WO2024011761A1 - 来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用 - Google Patents

来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用 Download PDF

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WO2024011761A1
WO2024011761A1 PCT/CN2022/122698 CN2022122698W WO2024011761A1 WO 2024011761 A1 WO2024011761 A1 WO 2024011761A1 CN 2022122698 W CN2022122698 W CN 2022122698W WO 2024011761 A1 WO2024011761 A1 WO 2024011761A1
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seq
methyltransferase
ecgonone
amino acid
expression vector
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黄胜雄
杨静
王永江
黄建萍
罗剑英
颜一军
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中国科学院昆明植物研究所
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  • the invention belongs to the technical field of plant molecular biology, and specifically relates to ecgonone methyltransferases EnEMT1 and EnEMT2 from coca and their genes and applications.
  • Tropane alkaloids are a class of alkaloids containing 8-azabicyclo[3.2.1]octane as the skeleton. They are mainly distributed in the Erythroxylaceae, Solanaceae, and Convolvulaceae families. More than 300 tropane alkaloids have been identified from plants such as Convolvulaceae, Rhizophoraceae, and Brassicaceae. Plant-derived tropane alkaloids have very good physiological activity. For example, cocaine isolated from the coca family can block the conduction between nerve fibers and has postoperative vasoconstriction effect. It can be used as a medicine for the nose, throat and lower respiratory tract.
  • hyoscyamine and scopolamine isolated from the Solanaceae family can block parasympathetic nerves or inhibit the central nervous system, thereby being used for analgesia, antispasmodic, etc.
  • Tropine alkaloids have multiple chiral centers in their structure, and the synthesis process directly through chemical means is complex and costly. Most of the tropane alkaloids currently used for medical treatment are isolated from plants; while tropane alkaloids such as cocaine are The biosynthetic pathway of alkali is unclear, and the rate-limiting step in the in vivo synthesis of tropane alkaloids such as cocaine cannot be relieved, which limits the improvement of tropane alkaloid production in plants. In addition, the isolation of tropane alkaloids through plants is affected by various factors such as plant growth and climate change, which also limits the supply of tropane alkaloids such as cocaine.
  • the present invention provides Ectonone methyltransferases EnEMT1 and EnEMT2 derived from coca and their genes and applications.
  • Ecgonone methyltransferase is provided, a protein having one of the following amino acid residue sequences:
  • an econone methyltransferase gene having a polynucleotide having one of the following nucleotide sequences:
  • a recombinant expression vector containing an econone methyltransferase gene is provided.
  • the recombinant expression vector is a recombinant expression vector that expresses the econone methyltransferase by inserting the econone methyltransferase gene into a prokaryotic or eukaryotic expression vector.
  • the egonone methyltransferase gene was constructed into the pET28a vector.
  • a transgenic recombinant bacterium or transgenic cell line containing a germonone methyltransferase gene is provided.
  • transgenic recombinant bacteria are bacteria and fungi, including but not limited to Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae.
  • ecgonone methyltransferase in catalyzing the production of methylecgonone and its derivatives in vivo or in vitro.
  • an ecgonone methyltransferase in catalyzing the production of tropane alkaloids, including but not limited to cocaine, in vivo or in vitro.
  • a recombinant expression vector containing an econone methyltransferase gene to construct a methyl econone and its derivatives synthesis pathway in prokaryotes or eukaryotes that do not have a methyl econone and its derivatives biosynthetic pathway, Or the application of improving the production of methyleconone and its derivatives in prokaryotes or eukaryotes having a synthesis pathway of methyleconone and its derivatives.
  • transgenic recombinant bacteria or transgenic cell lines containing a budone methyltransferase gene in catalyzing the methylation of budone and its derivatives.
  • the invention discloses an econone methyltransferase, whose amino acid sequences are: EnEMT1 is shown in SEQ ID NO.4, EnEMT2 is shown in SEQ ID NO.8, and the nucleotides encoded by it are: EnEMT1 is shown in SEQ ID NO. As shown in ID NO.3, EnEMT2 is shown in SEQ ID NO.7.
