-F726T/N突变体重组酿酒酵母工程菌细胞系的转化与表达
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3b所示，在pESC-URA-AoCAS中只在18.60分钟左右检测到环阿屯醇(P2)；在pESC-URA-AoCAS-F726T中，在16.20分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-AoCAS-F726N中，在16.20分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在17.60分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，也可以由人工改造环阿屯醇合酶类(CAS)中的东方泽泻AoCAS产生。
实施例4.鸢尾(Iris tectorum Maxim.)环阿屯醇合酶(ItOSC3 ID:QQR13796)(突变体 (ItOSC3-F728T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的ItOSC3基因(ID:MT104570)，由商业公司合成核苷酸序列并构建pESC-URA-ItOSC3重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-ItOSC3-F728T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变728位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-ItOSC3-F728T-F
5’-GAAATTATGGGAGTCACCAACAGAAACTGCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-ItOSC3-F728T-R
3’-CATGCAATTTCTGTTGGTTACCCCCATTATTTC-5’
设计突变728位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-ItOSC3-F728N-F
5’-GAAATTATGGGAGTCAACAACAGAAACTGCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-ItOSC3-F728N-R
3’-CATGCAGTTTCTGTTGTTGACTCCCATAATTTC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3c所示，在pESC-URA-ItOSC3中只在18.50分钟左右检测到环阿屯醇(P2)；在pESC-URA-ItOSC3-F728T中，在14.00分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-ItOSC3-F728N中，在14.00分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在17.00分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，还可以由人工改造环阿屯醇合酶类(CAS)中的鸢尾ItOSC3产生。
实施例5.番茄(Solanum lycopersicum)环阿屯醇合酶(SlCAS ID:NP_001233784)突变体 (SlCAS-F726T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的SlCAS基因(ID:NM_001246855.2)，由商业公司合成核苷酸序列并构建pESC-URA-SlCAS重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-SlCAS-F726T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变726位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-SlCAS-F726T-F
5’-GGAGATCATGGGAGTCACCAATAGGAACTGCATGA-3’
3’-端引物为上述引物反向互补得到：pESC-URA-SlCAS-F726T-R
3’-TCATGCAGTTCCTATTGGTGACTCCCATGATCTCC-5’
设计突变726位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-SlCAS-F726N-F
5’-GGAGATCATGGGAGTCAACAATAGGAACTGCATGA-3’
3’-端引物为上述引物反向互补得到：pESC-URA-SlCAS-F726N-R
3’-TCATGCAGTTCCTATTGTTGACTCCCATGATCTCC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3d所示，在pESC-URA-SlCAS中只在18.70分钟左右检测到环阿屯醇(P2)；在pESC-URA-SlCAS-F726T中，在14.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-ItOSC3-F728N中，在14.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在17.20分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，还可以由人工改造环阿屯醇合酶类(CAS)中的番茄SlCAS产生。
实施例6.罗汉果(Siraitia grosvenorii)葫芦二烯醇合酶(SgCBQ ID：K7NBZ9)突变体 (SgCBQ-F729T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的SgCBQ基因(ID:MF596154.1)，由商业公司合成核苷酸序列并构建pESC-URA-SgCBQ重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-SgCBQ-F729T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变729位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-SgCBQ-F729T-F
5’-GGAGATCATGGGAGTCACTAATAAAAATTGC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-SgCBQ-F729T-R
3’-GCAATTTTTATTAGTGACTCCCATGATCTCC-5’
设计突变729位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-SgCBQ-F729N-F
5’-GGAGATCATGGGAGTCAATAATAAAAATTGC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-SgCBQ-F729N-R
3’-GCAATTTTTATTATTGACTCCCATGATCTCC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3e所示，在pESC-URA-SgCBQ中只在13.30分钟左右检测到葫芦二烯醇(P4)；在pESC-URA-SgCBQ-F729T中，在12.30分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-SgCBQ-F729N中，在12.30分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在14.70分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，还可以由人工改造葫芦二烯醇合酶类(CBQ)中的罗汉果SgCBQ产生。
