WO2023202122A1 - 一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用 - Google Patents

一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用 Download PDF

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WO2023202122A1
WO2023202122A1 PCT/CN2022/140230 CN2022140230W WO2023202122A1 WO 2023202122 A1 WO2023202122 A1 WO 2023202122A1 CN 2022140230 W CN2022140230 W CN 2022140230W WO 2023202122 A1 WO2023202122 A1 WO 2023202122A1
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谢恬
谌容
殷晓浦
胡添源
王铭
陈姝
胡雨涵
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杭州师范大学
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  • the invention belongs to the field of biotechnology, and particularly relates to a curcumin synthase, gene, vector, and engineering bacteria derived from Yujin and their application in preparing curcumin and its derivatives.
  • Curcumin is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae families. Its chemical formula is C 21 H 20 O 6 .
  • Curcumin has a good therapeutic effect on type II diabetes. Curcumin can also inhibit the viral activity of human immunodeficiency virus and has anti-AIDS effects. Curcumin is effective in treating cancer, rheumatism, inflammatory eye diseases, intestinal diseases, oral cancer and leukoplakia, and is highly safe and highly effective.
  • curcumin is more effective than demethoxygen. Curcumin and bisdemethoxycurcumin have higher activity in antioxidants and DNA repair, and are the main ingredients that exert medicinal effects. At the same time, because it is an orange-yellow crystalline powder with a slightly bitter taste and is insoluble in water, it is often used as a coloring agent in food production.
  • Curcuma wenyujin Y.H.Chen&C.Ling is a cultivated variety of Curcuma longa belonging to the family Zingiberaceae.
  • the fresh rhizome slices of Curcuma wenyujin are called "curcuma longa", which can promote qi, break blood stasis, and unblock meridians. It is used for symptoms of rheumatic arthralgia, abdominal pain, chest and rib pain, amenorrhea and abdominal pain, bruises and other symptoms of blood stasis and qi stagnation.
  • curcumin there are many kinds of chemical components in Yujin, mainly including sesquiterpenes, monoterpenes, diterpenes, curcuminoids, polysaccharides, etc.
  • the main production method of curcumin is plant extraction.
  • the Wenyujin plant only contains a very small amount of curcumin in the leaves and roots.
  • the curcumin in the leaves is The curcumin content is lower; at the same time, the curcumin content is easily affected by many factors such as variety, leaf and rhizome quality, and planting environment.
  • curcumin is mixed with many curcuminoids with similar structures.
  • Curcumin Synthase can catalyze cinnamoyldiketoacetylcysteamine (a coenzyme A ester mimic) and feruloyl-CoA to generate curcuminoids in vitro.
  • CURS Curcumin Synthase
  • DCS diketo-CoA synthase
  • Monodemethoxycurcumin was obtained by co-incubation of diketo-CoA synthase (DCS) and CURS in the presence of feruloyl-CoA, coumaroyl-CoA and malonyl-CoA.
  • DCS diketo-CoA synthase
  • CURS CURS
  • the reported activity of CURS is not high. Therefore, studying the cloning and expression of high-quality curcumin synthase genes will be helpful in promoting the biosynthesis of curcumin, and using metabolic engineering or biosynthesis-related technologies to produce curcumin cell factories. of great significance.
  • the first object of the present invention is to solve the problem of low activity of existing curcumin synthase CURS and disclose a curcumin synthase derived from Yujin.
  • the curcumin synthase belongs to type III polyketide synthases (PKSs). It has good activity in catalyzing the substrate carboxylic acid to generate curcumin, monodemethoxycurcumin and tetrahydrobisdemethoxycurcumin.
  • PPSs polyketide synthases
  • the second object of the present invention is to disclose a curcumin synthase encoding gene derived from Wenyujin.
  • the nucleotide sequence of the encoding gene is as shown in SEQ.ID NO.1.
  • the third object of the present invention is to disclose a recombinant vector constructed from the curcumin synthase encoding gene derived from Yujin.
  • the recombinant vector is pYES2::CwPKS, which is obtained by connecting the curcumin synthase encoding gene shown in SEQ.ID NO.1 and the pEASY-BluntZero vector.
  • the fourth object of the present invention is to disclose the recombinant genetically engineered bacteria prepared by transformation with the above recombinant vector.