  • econone methyltransferase After prokaryotic expression of econone methyltransferase, it can catalyze econone to generate methyl econone.
  • the discovery of ecgonone methyltransferase provides more basic components for the synthetic biology of natural products, and provides guidance and basis for the rational design of this type of enzymes, which has good industrial prospects.
  • Figure 1 is an enzymatic chemical reaction equation catalyzed by EnEMT1 and EnEMT2 provided by the embodiment of the present invention
  • Figure 2 is the plasmid map of EnEMT1-pET28a provided by the embodiment of the present invention.
  • Figure 3 is the plasmid map of EnEMT2-pET28a provided by the embodiment of the present invention.
  • Figure 4 is a diagram of SDS-polyacrylamide gel electrophoresis results of EnEMT1 and EnEMT2 proteins provided by the embodiment of the present invention
  • Figure 5 is an LC-MS analysis diagram of EnEMT1 and EnEMT2 catalyzing ecgonone to generate methyl ecgonone provided by the embodiment of the present invention.
  • EMT ecgonone methyltransferase
  • RNA quality Take an appropriate amount of coca leaf bud tissue, grind it in liquid nitrogen, and extract total RNA using the Biotek Polysaccharide Polyphenol Plant Total RNA Rapid Extraction Kit according to the instructions. Use a Thermo Scientific NanoDrop spectrophotometer to detect RNA concentration and quality, and use agarose gel electrophoresis to detect RNA quality.
  • EnEMT1 and EnEMT2 genes were amplified and sequenced through PCR.
  • the nucleotide sequences of the EnEMT1 and EnEMT2 genes were obtained as follows.
  • EnEMT1 is as shown in SEQ ID NO.3, and the start codon is ATG.
  • the stop codon is TGA;
  • the translated protein coding sequence is as shown in SEQ ID NO.4;
  • EnEMT2 is as shown in SEQ ID NO.7, the start codon is ATG, and the stop codon is TGA;
  • the translated protein coding sequence is as SEQ Shown as ID NO.8.
  • the EnEMT1 forward primer has a homology arm sequence as shown in SEQ ID NO.9
  • the reverse primer has a homology arm sequence as shown in SEQ ID NO.10
  • the EnEMT2 forward primer has a homology arm sequence as shown in SEQ ID NO.9.
  • the reverse primer has a homology arm sequence as shown in SEQ ID NO.12.
  • the primer sequences are as follows:
  • EnEMT1-pET28a and EnEMT2-pET28a plasmids were transformed into the prokaryotic expression strain BL21 (DE3), spread on LB solid plates containing kanamycin, single colonies were picked for culture, the plasmids were extracted for verification by enzyme digestion, and positive clones were screened. , obtained prokaryotic expression engineering bacteria EnEMT1-pET28a-BL21 (DE3) and EnEMT2-pET28a-BL21 (DE3). Take 20 ⁇ L of bacterial solution and inoculate it into 20 mL LB liquid medium containing 50 ⁇ g/L kanamycin, and cultivate overnight at 37°C and 200 rpm.
  • the enzymatic chemical reaction equation catalyzed by EnEMT1 and EnEMT2 is shown in Figure 1.
  • the system of the in vitro enzymatic reaction catalyzed by EnEMT1 and EnEMT2 is as follows: 50mM potassium phosphate buffer salt (pH 7.5), 1mM ecgonone, 1mM SAM, 300ng ⁇ L -1 EnEMT1 or EnEMT2 protein, with a total volume of 100 ⁇ L, at 30°C. After incubation for 1 hour, add an equal amount of acetonitrile to terminate the reaction.
  • the enzyme reaction product was analyzed by LC-MS.
  • the LC-MS instrument was Agilent 1290/6530 system, the chromatographic column was YMC-Triart C 18 (ID4.6 ⁇ 250mm), the column temperature was 30°C, the flow rate was 1mL/min, and the mobile phase
  • the elution procedure is: 90% phase A (water containing 0.1% formic acid), 10% phase B (acetonitrile), isocratic elution for 6 minutes, the mass spectrometer is an electrospray ion source (ESI), and a positive ion mode scan is performed. Range (m/z): 50-400.