实施例7.黄瓜(Cucumis sativus)葫芦二烯醇合酶(CsCBQ ID:NP_001292630)突变体 (CsCBQ-F755T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的CsCBQ基因(ID:NM_001305701.1)，由商业公司合成核苷酸序列并构建pESC-URA-CsCBQ重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-CsCBQ-F755T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变755位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-CsCBQ-F755T-F
5’-GGAGATCATGGGAGTGACCAACAAAAATTGTATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-CsCBQ-F755T-R
3’-CATACAATTTTTGTTGGTCACTCCCATGATCTCC-5’
设计突变755位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-CsCBQ-F755N-F
5’-GGAGATCATGGGAGTGAACAACAAAAATTGTATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-CsCBQ-F755N-R
3’-GGAGATCATGGGAGTGAACAACAAAAATTGTATG-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3f所示，在pESC-URA-CsCBQ中只在13.50分钟左右检测到葫芦二烯醇(P4)；在pESC-URA-CsCBQ-F755T中，在12.30分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-SgCBQ-F729N中，在12.30分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在15.00分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，还可以由人工改造葫芦二烯醇合酶类(CBQ)中的黄瓜CsCBQ产生。
实施例8.西葫芦(Cucurbita pepo)葫芦二烯醇合酶(CpCBQ ID:XP_022992031)突变体(CpCBQ-F734T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的CpCBQ基因(ID:AB116238.1)，由商业公司合成核苷酸序列并构建pESC-URA-CpCBQ重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-CpCBQ-F734T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变734位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-CpCBQ-F734T-F
5’-GGAGATCATGGGAGTGACCAATAAGAACTGCATGATC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-CpCBQ-F734T-R
3’-GATCATGCAGTTCTTATTGGTCACTCCCATGATCTCC-5’
设计突变734位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-CpCBQ-F734N-F
5’-GGAGATCATGGGAGTGAACAATAAGAACTGCATGATC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-CpCBQ-F734N-R
3’-GATCATGCAGTTCTTATTGTTCACTCCCATGATCTCC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3g所示，在pESC-URA-CpCBQ中只在12.50分钟左右检测到葫芦二烯醇(P4)；在pESC-URA-CpCBQ-F734T中，在10.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-CpCBQ-F734N中，在10.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在13.00分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17)，24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的AoPDS蛋白产生外，还可以由人工改造葫芦二烯醇合酶类(CBQ)中的西葫芦CpCBQ产生。
实施例9.酿酒酵母(Saccharomyces cerevisiae)羊毛甾醇合酶(ERG7 ID:KAF1903435)突变体 (ERG7-F691T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的ERG7基因(ID:U41368.1)，由商业公司合成核苷酸序列并构建pESC-URA-ERG7重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-ERG7-F691T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变691位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-ERG7-F691T-F
5’-CGATGGAAGGGATCACCAACAAGAATGTTGCC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-ERG7-F691T-R
3’-GGCAACATTCTTGTTGGTGATCCCTTCCATCG-5’
设计突变691位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-ERG7-F691N-F
5’-CGATGGAAGGGATCAACAACAAGAATGTTGCC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-ERG7-F691N-R
3’-GGCAACATTCTTGTTGTTGATCCCTTCCATCG-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3h所示，在pESC-URA-ERG7中只在17.00分钟左右检测到羊毛甾醇(P5)；在pESC-URA-ERG7-F691T中，在14.15分钟左右检测到protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-ERG7-F691N中，在14.15分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在16.60分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17)，24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的AoPDS蛋白产生外，还可以由人工改造羊毛甾醇合酶类(LAS)中的酿酒酵母ERG7产生。