  • the recombinant genetically engineered bacterium is Saccharomyces cerevisiae BY4741/pESC-LEU::At4CL-ClDCS/pYES2::CwPKS, which is obtained by transforming the recombinant vectors pESC-LEU::At4CL-ClDCS and pYES2::CwPKS into Saccharomyces cerevisiae BY4741.
  • Expression of the recombinant vector pESC-LEU::At4CL-ClDCS in yeast cells can convert ferulic acid into ferulyldiketo-CoA, providing precursors for the synthesis of curcumin and monodemethoxycurcumin;
  • recombinant vector pESC -LEU::At4CL-ClDCS expression in yeast cells can convert dihydrocoumaric acid into dihydrocoumaryldiketo-CoA, providing the precursor for tetrahydrobisdemethoxycurcumin.
  • the fifth object of the present invention is to disclose the application of curcumin synthase derived from Wen Yujin in the preparation of curcumin and its derivatives, specifically to catalyze ferulic acid to generate curcumin, monodemethoxycurcumin and tetrahydrobisde. Methoxycurcumin Bio.
  • the amino acid sequence provided by the invention is a curcumin synthase (i.e., CwPKS) derived from Yujin as shown in SEQ.ID NO.2. It has been experimentally proven that the protein with the above structure belongs to a polyketide synthase, and in feruloyl-CoA In the presence of malonyl-CoA, diketo-CoA synthase (DCS) and curcumin synthase CwPKS are co-incubated to obtain curcumin.
  • CwPKS curcumin synthase
  • Monodemethoxycurcumin was obtained by co-incubation of diketo-CoA synthase (DCS) and CwPKS in the presence of feruloyl-CoA, coumaroyl-CoA and malonyl-CoA. In the presence of dihydrocoumaryl-CoA and malonyl-CoA, diketo-CoA synthase (DCS) and CwPKS were co-incubated to obtain tetrahydrobisdemethoxycurcumin. It is not difficult to imagine that, without changing the protein properties, appropriately changing the amino acid sequence will still have the properties of the curcumin synthase of the present invention. For example, a conservative variant polypeptide of the amino acid sequence SEQ ID NO. 2, or an active fragment thereof, or a derivative thereof.
  • the present invention provides a curcumin synthase encoding gene (i.e., CwPKS gene) derived from Yujin.
  • CwPKS gene a curcumin synthase encoding gene derived from Yujin.
  • the nucleotide sequence of the encoding gene is as shown in SEQ ID No. 1.
  • the invention provides a recombinant vector containing the gene encoding the curcumin synthase of Curcuma longifolia. Further, the recombinant vector is prepared as follows: connect the curcumin synthase encoding gene to the pYES2 vector to obtain the ligation product pYES2::CwPKS, which is a recombinant vector containing the curcumin synthase encoding gene of Wenyujin.
  • the present invention also provides a recombinant genetically engineered bacterium containing the gene or recombinant vector encoding the curcumin synthase encoding enzyme.
  • the recombinant genetically engineered bacteria are prepared as follows: co-transformation of pYES2::CwPKS, pESC-LEU::At4CL-ClDCS plasmids, that is, 2 to 3 ⁇ L of plasmid is added to yeast competent cells, 500 ⁇ L of EZ-Solution3 reagent is added and mixed, Incubate in water bath at 30°C for 45 minutes, mix by inverting up and down every 15 minutes, take 100 ⁇ L on Leu-Ura double glucose-deficient solid medium, spread evenly, and culture in a 30°C incubator for 2 to 3 days.
  • the present invention provides an application of the curcumin synthase derived from Curcuma longum in preparing curcumin compounds.
  • the application is: using the above-mentioned curcumin synthase-derived curcumin synthase-containing genetically engineered bacterial cells in 3mL Leu- Ura double glucose-deficient liquid medium, and culture in a shake flask at 30°C, 220 rpm for 24 hours in a dark environment; dilute to a total volume of 5 mL, calculate the volume of bacterial solution to be added, and add Leu-Ura double glucose-deficient liquid medium to a total volume of 5 mL.