  • the invention discloses an econone methyltransferase, whose amino acid sequences are: EnEMT1 is shown in SEQ ID NO.4, EnEMT2 is shown in SEQ ID NO.8, and the nucleotides encoded by it are: EnEMT1 is shown in SEQ As shown in ID NO.3, EnEMT2 is shown in SEQ ID NO.7.
  • EnEMT1 is shown in SEQ As shown in ID NO.3
  • EnEMT2 is shown in SEQ ID NO.7.

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Abstract

本发明公开了来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用。EnEMT1氨基酸序列如SEQ ID NO.4所示，核苷酸序列如SEQ ID NO.3所示；EnEMT2氨基酸序列如SEQ ID NO.8所示，核苷酸序列如SEQ ID NO.7所示。经大肠杆菌表达后的蛋白质能够催化芽子酮生成甲基芽子酮。芽子酮甲基转移酶的发现为天然产物的合成生物学提供了更多基础元件，并且为这类酶的理性设计提供了指导和依据，具有很好的工业化前景。

Description

来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用 技术领域
本发明属于植物分子生物学技术领域，具体涉及来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用。
背景技术
托品烷生物碱(tropane alkaloids)是一类含有8-氮杂双环[3.2.1]辛烷为骨架的生物碱，主要分布于古柯科(Erythroxylaceae)，茄科(Solanaceae)，旋花科(Convolvulaceae)，红树科(Rhizophoraceae)，十字花科(Brassicaceae)等植物，现已鉴定出300多种托品烷生物碱。植物源托品烷生物碱具有很好的生理活性，如：古柯科中分离得到的可卡因能阻断神经纤维之间的传导及具有术后血管收缩作用，可作为鼻、喉咙和下呼吸道等区域外科手术的局部麻醉药；茄科中分离得到的莨菪碱和东莨菪碱可阻断副交感神经或抑制中枢神经系统等，从而用于止痛、解痉等。
托品烷生物碱因其结构具有多个手性中心，直接通过化学手段合成工艺复杂且成本高，现阶段医疗使用的托品烷生物碱多是从植物中分离；而可卡因等托品烷生物碱的生物合成途径不清楚，无法解除可卡因等托品烷生物碱体内合成的限速步骤，限制了植物中托品烷生物碱产量的提高。此外，通过植物分离托品烷生物碱受到植物生长、气候变动等各种因素影响，也限制了可卡因等托品烷生物碱的供应。现代分子生物学和合成生物学的发展，使得可卡因等药用天然产物脱离原植物，在微生物中生产成为了可能。可卡因生物合成途径的解析及生物合成基因的挖掘作为其进行代谢工程的基础，是微生物生产中最重要的基本元件。
基于现有同位素前体标记实验等推测可卡因的生物合成路径如下：由鸟氨酸起始，经历N-甲基吡咯阳离子、4-(1-甲基-2-吡咯)-3-氧代丁酸、甲基芽子酮等中间体；最后，甲基芽子碱在CS酶的作用下与苯甲酰CoA缩合形成可卡因。但在现有技术中，没有相关甲基芽子酮合成的酶EnEMT1和EnEMT2及其编码基因的鉴定，以及芽子酮甲基转移酶(EnEMT1和EnEMT2)相关应用的报道。
发明内容
针对现有技术中的上述不足，本发明提供来自古柯的芽子酮甲基转移酶EnEMT1和EnEMT2及其基因和应用。
为了达到上述发明目的，本发明采用的技术方案为：
提供芽子酮甲基转移酶，其具有下述氨基酸残基序列之一的蛋白质：
a)具有SEQ ID NO.4或SEQ ID NO.8所示的氨基酸残基序列的蛋白质；
