Cytochrome p450 oxidase and its encoding gene and use thereof

Abstract

The present application relates to a cytochrome P450 oxidase, and a gene encoding the cytochrome P450 oxidase, the enzyme is involved in the final key step of the biosynthesis of important active ingredients of Tripterygium wilfordii, triptolide and / or neo-triptolide, and can be used for biosynthesis of triptolide and / or neo-triptolide.

Description

Technical Field

[0001] This invention relates to a cytochrome P450 oxidase and the gene encoding the enzyme, which is involved in the final key step of the biosynthesis of the important active components of Tripterygium wilfordii, Tripterygium wilfordii oxytocin and / or neo-Tripterygium wilfordii oxytocin, and belongs to the field of medicinal plant genetic engineering. Background Technology

[0002] Tripterygium wilfordii Hook.f. is the plant source of the traditional Chinese medicine Tripterygium wilfordii, widely used in the treatment of rheumatoid arthritis and inflammation. Diterpenoids are the main active components of Tripterygium wilfordii, including triptolide and tripterifordin. In the 1990s, Chen et al. discovered that the ethanol extract of Tripterygium wilfordii root exhibited significant anti-HIV activity. Further chemical separation and structural identification revealed that the kauriane-type diterpenoid lactones—tripterifordin and neotripterifordin—possessed significant anti-HIV-1 activity, showing great potential for development into anti-AIDS drugs. Currently, the acquisition of these two compounds mainly relies on extraction and isolation from the original plant. However, the content of diterpenoid secondary metabolites in Tripterygium wilfordii is extremely low, greatly limiting their application and development. By mining pathway genes to elucidate the biosynthetic pathways of terpenoid components in Tripterygium wilfordii, and utilizing synthetic biology strategies to achieve efficient heterologous production of target components, has become an environmentally friendly, efficient, and safe way to obtain natural resources.

[0003] Diterpenoids in plants are converted into the universal substrates of terpenoids, isopentenyl pyrophosphate (IPP) and its isomer, dimethylpropenyl pyrophosphate (DMAPP), via the cytoplasmic mevalonic acid (MVA) pathway and the plastid 2-methylerythritol-4-phosphate (MEP) pathway. Under the action of GGPPS, three IPP molecules are linked end-to-end with one DMAPP molecule to form GGPP. Then, through the catalysis of specific diterpenoid synthases, a kauriane-type diterpenoid skeleton is formed. It has been confirmed that TwCPS3 and TwKSL2 can catalyze the formation of the diterpenoid skeleton of Tripterygium wilfordii (Hansen NL, Heskes AM, Hamberger B, et al. The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily[J].The Plant Journal,2016,89(3)). Su et al. discovered that TwKO can catalyze the C-19 oxidation of 16-hydroxy-enantio-kaurethane to 16-hydroxy-enantio-kaurethane alcohol, which can further be oxidized to 16-hydroxy-enantio-kaurethane acid. The biological processes of C-20 oxidation and lactone ring formation of 16-hydroxy-enantio-kaurethane acid urgently need further investigation, such as... Figure 1 As shown.

[0004] Most post-oxidative modifications of terpenoids are accomplished by cytochrome P450-dependent monooxygenases (CYP450s). CYP450s are a large family of proteins in nature, capable of catalyzing various reactions of substrates under mild conditions, including hydroxylation of CH bonds, epoxidation of C=C double bonds, carboxylation of methyl groups, and ring rearrangement. Like versatile dancers, they can recognize various types of substrates, including not only terpenoids but also fatty acids, sterols, flavonoids, and alkaloids. Furthermore, the types of reactions catalyzed by CYP450s are diverse. However, research progress on CYP450s has been relatively slow, largely due to the lack of correlation between CYP450 sequences and functions. Even subtle differences in amino acid sequences can lead to completely different catalytic activities, making the function of CYP450s extremely difficult to predict and greatly limiting the development and application of plant CYP450 gene resources.

[0005] However, in the existing technology, no publicly disclosed or reported CYP450 oxidase has been found that directly participates in the biosynthesis of Tripterygium wilfordii, which can achieve the C-20 oxidation and lactone cyclization of 16-hydroxy-enantiomeric acid. Summary of the Invention

[0006] This invention successfully obtained a novel CYP450 oxidase by measuring the distribution differences of Tripterygium wilfordii in different tissues of plants and combining the tissue transcriptome data of candidate genes. The corresponding CYP450 oxidase gene was cloned, and experiments confirmed that it is the final key enzyme in the biosynthesis of Tripterygium wilfordii or / and neo-Tripterygium wilfordii.