  • the volume reaches 5 mL, and is cultured in a shake flask at 30°C, 220 rpm in the dark for 48 hours; add Leu-Ura double glucose-deficient liquid medium until the total volume reaches 20 mL, and incubate in a shake flask at 30°C, 220 rpm in the dark for 48 hours; After the glucose growth phase is over, enter the galactose induction phase. Centrifuge the fermentation broth from the previous phase at 3000 ⁇ g for 2 minutes at 4°C. Discard the supernatant and add 20 mL of Leu-Ura double-deficient galactose liquid medium. Repeat.
  • the present invention invents and applies a method for synthesizing curcumin, monodemethoxycurcumin, and tetrahydrobisdemethoxycurcumin in Saccharomyces cerevisiae using curcumin synthase derived from Wenyujin. It was found that the curcumin synthase derived from Wenyujin can catalyze the precursor carboxylic acid to generate a large amount of curcumin compounds in a yeast expression system, thereby quickly and in large quantities preparing the compounds in yeast, which is of great importance when applied to the synthesis of curcumin. significance.
  • Figure 1 is an LC-MS diagram of the CwPKS catalyzed reaction product, that is, LC-MS analysis of the fermentation products of engineered yeast fed with different precursors.
  • curcumin synthase gene, vector, engineering bacteria and application derived from Curcuma longum of the present invention will be further described below with reference to specific examples.
  • the pESC-LEU::At4CL-ClDCS recombinant plasmid was constructed through existing technologies, containing CoA ligase (4CL, (Arabidopsis thaliana p-coumaroyl:CoA ligase; ID: AY376729)) and diketone-CoA synthetase (Curcuma longa diketide -CoA synthesis; ID: AB495006.1).
  • the nucleotide sequences of At4CL and ClDCS were derived from the NCBI database. They were synthesized by Nanjing Qingke Biotechnology Company after optimization of Saccharomyces cerevisiae codon preference, and constructed into the pESC-Leu vector through gene assembly.
  • a Type III PKS gene (see SEQ.ID NO.1) screened in our laboratory was completely synthesized by Nanjing Qingke Biotechnology Company, and then constructed into the pYES2 expression vector to obtain pYES2:: CwPKS recombinant plasmid.
  • Saccharomyces cerevisiae is chemically transformed by using ZYMO RESEARCH’s Frozen-EZ Yeast Transformation II Kit. And the transformed yeast competent cells were gradient frozen.
  • Co-transformation of pESC-LEU::At4CL-ClDCS and pYES2::CwPKS plasmids Add 2 to 3 ⁇ L of plasmid to yeast competent cells, add 500 ⁇ L of EZ-Solution3 reagent and mix well, bathe in a 30°C water bath for 45 minutes, and mix by inverting up and down every 15 minutes. Homogenize once, take 100 ⁇ L on Leu-Ura double glucose-deficient solid medium, spread evenly, and culture in a 30°C incubator for 2 to 3 days.
  • Add 200 ⁇ L of activated bacterial liquid to 20mL Leu-Ura double glucose-deficient liquid medium at the ratio of bacterial liquid: culture medium 1:100.
  • the fermentation bacteria liquid was broken (JN-02C low-temperature and high-pressure continuous flow cell crusher, 4°C, 1800bar).
  • the broken fermentation liquid was mixed with an equal volume of ethyl acetate, and ultrasonic extraction was performed for 30 minutes.
  • the extract was heated to 3000 Centrifuge at a speed of The ethyl acetate phase was rotary evaporated to dryness, then reconstituted with 1 mL of acetonitrile.
  • the reconstituted liquid was filtered with a 0.22 ⁇ m nylon filter, and the filtrate was stored in a sampling bottle.
  • the curcumin content in the reconstituted liquid was detected using a liquid chromatography-mass spectrometry (liquid chromatography-single quadrupole mass spectrometer, Agilent 1260Infinity II/6125) detection method.
  • 430nm UV wavelength and MS chart were used to characterize curcumin.
  • 280nm wavelength and MS chart (m/z 371) to characterize tetrahydrocurcumin
  • the injection volume was 10 ⁇ L.
  • the yeast engineered strain carrying CwPKS expression can utilize the substrate ferulic acid to generate curcumin; utilize the substrates ferulic acid and coumaric acid to generate bisdemethoxycurcumin; utilize the substrate dihydrocoumar acid, producing tetrahydrobisdemethoxycurcumin. No product was formed when coumaric acid was added alone.