实施例10.人(Homo sapiens)羊毛甾醇合酶(HsLAS ID:BAA09875)突变体(HsLAS-F696T/N) 重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的HsLAS基因(ID:D63807.1)，由商业公司合成核苷酸序列并构建pESC-URA-HsLAS重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-HsLAS-F696T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变696位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-HsLAS-F696T-F
5’-GGAAAACATTGCTGGGGTCACCAACAAGTCCTGTGCCATC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-HsLAS-F696T-R
3’-GATGGCACAGGACTTGTTGGTGACCCCAGCAATGTTTTCC-5’
设计突变696位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-HsLAS-F696N-F
5’-GGAAAACATTGCTGGGGTCAACAACAAGTCCTGTGCCATC-3’
3’-端引物为上述引物反向互补得到：pESC-URA-HsLAS-F696N-R
3’-GATGGCACAGGACTTGTTGTTGACCCCAGCAATGTTTTCC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3i所示，在pESC-URA-HsLAS中只在17.75分钟左右检测到羊毛甾醇(P5)；在pESC-URA-HsLAS-F696T中，在16.25分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-HsLAS-F696N中，在16.25分钟左右检测到protosta-13(17),24-dien-3β-ol(P1)、在18.75分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17)，24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的AoPDS蛋白产生外，还可以由人工改造羊毛甾醇合酶类(LAS)中的人HsLAS产生。
实施例11.灵芝(Ganoderma lucidum)羊毛甾醇合酶(GlLAS ID:APO20484)突变体(GlLAS-F692T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的GlLAS基因(ID:GQ169528.1)，由商业公司合成核苷酸序列并构建pESC-URA-GlLAS重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-GlLAS-F692T/N，所述突变体构建步骤同实施例3，仅使用引物不同。。
所用突变692位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-GlLAS-F692T-F
5’-GGAAGCGATAGAGGGCGTGACCAACAAGAACGTCGCGATATCG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-GlLAS-F692T-R
3’-CGATATCGCGACGTTCTTGTTGGTCACGCCCTCTATCGCTTCC-5’
设计突变692位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-GlLAS-F692N-F
5’-GGAAGCGATAGAGGGCGTGAACAACAAGAACGTCGCGATATCG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-GlLAS-F692N-R
3’-CGATATCGCGACGTTCTTGTTGTTCACGCCCTCTATCGCTTCC-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3j所示，在pESC-URA-GlLAS中只在17.60分钟左右检测到羊毛甾醇(P5)；在pESC-URA-GlLAS-F692T中，在14.80分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-GlLAS-F692N中，在14.80分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在17.40分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17)，24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的AoPDS蛋白产生外，还可以由人工改造羊毛甾醇合酶类(LAS)中的灵芝GlLAS产生。
实施例12.水稻(Oryza sativa Japonica Group)帕克醇合酶(OsOSC7 ID:XP_015617632)突变体 (OsOSC7-F726T/N)重组酿酒酵母工程菌细胞系的构建与表达
突变重组载体构建方法采用全质粒克隆方法。根据公开的OsOSC7基因(ID:XM_015762146.2)，由商业公司合成核苷酸序列并构建pESC-URA-OsOSC7重组载体质粒(具体构建步骤参照实施例1)。以其为模板克隆pESC-URA-OsOSC7-F726T/N，所述突变体构建步骤同实施例3，仅使用引物不同。
所用突变726位置的苯丙氨酸为苏氨酸(T)的全质粒突变引物如下：
5’-端引物：pESC-URA-OsOSC7-F726T-F
5’-CACAGCAAGAAATTGTTGGAGTCACCCTCCAGACTGCCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-OsOSC7-F726T-R
3’-CATGGCAGTCTGGAGGGTGACTCCAACAATTTCTTGCTGTG-5’
设计突变726位置的苯丙氨酸为天冬酰胺(N)全质粒突变引物如下：
5’-端引物：pESC-URA-OsOSC7-F726N-F
5’-CACAGCAAGAAATTGTTGGAGTCAACCTCCAGACTGCCATG-3’
3’-端引物为上述引物反向互补得到：pESC-URA-OsOSC7-F726N-R
3’-CATGGCAGTCTGGAGGTTGACTCCAACAATTTCTTGCTGTG-5’
重组突变质粒转化CEN.PK2-1D酵母细胞的方法、突变体重组酿酒酵母工程菌细胞系的表达和产物检测方法同实施例1和实施例2，结果如图3k所示，在pESC-URA-OsOSC7中只在16.00分钟左右检测到帕克醇parkeol(P6)；在pESC-URA-OsOSC7-F726T中，在12.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)；在pESC-URA-OsOSC7-F726N中，在12.50分钟左右检测到 protosta-13(17),24-dien-3β-ol(P1)、在15.00分钟左右检测到protosta-17(20),24-dien-3β-ol(P3)。这说明本发明所述protosta-13(17),24-dien-3β-ol和protosta-17(20),24-dien-3β-ol的生物合成方法除自然存在的 AoPDS蛋白产生外，还可以由人工改造帕克醇合酶类中的水稻OsOSC7产生。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 广州中医药大学（广州中医药研究院）
<120> 生产原萜烷三萜的三萜环化酶及其编码序列、表达载体和应用
<160> 50
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2280
<212> DNA
<213> 泽泻科东方泽泻(Alisma orientale Sam.Juzep.)