[0007] Therefore, in a first aspect, the present invention provides an isolated protein, said isolated protein being a cytochrome p450 oxidase (hereinafter referred to as TW19G00978), a key enzyme involved in the biosynthesis of triptolide and / or neotriptolide, said enzyme having the amino acid sequence shown in SEQ ID NO: 2, or a peptide having one or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 2, which has the same function.

[0008] In a second aspect, the present invention provides a polynucleotide (or encoding gene, hereinafter referred to as TW19G00978) encoding the enzyme described herein, wherein the polynucleotide has the nucleotide sequence shown in SEQ ID NO: 1.

[0009] A third aspect of the present invention provides an expression vector comprising the polynucleotide described in the second aspect of the present invention, wherein the expression vector may be, for example, an expression vector selected from the pESC series.

[0010] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids and granules. In producing the cytochrome p450 oxidase TW19G00978 of this invention, the nucleotide sequence encoding the TW19G00978 gene can be operably linked to an expression regulatory sequence, thereby forming an expression vector for synthesizing triptolide and / or neo-triptolide. The term "operably linked" refers to DNA segments arranged in such a way that they can function in a coordinated manner for their intended purpose, such as initiating transcription in a promoter and proceeding through the coding segment to the terminator. It also refers to a situation where certain portions of a linear DNA sequence can influence the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and participates in polypeptide secretion, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter control sequence is transcribed, then it is operably linked to the coding sequence; if the ribosome binding site is positioned to enable translation, then it is operably linked to the coding sequence. Generally, "operationally connected" means adjacent, while for a secretory leader sequence it means adjacent within the reading frame.

[0011] In a fourth aspect, the present invention provides a recombinant host bacterium comprising the polynucleotide described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention, wherein the host bacterium is a yeast, such as BY series yeast or WAT series yeast.

[0012] The host bacteria described in this invention comprise a polynucleotide molecule encoding the cytochrome P450 oxidase TW19G00978 or a variant thereof, or a nucleotide molecule capable of hybridizing with the polynucleotide molecule under stringent conditions, or comprising the expression vector described above. The host cell is selected from bacteria, prokaryotic cells (such as Escherichia coli), fungal cells, yeast cells, insect cells, mammalian cells, or plant cells, preferably yeast cells or plant cells.

[0013] Particularly significant yeasts include *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Pichia methanolica*. Methods for transforming *Saccharomyces cerevisiae* cells with exogenous DNA and preparing recombinant peptides therefrom are disclosed, for example, in Kawassaki, U.S. Patents US4599311, US4931373, US4870008, US5037743, US4845075, etc. Transformed cells are selected by choosing a phenotype determined by a selectable marker, typically drug resistance or growth ability in the absence of a specific nutrient (e.g., leucine). Preferred vector systems for *Saccharomyces cerevisiae* may include the pESC series of expression vectors. Suitable promoters and terminators for yeast include those derived from glycolysis genes (US4599311, US4615974, and US4977092) and alcohol dehydrogenases. Transformation systems for other yeasts, including Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces brittlewallis, Pichia pastoris, Pichia methanolica, Pichia guilloché, and Candida maltose, are also known in the art.

[0014] Transformed or transfected host cells are cultured in a medium containing nutrients and other components essential for the growth of the selected host cells, using conventional methods. A variety of suitable media, including those with known components and complex media, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins, and minerals. If desired, the media may also contain components such as growth factors or serum. Growth media are generally selected for cells containing exogenously added DNA by means of, for example, drug screening or the absence of essential nutrients supplemented by selection markers that can be carried by expression vectors or co-transfected into host cells. Sufficient aeration is provided to the liquid culture using conventional methods, such as shaking Erlenmeyer flasks or aeration in fermenters.