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Abstract

提供一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用。该姜黄素合成酶的氨基酸序列如SEQ.IDNO.2 所示。其编码基因序列如SEQ.ID NO.1所示。构建的重组载体为pYES2::CwPKS。重组基因工程菌为Saccharomyces cerevisiae BY4741/pESC::LEU::At4CL-CIDCS/pYES2::CwPKS。该基因工程菌具有利用阿魏酸生成的姜黄素、利用阿魏酸和香豆酸生成单去甲氧基姜黄素，利用二氢香豆酸生成四氢双去甲氧基姜黄素的能力；能在酵母细胞中快速，大量地制备所述的催化剂，用于姜黄素类化合物的大量合成。

Description

一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用
本申请要求于2022年04月18日提交中国专利局、申请号为CN202210405041.3、发明名称为“一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，特别涉及一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及其制备姜黄素及其衍生物的应用。
背景技术
姜黄素(Curcumin)是从姜科、天南星科中的一些植物的根茎中提取的一种二酮类化合物，化学式为C 21H 20O 6。在药品领域，姜黄素对II型糖尿病具有较好的治疗作用。姜黄素还可以抑制人类免疫缺陷病毒的病毒活性，具有抗艾滋病功效。姜黄素对治疗癌症、风湿、炎性眼病、肠道疾病以及口腔癌和黏膜白斑病方面均有效果，且安全性高，药效明显，在天然姜黄素类化合物中，姜黄素比去甲氧基姜黄素和双去甲氧基姜黄素在抗氧化及修复DNA等方面具有更高的活性，是发挥药效的最主要成分。同时因其为橙黄色结晶粉末，味稍苦，不溶于水的特性常在食品生产中用作着色剂。
温郁金(CurcumawenyujinY.H.Chen&C.Ling)是姜科姜黄属郁金的栽培品种，温郁金的新鲜根茎切片称“片姜黄”，能行气破瘀，通经络。用于风湿痹痛，心腹积痛、胸肋疼痛，经闭腹痛，跌打损伤等血瘀气滞的症候。温郁金中含有的化学成分种类繁多，主要有倍半萜、单萜类、二萜类、姜黄素类、多糖类等。目前，姜黄素的生产方法主要为植物提取法，但从天然植物温郁金中提取分离姜黄素仍存在许多技术难点：第一，温郁金植株仅在叶片和块根中含有极少量姜黄素，其中叶片中的姜黄素含量更低；同时姜黄素的含量易受品种、叶片和根茎质量、种植环境等诸多因素影响。第二，姜黄素与许多结构相似的姜黄素类化合物混杂在一起，必须通过精密分馏、分子蒸馏进行初分，再通过薄层色谱和柱层析，经过多次分离才能得到纯品，因此姜黄素的提取工艺要求高、难度大。总之，这种方法获得的姜黄素产量有限，后续分离过程昂贵、耗时。
采用酶催化方法进行姜黄素的生物合成具有一定优势，例如反应条件温和、 选择性高、产物专一性高。而进行酶催化的首要条件是优质的生物催化剂的获得。姜黄素合成酶(Curcumin Synthase,CURS)能催化肉桂酰二酮基乙酰半胱胺(一种辅酶A酯的模拟物)和阿魏酰-CoA体外生成类姜黄素。在阿魏酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和CURS共孵育得到姜黄素。在阿魏酰-CoA、香豆酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和CURS共孵育得到单去甲氧基姜黄素。在二氢香豆酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和CURS共孵育得到四氢双去甲氧基姜黄素。但是已报道的CURS的活性不高，因此，研究优质的姜黄素合成酶基因的克隆、表达，对促进姜黄素的生物合成，以及利用代谢工程或者生物合成相关技术进行姜黄素的细胞工厂生产，具有重要的意义。
发明内容
本发明的第一个目的是针对现有姜黄素合成酶CURS的活性不高的问题，公开一种温郁金来源的姜黄素合成酶，该姜黄素合成酶属于III型聚酮合酶(PKSs)，具有很好的催化底物羧酸生成姜黄素、单去甲氧基姜黄素及四氢双去甲氧基姜黄素的活性。所述酶的氨基酸序列如SEQ.ID NO.2所示。
本发明的第二个目的在于公开了一种温郁金来源的姜黄素合成酶编码基因，所述编码基因的核苷酸序列如SEQ.ID NO.1所示。