b)将SEQ ID NO.4或SEQ ID NO.8中的氨基酸残基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a)酶功能的由a)衍生的氨基酸序列；
c)与a)或b)限定的氨基酸序列具有80％以上的同源性，且具有a)酶功能的由a)衍生的蛋白质；
d)序列中含有a)、b)或c)氨基酸序列的衍生蛋白质。
提供芽子酮甲基转移酶基因，其具有下述核苷酸序列之一的多核苷酸：
a)核苷酸序列如SEQ ID NO.3或SEQ ID NO.7所示的多核苷酸；
b)编码如SEQ ID NO.4或SEQ ID NO.8所示的氨基酸序列的多核苷酸；
c)与a)或b)限定的核苷酸序列具有80％以上的同源性且编码具有权利要求1所述芽子酮甲基转移酶功能的多核苷酸；
d)与a)、b)或c)序列互补的多核苷酸。
提供包含芽子酮甲基转移酶基因的重组表达载体。
进一步地，重组表达载体为将芽子酮甲基转移酶基因插入原核或真核表达载体得到表达芽子酮甲基转移酶的重组表达载体。
进一步地，将芽子酮甲基转移酶基因构建到pET28a载体而得。
提供包含芽子酮甲基转移酶基因的转基因重组菌或转基因细胞系。
进一步地，转基因重组菌为细菌和真菌，细菌和真菌包括但不限于大肠杆菌、枯草芽孢杆菌、毕赤酵母、酿酒酵母。
提供芽子酮甲基转移酶在体内或体外催化生成甲基芽子酮及其衍生物的应用。
提供芽子酮甲基转移酶在体内或体外催化生产托品烷生物碱中的应用，托品烷生物碱包括但不限于可卡因。
提供包含芽子酮甲基转移酶基因的重组表达载体在不具有甲基芽子酮及其衍生物生物合成途径的原核生物或真核生物中构建甲基芽子酮及其衍生物合成途径，或是在提升甲基芽子酮及其衍生物在具有甲基芽子酮及其衍生物合成途径的原核生物或真核生物产量中的应用。
提供包含芽子酮甲基转移酶基因的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中构建托品烷生物碱的生物合成途径中或是在提升托品烷生物碱在具有托品烷生物碱生物合成途径的原核生物或真核生物产量中的应用。
提供包含芽子酮甲基转移酶基因的转基因重组菌或转基因细胞系在催化芽子酮及其衍生物甲基化中的应用。
本发明的有益效果为：
本发明公开了芽子酮甲基转移酶，其氨基酸序列分别是，EnEMT1如SEQ ID  NO.4所示，EnEMT2如SEQ ID NO.8所示，其编码的核苷酸分别是，EnEMT1如SEQ ID NO.3所示，EnEMT2如SEQ ID NO.7所示，将芽子酮甲基转移酶进行原核表达后能够催化芽子酮生成甲基芽子酮。芽子酮甲基转移酶的发现为天然产物的合成生物学提供了更多基础元件，并且为这类酶的理性设计提供了指导和依据，具有很好的工业化前景。
附图说明
图1为本发明实施例提供的EnEMT1和EnEMT2催化的酶化学反应方程式；
图2为本发明实施例提供的EnEMT1-pET28a的质粒图谱；
图3为本发明实施例提供的EnEMT2-pET28a的质粒图谱；
图4为本发明实施例提供的EnEMT1和EnEMT2蛋白的SDS-聚丙烯酰胺凝胶电泳结果图；
图5为本发明实施例提供的EnEMT1和EnEMT2催化芽子酮生成甲基芽子酮的LC-MS分析图。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1
芽子酮甲基转移酶(ecgonone methyltransferase，EMT)基因的克隆。
(1)古柯嫩芽总RNA的提取及cDNA第一链的合成。
取适量古柯叶芽组织，在液氮中研磨，用biotek多糖多酚植物总RNA快速 提取试剂盒按说明书提取总RNA。用Thermo Scientific NanoDrop分光光度计检测RNA浓度及质量，并用琼脂糖凝胶电泳检测RNA质量。
用诺唯赞公司HiScript III 1st Strand cDNA Synthesis Kit反转录试剂盒，按照其产品说明书指示，以总RNA为模板，合成cDNA。
(2)EnEMT1和EnEMT2基因的克隆。
设计特异性引物，具体引物序列如下：