[0015] In this invention, the full-length polynucleotide sequence or fragment thereof encoding the cytochrome p450 oxidase TW19G00978 can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed nucleotide sequences, particularly the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared using conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, followed by splicing the amplified fragments together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. Furthermore, mutants can be introduced into the protein sequences of this invention through chemical synthesis. Besides being produced by recombinant methods, fragments of the protein of this invention can also be produced by solid-phase technology through direct peptide synthesis (Stewart et al., Solid-Phase Pedtide Synthesis, J. Am. Chem. Soc. 85: 2149-2154, 1963). In vitro protein synthesis can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems 431A peptide synthesizer (Foster City, CA). The individual fragments of the protein of this invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

[0016] In this invention, the cloned TW19G00978 gene was used to construct an expression vector. Catalytic experiments confirmed that it has the biological function of catalyzing the C-20 hydroxylation of 16-hydroxy-enantio-kauridine, which then undergoes lactone cyclization with the C-19 carboxyl group to generate triptolide.

[0017] The TW19G00978 gene was integrated into the chromosome of *Saccharomyces cerevisiae* using synthetic biology techniques, and then the expression plasmid of TW19G00978 was introduced into it. The results showed that not only was triptolide detected in the fermentation broth, but also its isomer, neotriptolide, was detected. This represents the first time that heterologous bioproduction of these two genes has been achieved in *Saccharomyces cerevisiae*, possessing extremely important application value. This gene was cloned for the first time from *Tripterygium wilfordii*, and it involves the final key step in the biosynthesis of triptolide and / or neotriptolide.

[0018] Therefore, in a fifth aspect, the present invention provides the use of cytochrome P450 oxidase TW19G00978, or the gene encoding cytochrome P450 oxidase TW19G00978, or an expression vector containing the gene TW19G00978, and a host bacterium containing the expression vector, in regulating and / or synthesizing triptolide and / or neotriptolide.

[0019] The Tripterygium wilfordii and / or neo-Tripterygium wilfordii described in this invention can be prepared by means of a biosynthesis method, the method comprising: introducing the gene encoding cytochrome P450 oxidase TW19G00978 into Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae, fermenting the recombinant Saccharomyces cerevisiae to obtain Tripterygium wilfordii and / or neo-Tripterygium wilfordii, wherein the Saccharomyces cerevisiae is, for example, the BY4741 yeast strain.

[0020] The present invention provides a method for the biosynthesis of Tripterygium wilfordii oxytocin and / or neo-Tripterygium wilfordii oxytocin: For example, it can utilize "modular yeast chromosome integration technology" (Li S, et al. Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae[J]. Biotechnol Biofuels, 2016, 9:232.) to integrate the TW19G00978 expression cassette encoding the cytochrome p450 oxidase TW19G00978 gene into yeast, and produce Tripterygium wilfordii oxytocin and / or neo-Tripterygium wilfordii oxytocin through yeast fermentation. This invention has significant theoretical and practical implications for cultivating high-quality medicinal plant varieties, especially for cultivating Tripterygium wilfordii varieties with high Tripterygium wilfordii oxytocin or neo-Tripterygium wilfordii oxytocin content. Attached Figure Description

[0021] Figure 1 This refers to the biosynthetic pathway of Tripterygium wilfordii.

[0022] Figure 2 The distribution of Tripterygium wilfordii fudin in different tissues of Tripterygium wilfordii plant is shown. ** indicates P<0.01, and * indicates P<0.05.

[0023] Figure 3 This is a schematic diagram of the TW19G00978 gene fragment encoding the cytochrome P450 oxidase gene. The left band is the DNA marker, and the right band is the TW19G00978 gene encoding the cytochrome P450 oxidase gene.

[0024] Figure 4 The image shows the microsomal enzymatic UPLC / Q-TOF MS plot of cytochrome P450 oxidase TW19G0097. CK represents the empty vector control group, and TW19G00978 represents the group containing the encoding gene TW19G00978.