本发明的第三个目的在于公开了温郁金来源的姜黄素合成酶编码基因构建的重组载体。所述重组载体为pYES2::CwPKS，是由SEQ.ID NO.1所示的姜黄素合成酶编码基因与pEASY-BluntZero载体连接得到。
本发明第四个目的在于公开了上述重组载体转化制备的重组基因工程菌。所述重组基因工程菌为Saccharomyces cerevisiae BY4741/pESC-LEU::At4CL-ClDCS/pYES2::CwPKS，是由重组载体pESC-LEU::At4CL-ClDCS及pYES2::CwPKS转化至Saccharomyces cerevisiae BY4741中得到。重组载体pESC-LEU::At4CL-ClDCS在酵母细胞中的表达可将阿魏酸生成阿魏酰二酮基辅酶A，为姜黄素和单去甲氧基姜黄素合成提供前体；重组载体pESC-LEU::At4CL-ClDCS在酵母细胞中的表达可将二氢香豆酸生成二氢香豆酰二酮基辅酶A，为四氢双去甲氧基姜黄素提供前体。
本发明第五个目的在于公开了温郁金来源的姜黄素合成酶在制备姜黄素及其衍生物中的应用，具体是催化阿魏酸生成姜黄素、单去甲氧基姜黄素及四氢 双去甲氧基姜黄素生物。
其中，所述的应用为：将重组基因工程菌Saccharomyces cerevisiae BY4741/pESC-LEU::At4CL-ClDCS/pYES2::CwPKS，以菌液：培养基＝1：100的比例向3mL Leu-Ura双缺葡萄糖液体培养基中加入30μL的菌液，与30℃、220rpm黑暗环境下摇瓶培养24小时；活化后进入发酵的第一阶段，即葡萄糖生长阶段，以菌液：培养基＝1：100的比例向20mL Leu-Ura双缺葡萄糖液体培养基中加入200μL活化后的菌液，与30℃、220rpm黑暗环境下摇瓶培养48小时；葡萄糖生长阶段结束后进入发酵的第二阶段，即半乳糖诱导阶段，将第一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并加入20μL阿魏酸溶液(200mM)，与30℃、220rpm黑暗环境下摇瓶培养48小时。
本发明提供的氨基酸序列如SEQ.ID NO.2所示的一种温郁金来源的姜黄素合成酶(即CwPKS)，经实验证明，上述结构的蛋白质属于聚酮合酶，在阿魏酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和姜黄素合成酶CwPKS共孵育得到姜黄素。在阿魏酰-CoA、香豆酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和CwPKS共孵育得到单去甲氧基姜黄素。在二氢香豆酰-CoA和丙二酰-CoA存在下，二酮基-CoA合成酶(DCS)和CwPKS共孵育得到四氢双去甲氧基姜黄素。不难想到的是，在不改变蛋白质特性的情况，适当改变氨基酸序列，仍具有本发明姜黄素合成酶的特性。比如，氨基酸序列SEQ ID NO.2的保守性变异多肽，或其活性片段、或其衍生物。
本发明提供一种温郁金来源的姜黄素合成酶编码基因(即CwPKS基因)，所述编码基因核苷酸序列如SEQ ID No.1所示。
本发明提供一种含有所述温郁金姜黄素合成酶编码基因的重组载体。进一步，所述重组载体按如下方法制备：将姜黄素合成酶编码基因与pYES2载体连接，获得连接产物pYES2::CwPKS，即为含有温郁金姜黄素合成酶编码基因的重组载体。将重组载体pYES2::CwPKS转化Saccharomyces cerevisiae BY4741细胞中，在含有URA3缺陷的葡萄糖液体培养基中，并在30℃、220rpm黑暗环境下摇瓶培养24小时，即获得含有温郁金姜黄素合成酶编码基因的重组载体。
本发明还提供一种含有所述温郁金姜黄素合成酶编码基因或重组载体的重 组基因工程菌。所述重组基因工程菌按如下方法制备：pYES2::CwPKS、pESC-LEU::At4CL-ClDCS质粒的共转化，即2～3μL质粒加入酵母感受态细胞中，加入500μL EZ-Solution3试剂混匀，30℃水浴45min，每隔15min上下颠倒混匀一次，取100μL在Leu-Ura双缺葡萄糖固体培养基上，涂布均匀，于30℃培养箱中培养2～3天。