<223> AoPDS编码序列
atgtggaggc tgaagattgc ggacggcctg caggacccct ggctgcggac aaagaataat 60
catgttggcc ggcagacctg ggagtacgac cccaacctag gcacagacga agagcgggct 120
gccgtggagg ctgcccgccg ggccttcacc gagaacaggt tcgagaagaa gcagagcggt 180
gatatgctct tgcgcctcca gtttgcgaag gagaatccag ttggaatgat accgccgcag 240
gttatcctgg gggatgacga agatatcaca gaagaggccg tcacggcaac gctgagaagg 300
gcgatcagtt tcttctccac ccttcaggga catgatggcc actggccagg ggattatgga 360
gcccccctgt tccttgtgcc tggcttggtt attattctgt atatcactgg cgcaatgaac 420
actgtgctat ccgctgagca taggatcgag atgtgccgct atatttacaa ccatcagaac 480
gaggacgggg gttggggcct tcatatcgag ggacctagca taatgtttgg gtctgcgctg 540
tcatatgttt gtttgcggtt gttgggtgag gacgccaatg gtgggaatgg ggcaatggag 600
aaagcaaggc ggtgggttct agatcacggt ggtgctacta acacaccctc gtgggggaaa 660
ctgtatctgt cggtgcttgg agtttatgat tggtcgggta acaaccccat tccacctgaa 720
gcatggttgc tacctaattt tcttcctttt catccaggga aaatgtggtg ccactgcagg 780
caggtttatt caccaatgtc ctatttgtat ggaaagagat ttgttggtaa aattactcca 840
ttgattctat cgttaaggaa ggagatttac aatgtttcct atgacaagat taattggaat 900
cgtgctcgaa atatgtgtgc aaaggaagat gtaatttatc cgcatccctt ggtacaagat 960
attgtgtggg catgtctcga taaagttgtt gagcctatgt taatgagctg gcctggaacc 1020
ttgttgaggg aaaaggccct ctcaacagta atgcaacata tccattacga ggatgaaaac 1080
acccgctaca tctgtcttgg gcctgtcaat aaggttctga atacgctttg ttgttgggta 1140
gaaaatccta actctgtggc attcaagttg catctaagta ggatacatga ttacttttgg 1200
attgctgaag atggcatgaa atctcagtcc gttaatggaa gtcaatcgtg ggatacagct 1260
tttgccatcc aagccattat ctcatctaag ctttcagagg aatatggtcc tactcttaag 1320
aaagctcata gcttcataaa aaactctcaa attttagata attgccctgg tgatcaatca 1380
ttctggtatc gccacatttc gaaaggtgca tggacctttt caactgcaga tcacggatgg 1440
cctgtttccg actgcactgc agaaggcttg cgtgcttctc tcttgctctc ttgcatttca 1500
tctgaaatcg tcggtgaacc acttgaagcc acgcgtttgt atgacgcggt taatgtcgtt 1560
ttgtcttata tgaaccctaa tggtggtctt gcgagttatg agttgacaag gtcttatccc 1620
tggatggaga tgatcaatcc aacggtttca tttcgtagca tcattattga tcaccaatac 1680
gtggagtgca cttcagcagc tattcaggca ttaactacat ttagaaaact atttcctgga 1740
caccggagaa aagatataga taacttcatt aagaaagctt tgcattttat tgaaagtatt 1800
caacaggatg atggctcatg gtatggttct tggggtatat gcttcactta cggcatctgg 1860
tttggggtga agggtcttat tgctgctgga agaacttaca aaaatagtcc tgccatccaa 1920
aaagcctgtc aatttctatt gtcaaagcaa cttccatcag ggggttgggg cgaatgctat 1980
ttatcatgtc aagacaaggt ctacacaaat ttcaaaggga acaaatccca tctggttaac 2040
acaggatggg ctatgcttgc tcttattgat gctggacagg cagaaagaga ccctgacccg 2100
ctgcatcggg cagccaagtt tttgatcaac tcccaaatgc agaatggaga cttcccacag 2160
caagaaactg taggggtcac taacagagat ctcttggtca cttatgctgc atatcggaac 2220
atctttccaa tttgggcact cggtgaatat cgtagcaggg ttttacagtc cctcaaatag 2280
<210> 2
<211> 759
<212> PRT
<213> 泽泻科东方泽泻(Alisma orientale Sam.Juzep.)