EnEMT1-F:5’-atggcaattgaacaagtgcttcacat-3’(SEQ ID NO.1)
EnEMT1-R:5’-tcaatccaaatcaactgcagttctt-3’(SEQ ID NO.2)
EnEMT2-F:5’-atggcaattgatcaagtacttcacatg-3’(SEQ ID NO.5)
EnEMT2-R:5’-tcaatccatatcaactacagttctc-3’(SEQ ID NO.6)
通过PCR以嫩芽组织的cDNA为模板，扩增EnEMT1和EnEMT2的基因并测序，获得EnEMT1和EnEMT2基因的核苷酸序列如下，EnEMT1如SEQ ID NO.3所示，起始密码子为ATG，终止密码子为TGA；翻译出蛋白编码序列如SEQ ID NO.4所示；EnEMT2如SEQ ID NO.7所示，起始密码子为ATG，终止密码子为TGA；翻译出蛋白编码序列如SEQ ID NO.8所示。
实施例2
原核表达验证EnEMT1和EnEMT2基因功能。
在酶切位点引入EnEMT1和EnEMT2基因。为EnEMT1和EnEMT2基因设计引物，EnEMT1正向引物带有同源臂序列如SEQ ID NO.9所示，反向引物带有同源臂序列如SEQ ID NO.10所示；EnEMT2正向引物带有同源臂序列如SEQ ID NO.11所示，反向引物带有同源臂序列如SEQ ID NO.12所示。通过PCR扩增得到两端带有同源臂序列的EnEMT1和EnEMT2基因后，用重组酶(ClonExpress Ultra One Step Cloning Kit)分别将EnEMT1和EnEMT2的编码区 完整序列连接质粒pET28a，获得重组表达载体EnEMT1-pET28a和EnEMT2-pET28a。如图2-3所示。
引物序列如下：
EnEMT1-AF:5’-tggtgccgcgcggcagccatatggcaattgaacaagtgcttc-3’(SEQ ID NO.9)
EnEMT1-AR:5’-cggagctcgaattcggatcctcaatccaaatcaactgcagtt-3’(SEQ ID NO.10)
EnEMT2-AF:5’-tggtgccgcgcggcagccatatggcaattgatcaagtacttc-3’(SEQ ID NO.11)
EnEMT2-AR:5’-cggagctcgaattcggatcctcaatccatatcaactacagtt-3’(SEQ ID NO.12)
将构建好的EnEMT1-pET28a和EnEMT2-pET28a质粒转化原核表达菌株BL21(DE3)，涂布含有卡那霉素的LB固体平板，挑取单菌落培养，提取质粒进行酶切验证，筛选阳性克隆子，获得原核表达工程菌EnEMT1-pET28a-BL21(DE3)和EnEMT2-pET28a-BL21(DE3)。取菌液20μL接种到含50μg/L卡那霉素的20mL LB液体培养基中，37℃，200rpm过夜培养。再按1：100比例接种量分别接种到2L LB液体培养基中37℃，200rpm进行培养，培养至OD 600＝0.8左右时，冰浴十分钟，加入IPTG至终浓度0.2mM。在16℃，200rpm条件下继续培养18h。将收获的菌液4000rpm离心，去掉上清。再把菌体用蛋白纯化buffer重悬，并在超声破碎后，用镍柱进行蛋白纯化获得EnEMT1和EnEMT2蛋白。得到的蛋白用SDS-聚丙烯酰胺凝胶电泳检测，如图3所示。
EnEMT1和EnEMT2催化的酶化学反应方程式如图1所示，EnEMT1和EnEMT2催化的体外酶反应的体系如下：50mM磷酸钾缓冲盐(pH 7.5)，1mM 芽子酮，1mM SAM，300ng μL -1EnEMT1或EnEMT2蛋白，总体积100μL，30℃，孵育1h后，加等量乙腈终止反应。
酶反应产物用LC-MS进行分析，液质联用仪为agilent 1290/6530 system，色谱柱为YMC-Triart C 18(I.D.4.6×250mm)，柱温30℃，流速1mL/min，流动相洗脱程序为：90％A相(含0.1％甲酸的水)，10％B相(乙腈)，等度洗脱6min，质谱仪为电喷雾离子源(ESI)，进行正离子模式扫描，扫描范围(m/z)：50-400。