本发明提供一种所述温郁金来源的姜黄素合成酶在制备姜黄素类化合物中的应用，所述的应用为：以上述含温郁金来源的姜黄素合成酶编码基因工程菌菌体于3mL Leu-Ura双缺葡萄糖液体培养基中，并在30℃、220rpm黑暗环境下摇瓶培养24小时；稀释至总体积为5mL计算加入菌液的体积，并加入Leu-Ura双缺葡萄糖液体培养基至总体积达到5mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到20mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时；葡萄糖生长阶段结束后，进入半乳糖诱导阶段，将前一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并分别加入前体20μL阿魏酸溶液(200mM)，阿魏酸溶液(200mM)和香豆酸(200mM)组合，二氢香豆酸(200mM)，共三组，三个重复，于30℃、220rpm黑暗环境下摇瓶培养48小时。
本发明具有以下有益效果：
本发明对温郁金来源的姜黄素合成酶在酿酒酵母中进行姜黄素、单去甲氧基姜黄素、四氢双去甲氧基姜黄素合成方法的发明与应用。发现温郁金来源的姜黄素合成酶可在酵母表达体系中催化前体羧酸生成大量的姜黄素化合物，从而在酵母中快速，大量地制备所述的化合物，应用于姜黄素的合成，具有重要的意义。
附图说明
图1为CwPKS催化反应产物的LC-MS图，即LC-MS分析不同前体饲喂的工程酵母菌的发酵产物。
具体实施方式
为使本发明的技术方案便于理解，以下结合具体实施例对本发明一种温郁金来源的姜黄素合成酶、基因、载体、工程菌及应用作进一步的说明。
1、工程菌的设计
通过现有技术构建了pESC-LEU::At4CL-ClDCS重组质粒，含有CoA连接酶(4CL，(Arabidopsis thaliana p-coumaroyl:CoA ligase；ID:AY376729))和二酮-CoA合成酶(Curcuma longa diketide-CoA synthase；ID:AB495006.1)。At4CL和ClDCS的核苷酸序列来源于NCBI数据库，经过酿酒酵母密码子偏好性优化后由南京擎科生物技术公司合成，并通过基因组装构建到pESC-Leu载体上。
将本实验室筛选到的一条TypeⅢPKS基因(见SEQ.ID NO.1)进行酿酒酵母宿主优化后，由南京擎科生物技术公司进行序列全合成，继而构建到pYES2表达载体上，获得pYES2::CwPKS重组质粒。
酿酒酵母菌通过使用ZYMO RESEARCH的Frozen-EZ Yeast Transfromation II试剂盒对酵母细胞进行化学转化。并对转化后的酵母感受态细胞进行梯度冻存。
pESC-LEU::At4CL-ClDCS、pYES2::CwPKS质粒的共转化：取2～3μL质粒加入酵母感受态细胞中，加入500μL EZ-Solution3试剂混匀，30℃水浴45min，每隔15min上下颠倒混匀一次，取100μL在Leu-Ura双缺葡萄糖固体培养基上，涂布均匀，于30℃培养箱中培养2～3天。
2、定性发酵：对上述所设计的工程菌是否有合成姜黄素的功能进行确定，采取阿魏酸饲喂的定性发酵的方法，以发酵后发酵液是否变为黄色为判断依据，初步确定是否有功能。
首先对酵母工程菌进行活化，以菌液：培养基＝1：100的比例向3mL Lea-Ura双缺葡萄糖液体培养基中加入30μL的菌液，于30℃、220rpm黑暗环境下摇瓶培养24小时；活化后进入发酵的第一阶段，即葡萄糖生长阶段，以菌液：培养基＝1：100的比例向20mL Leu-Ura双缺葡萄糖液体培养基中加入200μL活化后的菌液，于30℃、220rpm黑暗环境下摇瓶培养48小时；葡萄糖生长阶段结束后进入发酵的第二阶段，即半乳糖诱导阶段，将第一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并加入20μL阿魏酸溶液(200mM)，与30℃、220rpm黑暗环境下摇瓶培养48小时。
为探索CwPKS的底物选择性，如上所述，添加阿魏酸和香豆酸的组合，以及二氢阿魏酸和阿魏酸，二氢香豆酸和香豆酸的组合，分析其产物特性。
3、姜黄素类化合物的提取
首先对发酵菌液进行菌体破碎(JN-02C低温高压连续流细胞破碎机，4℃，1800bar)，已破碎的发酵液与等体积的乙酸乙酯混匀，超声提取30min，提取液以3000×g的转速离心5min，产生明显分层，将上层乙酸乙酯相移入新管中，水相管中再加入等体积的乙酸乙酯混匀，重复上述操作直至下层水相没有颜色，所得的乙酸乙酯相旋蒸至干燥，用1mL乙腈复溶，用0.22μm尼龙滤膜过滤复溶液体，滤液保存于进样瓶中。
5、姜黄素类化合物的检测