<223> AoPDS
Met Trp Arg Leu Lys Ile Ala Asp Gly Leu Gln Asp Pro Trp Leu Arg
1 5 10 15
Thr Lys Asn Asn His Val Gly Arg Gln Thr Trp Glu Tyr Asp Pro Asn
20 25 30
Leu Gly Thr Asp Glu Glu Arg Ala Ala Val Glu Ala Ala Arg Arg Ala
35 40 45
Phe Thr Glu Asn Arg Phe Glu Lys Lys Gln Ser Gly Asp Met Leu Leu
50 55 60
Arg Leu Gln Phe Ala Lys Glu Asn Pro Val Gly Met Ile Pro Pro Gln
65 70 75 80
Val Ile Leu Gly Asp Asp Glu Asp Ile Thr Glu Glu Ala Val Thr Ala
85 90 95
Thr Leu Arg Arg Ala Ile Ser Phe Phe Ser Thr Leu Gln Gly His Asp
100 105 110
Gly His Trp Pro Gly Asp Tyr Gly Ala Pro Leu Phe Leu Val Pro Gly
115 120 125
Leu Val Ile Ile Leu Tyr Ile Thr Gly Ala Met Asn Thr Val Leu Ser
130 135 140
Ala Glu His Arg Ile Glu Met Cys Arg Tyr Ile Tyr Asn His Gln Asn
145 150 155 160
Glu Asp Gly Gly Trp Gly Leu His Ile Glu Gly Pro Ser Ile Met Phe
165 170 175
Gly Ser Ala Leu Ser Tyr Val Cys Leu Arg Leu Leu Gly Glu Asp Ala
180 185 190
Asn Gly Gly Asn Gly Ala Met Glu Lys Ala Arg Arg Trp Val Leu Asp
195 200 205
His Gly Gly Ala Thr Asn Thr Pro Ser Trp Gly Lys Leu Tyr Leu Ser
210 215 220
Val Leu Gly Val Tyr Asp Trp Ser Gly Asn Asn Pro Ile Pro Pro Glu
225 230 235 240
Ala Trp Leu Leu Pro Asn Phe Leu Pro Phe His Pro Gly Lys Met Trp
245 250 255
Cys His Cys Arg Gln Val Tyr Ser Pro Met Ser Tyr Leu Tyr Gly Lys
260 265 270
Arg Phe Val Gly Lys Ile Thr Pro Leu Ile Leu Ser Leu Arg Lys Glu
275 280 285
Ile Tyr Asn Val Ser Tyr Asp Lys Ile Asn Trp Asn Arg Ala Arg Asn
290 295 300
Met Cys Ala Lys Glu Asp Val Ile Tyr Pro His Pro Leu Val Gln Asp
305 310 315 320
Ile Val Trp Ala Cys Leu Asp Lys Val Val Glu Pro Met Leu Met Ser
325 330 335
Trp Pro Gly Thr Leu Leu Arg Glu Lys Ala Leu Ser Thr Val Met Gln
340 345 350
His Ile His Tyr Glu Asp Glu Asn Thr Arg Tyr Ile Cys Leu Gly Pro
355 360 365
Val Asn Lys Val Leu Asn Thr Leu Cys Cys Trp Val Glu Asn Pro Asn
370 375 380
Ser Val Ala Phe Lys Leu His Leu Ser Arg Ile His Asp Tyr Phe Trp
385 390 395 400
Ile Ala Glu Asp Gly Met Lys Ser Gln Ser Val Asn Gly Ser Gln Ser
405 410 415
Trp Asp Thr Ala Phe Ala Ile Gln Ala Ile Ile Ser Ser Lys Leu Ser
420 425 430
Glu Glu Tyr Gly Pro Thr Leu Lys Lys Ala His Ser Phe Ile Lys Asn
435 440 445
Ser Gln Ile Leu Asp Asn Cys Pro Gly Asp Gln Ser Phe Trp Tyr Arg
450 455 460
His Ile Ser Lys Gly Ala Trp Thr Phe Ser Thr Ala Asp His Gly Trp
465 470 475 480
Pro Val Ser Asp Cys Thr Ala Glu Gly Leu Arg Ala Ser Leu Leu Leu
485 490 495
Ser Cys Ile Ser Ser Glu Ile Val Gly Glu Pro Leu Glu Ala Thr Arg
500 505 510
Leu Tyr Asp Ala Val Asn Val Val Leu Ser Tyr Met Asn Pro Asn Gly
515 520 525
Gly Leu Ala Ser Tyr Glu Leu Thr Arg Ser Tyr Pro Trp Met Glu Met
530 535 540
Ile Asn Pro Thr Val Ser Phe Arg Ser Ile Ile Ile Asp His Gln Tyr
545 550 555 560
Val Glu Cys Thr Ser Ala Ala Ile Gln Ala Leu Thr Thr Phe Arg Lys
565 570 575
Leu Phe Pro Gly His Arg Arg Lys Asp Ile Asp Asn Phe Ile Lys Lys
580 585 590
Ala Leu His Phe Ile Glu Ser Ile Gln Gln Asp Asp Gly Ser Trp Tyr
595 600 605