[0025] Figure 5 The image shows the fermentation detection results of the engineered yeast MW-1, which produces Tripterygium wilfordii and neo-Tripterygium wilfordii. CK represents the empty vector control group, and TW19G00978 represents the group containing the gene encoding TW19G00978. (Peaks a, b, c, and d are new products detected in the yeast fermentation broth containing the TW19G00978 gene. Peaks a and b have a precursor ion peak of 301.2 ppm in positive ion mode, and Peaks c and d have a precursor ion peak of 335.2 ppm in negative ion mode.) Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0029] The Tripterygium wilfordii Hook.f. suspension cells used in the following examples were obtained from the Laboratory of Molecular Pharmacognosy and Traditional Chinese Medicine Resources, Capital Medical University (Wang Jiadian, Zhao Yujun, Zhang Yifeng, et al. Effects of TwHMGR overexpression on the biosynthesis of triptolide and triptolide. Acta Pharmaceutica Sinica, 2018, 53(8):1225-1232). The Tripterygium wilfordii plants were provided by Datian Taoyuan State-owned Forest Farm, Yong'an City, Sanming City, Fujian Province. 16α-hydroxy-ent-kaurene acid was prepared in the Medicinal Chemistry Laboratory of Southwest University (Su P, Guan HY, Zhang YF, et al. Probing the single key amino acid responsible for the novel catalytic function of ent-kaurene oxidase supported by NADPH-Cytochrome P450 reductases in Tripterygium wilfordii[J]. Frontiers in Plant Science,2017,8:1756-1766.).

[0030] Example 1: Tissue distribution pattern of Tripterygium wilfordii extract in Tripterygium wilfordii plants

[0031] 1. Collection and sorting of Tripterygium wilfordii roots, stems, and leaves

[0032] Three fresh Tripterygium wilfordii plants were collected from Sanming City, Fujian Province. They were divided into three groups: roots, stems, and leaves. Each group had three biological replicates. The plants were flash-frozen in liquid nitrogen for 2 minutes. The samples were then pulverized at low temperature using a ball mill until they became a uniform powder and freeze-dried for 48 hours.

[0033] 2. Determination of Tripterygium wilfordii content

[0034] Accurately weigh 200 mg of dried sample powder, add 1 mL of 80% chromatographic grade methanol (v / v), and extract the sample overnight; extract the sample by ultrasonication at 100 W and 40 kHz for 1 h; centrifuge at 12000 × g for 10 min, and filter the supernatant through a 0.22 μm PTFE microporous membrane (Jinteng) to obtain the sample solution. Use UPLC / QQQ MS to determine the content of Tripterygium wilfordii in the sample solution.

[0035] UPLC / QQQ MS liquid chromatography conditions:

[0036] A Waters ACQUITY UPLC HSS T3 column was used for analysis. The mobile phase was 0.1% formic acid in water (A) and acetonitrile (B), with a flow rate of 0.4 mL / min. The flow rates were: 0 min - 50% B, 2 min - 50% B, 4 min - 95% B, 6.5 min - 95% B, 7.5 min - 50% B, and 9 min - 50% B. The injection volume was 2 μL. UPLC / QQQ MS mass spectrometry conditions were: positive ion mode, high-energy scan, precursor ion set to 319.0, cone voltage set to 10 V, and scan time set to 0.2 s. Faction ion 1 was set to 227.2 with a collision voltage of 18 V; faction ion 2 was set to 255.3 with a collision voltage of 17 V. The data acquisition MS range was 50-550 Da.

[0037] Results using GraphPadPrism 7.0 are as follows Figure 2 As shown,

[0038] The experimental results show that the content of Tripterygium wilfordii fucoidin is the highest in the stem, which is significantly higher than that in the leaves and roots. This provides a necessary basis for screening the key enzyme gene at position C-20 of Tripterygium wilfordii fucoidin biosynthesis by means of the tissue-specific distribution of gene expression.

[0039] Example 2: Cloning of candidate gene TW19G00978

[0040] 1. Primer design

[0041] Based on the annotation and screening of Tripterygium wilfordii transcriptome data, the gene ORF sequence fragment was obtained, and primers TW19G00978-F and TW19G00978-R were designed. The primer sequences are as follows:

[0042]

[0043] 2. Full-length PCR amplification

[0044] Using the cDNA obtained in step 1 as a template, PCR amplification was performed using primers TW19G00978-F and TW19G00978-R to obtain the PCR amplification product. PCR reaction conditions: 98℃ for 30s; 98℃ for 20s, 55℃ for 20s, 72℃ for 2min, 35 cycles; 72℃ for 7min, hold at 4℃. After the reaction, 6×DNA Loading Buffer was added to the system, mixed well, and then subjected to 1.5% agarose gel electrophoresis (160V, 15min) to detect the presence of the target band. If the target band appeared, it was recovered using the Gene JET Gel Extraction Kit according to the instructions. A schematic diagram of the TW19G00978 gene fragment is shown below. Figure 3 As shown.