采用液相质谱联用(液相色谱-单四极杆质谱仪，安捷伦1260Infinity II/6125)的检测方法检测复溶液体中姜黄素的含量。C18反相色谱柱，流动相A为0.1％的甲酸，流动相B为乙腈(加了0.1％的甲酸)，流量为0.4mL/min，B相梯度洗脱(0-6min 44％-44％，6-15min 44％-45％，15-20min 45％-46％，20-26min 46％-44％)，柱温30℃，430nm紫外波长及MS图(m/z＝367)对姜黄素进行定性，430nm紫外波长及MS图(m/z＝337)对姜黄素进行定性360nm波长及MS图(m/z＝369)对二氢姜黄素进行定性，280nm波长及MS图(m/z＝371)对四氢姜黄素进行定性，280nm波长及MS图(m/z＝311)对四氢双去甲氧基姜黄素进行定性，进样量为10μL。
图1结果表明：携带CwPKS表达的酵母工程菌可利用底物阿魏酸，生成姜黄素；利用底物阿魏酸和香豆酸，生成双去甲氧基姜黄素；利用底物二氢香豆酸，生成四氢双去甲氧基姜黄素。单独添加香豆酸没有产物生成。
以上所述，仅为本发明的较佳实施例，并非对本发明作任何形式上和实质上的限制，凡熟悉本专业的技术人员，在不脱离本发明技术方案范围内，当可利用以上所揭示的技术内容，而作出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对以上实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (21)

  1. 一种温郁金来源的姜黄素合成酶，其特征在于，所述姜黄素合成酶的氨基酸序列如SEQ.ID NO.2所示。
  2. 编码权利要求1所述温郁金来源的姜黄素合成酶的基因，其特征在于，所述基因的核苷酸序列如SEQ.ID NO.1所示。
  3. 一种表达权利要求1所述温郁金来源的姜黄素合成酶的重组载体，其特征在于，所述重组载体为pYES2::CwPKS，由SEQ.ID NO.1所示的编码姜黄素合成酶的基因与pYES2载体连接得到。
  4. 权利要求3所述的重组载体的构建方法，其特征在于，由如下步骤组成：
    将权利要求2所述的编码姜黄素合成酶的基因与pYES2载体连接，获得连接产物pYES2::CwPKS；
    将所述连接产物pYES2::CwPKS转化Saccharomyces cerevisiae BY4741细胞中，在含有URA3缺陷的葡萄糖液体培养基中，30℃、220rpm黑暗环境下摇瓶培养24小时，获得所述重组载体。
  5. 一种由权利要求3所述重组载体转化制备的重组基因工程菌，其特征在于，所述重组基因工程菌为Saccharomyces cerevisiae BY4741/pESC-LEU::At4CL-ClDCS/pYES2::CwPKS，是由重组载体pYES2::CwPKS及pESC-LEU::At4CL-ClDCS转化至Saccharomyces cerevisiae BY4741中得到。
  6. 权利要求5所述的重组基因工程菌的制备方法，其特征在于，由如下步骤组成：
    共转化pYES2::CwPKS和pESC-LEU::At4CL-ClDCS，即将2～3μL pYES2::CwPKS和pESC-LEU::At4CL-ClDCS的混合物加入酵母感受态细胞中，加入500μL EZ-Solution3试剂混匀，30℃水浴45min，每隔15min上下颠倒混匀一次，取100μL在Leu-Ura双缺葡萄糖固体培养基上，涂布均匀，30℃培养箱中培养2～3天，获得所述重组基因工程菌。
  7. 权利要求1所述温郁金来源的姜黄素合成酶或权利要求2所述的基因或权利要求3所述的重组载体或权利要求4所述的构建方法得到的重组载体或权利要求5所述的重组基因工程菌或权利要求6所述的制备方法得到的重组基因工程菌在制备姜黄素及其衍生物中的应用。
  8. 根据权利要求7所述的应用，所述衍生物包括双去甲氧基姜黄素和四氢双去甲氧基姜黄素中的一种或两种。
  9. 根据权利要求7所述的应用，其特征在于，所述的应用为：诱导培养权利要求4所述的重组基因工程菌，于所述诱导培养的过程中，在半乳糖诱导阶段加入底物阿魏酸，继续诱导培养，生成姜黄素。
  10. 根据权利要求8所述的应用，其特征在于，所述的应用为：诱导培养权利要求4所述的重组基因工程菌，于所述诱导培养的过程中，在半乳糖诱导阶段加入底物阿魏酸和香豆酸，继续诱导培养，生成双去甲氧基姜黄素。
  11. 根据权利要求8所述的应用，其特征在于，所述的应用为：诱导培养权利要求4所述的重组基因工程菌，于所述诱导培养的过程中，在半乳糖诱导阶段加入底物二氢香豆酸，继续诱导培养，生成四氢双去甲氧基姜黄素。
  12. 根据权利要求9～11任一项所述的应用，其特征在于，所述诱导培养的条件为30℃、220rpm黑暗环境。