Gly Ser Trp Gly Ile Cys Phe Thr Tyr Gly Ile Trp Phe Gly Val Lys
610 615 620
Gly Leu Ile Ala Ala Gly Arg Thr Tyr Lys Asn Ser Pro Ala Ile Gln
625 630 635 640
Lys Ala Cys Gln Phe Leu Leu Ser Lys Gln Leu Pro Ser Gly Gly Trp
645 650 655
Gly Glu Cys Tyr Leu Ser Cys Gln Asp Lys Val Tyr Thr Asn Phe Lys
660 665 670
Gly Asn Lys Ser His Leu Val Asn Thr Gly Trp Ala Met Leu Ala Leu
675 680 685
Ile Asp Ala Gly Gln Ala Glu Arg Asp Pro Asp Pro Leu His Arg Ala
690 695 700
Ala Lys Phe Leu Ile Asn Ser Gln Met Gln Asn Gly Asp Phe Pro Gln
705 710 715 720
Gln Glu Thr Val Gly Val Thr Asn Arg Asp Leu Leu Val Thr Tyr Ala
725 730 735
Ala Tyr Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly Glu Tyr Arg Ser
740 745 750
Arg Val Leu Gln Ser Leu Lys
755
<210> 3
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoPDS-F
cgtcaaggag aaaaaacccc ggatccatgt ggaggctgaa gattgcggac g 51
<210> 4
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoPDS-R
acttctgttc catgtcgacg cccgggctat ttgagggact gtaaaaccct g 51
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> GAL1-F
agtacggatt agaagccgcc g 21
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> GAL1-R
gggttttttc tccttgacgt ta 22
<210> 7
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoCAS-F726T-F
gaaataatgg gggtaaccaa cagaaattgc atg 33
<210> 8
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoCAS-F726T-R
catgcaattt ctgttggtta cccccattat ttc 33
<210> 9
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoCAS-F726N-F
gaaataatgg gggtaaacaa cagaaattgc atg 33
<210> 10
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AoCAS-F726N-R
catgcaattt ctgttgttta cccccattat ttc 33
<210> 11
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ItOSC3-F728T-F
gaaattatgg gagtcaccaa cagaaactgc atg 33
<210> 12
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ItOSC3-F728T-R
catgcaattt ctgttggtta cccccattat ttc 33
<210> 13
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ItOSC3-F728N-F
gaaattatgg gagtcaacaa cagaaactgc atg 33
<210> 14
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ItOSC3-F728N-R
catgcagttt ctgttgttga ctcccataat ttc 33
<210> 15
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SlCAS-F726T-F
ggagatcatg ggagtcacca ataggaactg catga 35
<210> 16
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SlCAS-F726T-R
tcatgcagtt cctattggtg actcccatga tctcc 35
<210> 17
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SlCAS-F726N-F
ggagatcatg ggagtcaaca ataggaactg catga 35
<210> 18
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SlCAS-F726N-R
tcatgcagtt cctattgttg actcccatga tctcc 35
<210> 19
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SgCBQ-F729T-F
ggagatcatg ggagtcacta ataaaaattg c 31
<210> 20
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> ppESC-URA-SgCBQ-F729T-R
gcaattttta ttagtgactc ccatgatctc c 31
<210> 21
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SgCBQ-F729N-F
ggagatcatg ggagtcaata ataaaaattg c 31