[0045] 3. Ligation of cloning vectors

[0046] Using the pEASY-Blunt Zero cloning vector kit, the gel-recovered product of the full-length clone was ligated to the cloning vector according to the instructions. The ligation product was transformed into Trans1-T1 competent cells, cultured, identified positive clones, and sequenced.

[0047] Sequencing results showed that the sequence of the PCR amplification product is shown in Sequence 1, and it was named TW19G00978. This product encodes a protein consisting of 506 amino acid residues, also named TW19G00978, and its amino acid sequence is shown in Sequence 2. The cloning vector was named B-zero-TW19G00978 plasmid and stored at -20°C.

[0048] Example 3: Biological Function Study of Tripterygium wilfordii TW19G00978

[0049] 1. Construction of expression vector

[0050] (1) Preparation of linearized empty vector

[0051] The pESC-Leu::TwCPR3 vector (Jiadian W, Ping S, Linhui G, et al. A cytochrome P450 CYP81AM1 from Tripterygium wilfordii catalysts the C-15 hydroxylation of dehydroabietic acid[J].Planta,254(5):95.) stored in the laboratory was digested with the restriction endonuclease NotI, and the linearized vector was recovered by gel digestion.

[0052] (2) Preparation of PCR products (target gene)

[0053] Using plasmid B-zero-TW19G00978 as a template, bidirectional primers containing the vector homologous arm sequence were designed for PCR amplification. PCR reaction conditions: 98℃ for 30s; 98℃ for 20s, 55℃ for 20s, 72℃ for 2min, 35 cycles; 72℃ for 7min, hold at 4℃. After the reaction, 6×DNALoading Buffer was added to the system, mixed well, and then detected by 1.5% agarose gel electrophoresis (160V, 15min) to check for the presence of the target band. If the target band appeared, it was recovered using the Gene JET Gel Extraction Kit according to the manufacturer's instructions.

[0054] The primer sequences are as follows:

[0055]

[0056] (3) Seamless splicing of linearized vector with gene fragment with homologous arm

[0057] The TW19G00978 fragment with homologous arms (0.01 pmols) was gently mixed with the linearized support pESC-Leu::TwCPR3 (NotI cut, 0.02 pmols) and reacted at 50 °C for 25 min.

[0058] (4) Transform the ligation product into clone competent cells

[0059] The ligation product was added to 50 μL of Trans1-T1 competent cells and sequenced for verification. The sequencing results were compared with the original sequence using Seqman software. The expression vector with correct sequencing was named "pESC-Leu::(TwCPR3+TW19G00978)" and the plasmid was stored at -20℃ for later use.

[0060] 2. TW19G00978 In vitro microsomal enzymatic assay

[0061] (1) Preparation of BY4741 competent yeast cells and transformation with yeast expression vector

[0062] BY4741 competent yeast cells were prepared using the Zymo Research Frozen-EZ Yeast Transformation Kit II. 1 μg of the expression vector plasmid pESC-Leu::(TwCPR3+TW19G00978) was transformed into the competent yeast cells. Yeast cells transformed with the empty vector pESC-Leu::TwCPR3 (without the target gene) served as a control. The cells were treated in the same way, plated on SD-Leu plates, and incubated at 30°C for 2-3 days until single colonies appeared.

[0063] (2) Preparation of TW19G00978 microparticles

[0064] 1) Pick a single colony and place it in 5 mL of SD-Leu + 2% Glc liquid medium, and incubate overnight at 30℃ and 200 rpm to activate it;

[0065] 2) The activated bacterial solution was prepared with an initial OD value. 600 0.05 μL was inoculated into 150 mL of SD-Leu + 2% Glc liquid medium and incubated at 30°C and 200 rpm until OD reached. 600 Centrifuge at 0.8-1 at room temperature for 4000×g for 5 min, then discard the supernatant;

[0066] 3) Resuspend the bacterial cells in 150ml LYP (1% Yeast Extract, 2% Peptone) + 2% Gal liquid medium and induce them at 30℃ and 200rpm for 16h.