  13. 根据权利要求9所述的应用，其特征在于，所述诱导培养的方法由如下步骤组成：
    将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中，并在30℃、220rpm黑暗环境下摇瓶培养24小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到5mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到20mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时，得第一阶段的发酵液；
    葡萄糖生长阶段结束后，进入半乳糖诱导阶段，将所述第一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并加入前体20μL阿魏酸溶液，于30℃、220rpm黑暗环境下摇瓶培养48小时。
  14. 根据权利要求13所述的应用，其特征在于，所述阿魏酸溶液中阿魏酸的浓度为200mM。
  15. 根据权利要求13所述的应用，其特征在于，所述将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中为将30μL权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中。
  16. 根据权利要求10所述的应用，其特征在于，所述诱导培养的方法由如下步骤组成：
    将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中，并在30℃、220rpm黑暗环境下摇瓶培养24小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到5mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到20mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时，得第一阶段的发酵液；
    葡萄糖生长阶段结束后，进入半乳糖诱导阶段，将所述第一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并加入前体20μL阿魏酸溶液和香豆酸溶液组合，于30℃、220rpm黑暗环境下摇瓶培养48小时。
  17. 根据权利要求16所述的应用，其特征在于，所述阿魏酸溶液中阿魏酸的浓度为200mM；所述香豆酸溶液中香豆酸的浓度为200mM。
  18. 根据权利要求16所述的应用，其特征在于，所述将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中为将30μL权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中。
  19. 根据权利要求11所述的应用，其特征在于，所述诱导培养的方法由如下步骤组成：
    将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中，并在30℃、220rpm黑暗环境下摇瓶培养24小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到5mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时；加入Leu-Ura双缺葡萄糖液体培养基至总体积达到20mL，并在30℃、220rpm黑暗环境下摇瓶培养48小时，得第一阶段的发酵液；
    葡萄糖生长阶段结束后，进入半乳糖诱导阶段，将所述第一阶段的发酵液以3000×g的转速于4℃离心2min，弃上清液，加入20mL Leu-Ura双缺半乳糖液体培养基，重悬菌体，并加入前体20μL二氢香豆酸溶液，于30℃、220rpm黑暗环境下摇瓶培养48小时。
  20. 根据权利要求19所述的应用，其特征在于，所述二氢香豆酸溶液中二氢香豆酸的浓度为200mM。
  21. 根据权利要求19所述的应用，其特征在于，所述将权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中为将30μL权利要求4所述重组基因工程菌的菌液接种于3mL Leu-Ura双缺葡萄糖液体培养基中。
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