<210> 22
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-SgCBQ-F729N-R
gcaattttta ttattgactc ccatgatctc c 31
<210> 23
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CsCBQ-F755T-F
ggagatcatg ggagtgacca acaaaaattg tatg 34
<210> 24
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CsCBQ-F755T-R
catacaattt ttgttggtca ctcccatgat ctcc 34
<210> 25
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CsCBQ-F755N-F
ggagatcatg ggagtgaaca acaaaaattg tatg 34
<210> 26
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CsCBQ-F755N-R
ggagatcatg ggagtgaaca acaaaaattg tatg 34
<210> 27
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CpCBQ-F734T-F
ggagatcatg ggagtgacca ataagaactg catgatc 37
<210> 28
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CpCBQ-F734T-R
gatcatgcag ttcttattgg tcactcccat gatctcc 37
<210> 29
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CpCBQ-F734N-F
ggagatcatg ggagtgaaca ataagaactg catgatc 37
<210> 30
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-CpCBQ-F734N-R
gatcatgcag ttcttattgt tcactcccat gatctcc 37
<210> 31
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ERG7-F691T-F
cgatggaagg gatcaccaac aagaatgttg cc 32
<210> 32
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ERG7-F691T-R
ggcaacattc ttgttggtga tcccttccat cg 32
<210> 33
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ERG7-F691N-F
cgatggaagg gatcaacaac aagaatgttg cc 32
<210> 34
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-ERG7-F691N-R
ggcaacattc ttgttgttga tcccttccat cg 32
<210> 35
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-HsLAS-F696T-F
ggaaaacatt gctggggtca ccaacaagtc ctgtgccatc 40
<210> 36
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-HsLAS-F696T-R
gatggcacag gacttgttgg tgaccccagc aatgttttcc 40
<210> 37
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-HsLAS-F696N-F
ggaaaacatt gctggggtca acaacaagtc ctgtgccatc 40
<210> 38
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-HsLAS-F696N-R
gatggcacag gacttgttgt tgaccccagc aatgttttcc 40
<210> 39
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-GlLAS-F692T-F
ggaagcgata gagggcgtga ccaacaagaa cgtcgcgata tcg 43
<210> 40
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-GlLAS-F692T-R
cgatatcgcg acgttcttgt tggtcacgcc ctctatcgct tcc 43
<210> 41
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-GlLAS-F692N-F
ggaagcgata gagggcgtga acaacaagaa cgtcgcgata tcg 43
<210> 42
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-GlLAS-F692N-R
cgatatcgcg acgttcttgt tgttcacgcc ctctatcgct tcc 43
<210> 43
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AjLAS-F691T-F
gtggtgttac caacaagtct tgcgc 25
<210> 44
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AjLAS-F691T-R
gcgcaagact tgttggtaac accac 25
<210> 45
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AjLAS-F691N-F
gtggtgttaa caacaagtct tgcgc 25
<210> 46
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-AjLAS-F691N-R