检测结果显示，EnEMT1和EnEMT2催化芽子酮生产甲基芽子酮(m/z:198.1125[M+H] +)，在分析条件下保留时间为3.7min如图4所示。
本发明公开了芽子酮甲基转移酶，其氨基酸序列分别是，EnEMT1如SEQ ID NO.4所示，EnEMT2如SEQ ID NO.8所示，其编码的核苷酸分别是，EnEMT1如SEQ ID NO.3所示，EnEMT2如SEQ ID NO.7所示，将芽子酮甲基转移酶进行原核表达后能够催化芽子酮生成甲基芽子酮。芽子酮甲基转移酶的发现为天然产物的合成生物学提供了更多基础元件，并且为这类酶的理性设计提供了指导和依据，具有很好的工业化前景。
于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见， 本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (12)

  1. 芽子酮甲基转移酶，其特征在于，具有下述氨基酸残基序列之一的蛋白质：
    a)具有SEQ ID NO.4或SEQ ID NO.8所示的氨基酸残基序列的蛋白质；
    b)将SEQ ID NO.4或SEQ ID NO.8中的氨基酸残基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a)酶功能的由a)衍生的氨基酸序列；
    c)与a)或b)限定的氨基酸序列具有80％以上的同源性，且具有a)酶功能的由a)衍生的蛋白质；
    d)序列中含有a)、b)或c)氨基酸序列的衍生蛋白质。
  2. 芽子酮甲基转移酶基因，其特征在于，具有下述核苷酸序列之一的多核苷酸：
    a)核苷酸序列如SEQ ID NO.3或SEQ ID NO.7所示的多核苷酸；
    b)编码如SEQ ID NO.4或SEQ ID NO.8所示的氨基酸序列的多核苷酸；
    c)与a)或b)限定的核苷酸序列具有80％以上的同源性且编码具有权利要求1所述芽子酮甲基转移酶功能的多核苷酸；
    d)与a)、b)或c)所述的序列互补的多核苷酸。
  3. 含有权利要求2所述芽子酮甲基转移酶基因的重组表达载体。
  4. 根据权利要求3所述的重组表达载体，其特征在于，所述重组表达载体为将芽子酮甲基转移酶基因插入原核或真核表达载体得到表达芽子酮甲基转移酶的重组表达载体。
  5. 根据权利要求3所述的重组表达载体，其特征在于，将芽子酮甲基转移酶基因构建到pET28a载体而得。
  6. 含有权利要求2所述芽子酮甲基转移酶基因的转基因重组菌或转基因细胞系。
  7. 根据权利要求6所述的芽子酮甲基转移酶基因的转基因重组菌或转基因细胞系，其特征在于，所述转基因重组菌为细菌和真菌，所述细菌和真菌包括但不限于大肠杆菌、枯草芽孢杆菌、毕赤酵母、酿酒酵母。
  8. 权利要求1所述芽子酮甲基转移酶在体内或体外催化生成甲基芽子酮及其衍生物的应用。
  9. 权利要求1所述芽子酮甲基转移酶在体内或体外催化生产托品烷生物碱中的应用，所述的托品烷生物碱包括但不限于可卡因。
  10. 权利要求3-5任一所述的重组表达载体在不具有甲基芽子酮及其衍生物生物合成途径的原核生物或真核生物中构建甲基芽子酮及其衍生物合成途径，或是在提升甲基芽子酮及其衍生物在具有甲基芽子酮及其衍生物合成途径的原核生物或真核生物产量中的应用。
  11. 权利要求3-5任一所述的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中构建托品烷生物碱的生物合成途径中或是在提升托品烷生物碱在具有托品烷生物碱生物合成途径的原核生物或真核生物产量中的应用。
  12. 权利要求6所述的转基因重组菌或转基因细胞系在催化芽子酮及其衍生物甲基化中的应用。
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