gcgcaagact tgttgttaac accac 25
<210> 47
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-OsOSC7-F726T-F
cacagcaaga aattgttgga gtcaccctcc agactgccat g 41
<210> 48
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-OsOSC7-F726T-R
catggcagtc tggagggtga ctccaacaat ttcttgctgt g 41
<210> 49
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-OsOSC7-F726N-F
cacagcaaga aattgttgga gtcaacctcc agactgccat g 41
<210> 50
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> pESC-URA-OsOSC7-F726N-R
catggcagtc tggaggttga ctccaacaat ttcttgctgt g 41
Claims (10)
1.一种生产原萜烷三萜的三萜环化酶，能够合成原萜烷型三萜，其特征在于是如下酶的至少一种：
(1)氨基酸如SEQ ID No:2所示的AoPDS酶，由760个氨基酸残基组成，其特征在于编码第727个氨基酸为苏氨酸；
(2)将环阿屯醇合酶的氨基酸序列中序列为“FNRNCMI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(3)将葫芦二烯醇合成酶的氨基酸序列中序列为“FNKNCMI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(4)将羊毛甾醇合酶的氨基酸序列中序列为“FNKNVAI”或“FNKSCAI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(5)将帕克醇合酶的氨基酸序列中序列为FLQTAMI”或“FNKSCAI”的氨基酸残基中苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶。
2.根据权利要求1所述的生产原萜烷三萜的三萜环化酶，其特征在于是如下酶的至少一种：
(1)将泽泻环阿屯醇合酶第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(2)将鸢尾环阿屯醇合酶第728位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(3)将番茄环阿屯醇合酶第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(4)将罗汉果葫芦二烯醇合成酶第729位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(5)将黄瓜葫芦二烯醇合成酶第755位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(6)将西葫芦葫芦二烯醇合成酶第734位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(7)将酿酒酵母羊毛甾醇合酶第691位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(8)将人羊毛甾醇合酶第696位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(9)将灵芝羊毛甾醇合酶第692位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶；
(10)将水稻帕克醇合酶第726位氨基酸由苯丙氨酸突变为苏氨酸或天冬酰胺得到的酶。
3.根据权利要求1或2所述的生产原萜烷三萜的三萜环化酶，其特征在于：
所述的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有合成原萜烷型三萜功能的酶；
所述的取代和/或缺失和/或添加具体为1至10个氨基酸残基的取代和/或缺失和/或添加。
4.编码权利要求1所述的AoPDS酶的编码基因，其特征在于：
所述编码基因如SEQ ID No:1所示。
5.编码权利要求1或2所述的生产原萜烷三萜的三萜环化酶的编码基因，其特征在于：
1)核苷酸序列依据密码子编码规则得到；
2)在严格条件下与1)限定的核苷酸序列杂交且编码所述蛋白的核苷酸序列；
3)与1)或2)限定的核苷酸序列具有90％以上的同源性，且编码相同功能蛋白质分子的核苷酸序列。
6.根据权利要求4或5所述的编码基因，其特征在于：
通过将所述的编码基因中的自5’端第1至末位碱基所示的核苷酸序列中缺失一个或者几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5’端和/或3’端连上纯化标签得到。
7.一种重组表达载体，其特征在于：
含有权利要求4～6任一项所述的编码基因；
所述的重组表达载体为使用现有的表达载体构建；
所述的重组表达载体在构建时，在其转录起始核苷酸前加上任何一种增强型启动子或组成型启动子；此外，还使用增强子，包括翻译增强子或转录增强子，这些增强子区域是ATG起始密码子或邻接区域起始密码子，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译；所述翻译控制信号和起始密码子的来源是天然的或是合成的；翻译起始区域为来自转录起始区域或结构基因。
8.一种转基因细胞系，其特征在于：
将权利要求7所述的重组表达载体导入细胞得到。
9.权利要求1～3任一项所述的生产原萜烷三萜的三萜环化酶在制备原萜烷型三萜中的应用。
10.一种三萜环化酶的构建方法，其特征在于：
使用环阿屯醇合酶、葫芦二烯醇合成酶、羊毛甾醇合酶、帕克醇合酶构建突变表达载体时，将其相对于权利要求1所述的AoPDS酶的氨基酸序列的第727位苏氨酸的苯丙氨酸替换为苏氨酸或天冬酰胺。
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| CN118126997B (zh) * | 2024-05-07 | 2024-08-16 | 广州中医药大学(广州中医药研究院) | 一种多功能三萜环化酶及其在制备三萜化合物中的应用 |
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