[0067] 4) Centrifuge at 4000×g for 3 min at room temperature, discard the supernatant, resuspend the bacterial cells in 15 mL TEK solution (50 mM Tris-HCl, 1 mM EDTA, pH 7.5, 0.1 M KCl), and let stand at room temperature for 5 min.

[0068] 5) Centrifuge at 4000×g for 3 min at 4℃, discard the supernatant, and resuspend the bacterial cells in 100 mL of pre-cooled TESB (50 mM Tris-HCl, 1 mM EDTA, pH 7.5, 0.1 M KCl, 0.6 M Sorbitol);

[0069] 6) Dissolve the bacterial resuspension in a pre-cooled ATS high-pressure cell disruptor at a pressure of >1000 psi for 7 min and collect the disrupted solution.

[0070] 7) Centrifuge at 12000×g for 15 min at 4℃, take the supernatant and add NaCl (to a final concentration of 0.15 mM) and PEG 4000 (to a final concentration of 0.1 g·mL). -1After mixing thoroughly, let stand in ice for 15 minutes to allow the microparticles to settle completely.

[0071] 8) Centrifuge at 12000×g for 15 min at 4℃, discard the supernatant, and dissolve the precipitated microparticles in 2 mL of pre-cooled TEG (TE solution containing 20% ​​glycerol) for subsequent enzymatic reactions.

[0072] (3) TW19G00978 Microsomal Enzymatic Reaction and Product Detection

[0073] The microsomal enzymatic system is as follows:

[0074]

[0075]

[0076] After adding all components to the above system, mix thoroughly and react at 30℃ and 100 rpm for 12 h. Extract three times with an equal volume of ethyl acetate, combine the extracts, dry under nitrogen, add 100 μL of chromatographic grade methanol, vortex for 30 s to redissolve, centrifuge at 12000 × g for 10 min, and analyze the product using UPLC / Q-TOF MS. The results are as follows: Figure 4 As shown.

[0077] UPLC / Q-TOF MS liquid phase conditions:

[0078] A Waters ACQUITY UPLC HSS T3 analytical column was used. The mobile phase was 0.1% formic acid in water (A) and acetonitrile (B), with a flow rate of 0.4 mL / min. The flow rates were: 0 min - 5% B, 2 min - 40% B, 3 min - 40% B, 13 min - 60% B, 14.5 min - 100% B, 15 min - 100% B, 15.5 min - 5% B, and 16 min - 95% B. The injection volume was 2 μL. UPLC / Q-TOF MS mass spectrometry conditions were: detection in both positive and negative ion modes; during high-energy scans, the ramp collision energy was set to 20–40 eV. The data acquisition MS range was 50–1500 Da.

[0079] The results showed that, compared to the control group, the experimental group using 16-hydroxy-enantioretinic acid as a substrate detected a new product peak (Peak c) in negative ion mode, with a retention time of 4.68 min and a molecular weight of 335.2173. Product c's molecular weight increased by 16 compared to the substrate, suggesting that this product originates from the hydroxylation of the substrate. Simultaneously, in positive ion mode, product a (Peak a) was detected, with a retention time of 6.31 min and a molecular weight of 319.2290, consistent with the peak of the Tripterygium wilfordii standard. This indicates that TW19G00978 is the key CYP450 gene involved in the final step of the Tripterygium wilfordii biosynthesis pathway.

[0080] Example 4: Construction of engineered yeast producing Tripterygium wilfordii and neo-Tripterygium wilfordii

[0081] 1. Pathway gene integration into BY-HZ16 yeast chromosome

[0082] (1) Cloning of promoter sequence, terminator, pathway gene, and head and tail homologous arm sequences.

[0083] Based on the promoter, terminator, pathway gene, and head-tail homologous arm sequence information, primers with recognition sites for the restriction endonuclease Bsal were designed as follows:

[0084]

[0085]

[0086] Using the original gene plasmids (Li S, Ding W, Zhang X, et al. Development of amodularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2016, 30(1):232-243.) as templates, PCR amplification was performed under the following conditions: 98℃ for 30s; 98℃ for 20s, 55℃ for 20s, 72℃ for 2min, 35 cycles; 72℃ for 7min, 4℃ hold. After the reaction, 6×DNALoadingBuffer was added to the system, mixed well, and then 1.5% agarose gel electrophoresis was performed (160V, 15min) to detect the presence of the target band. If the target band appeared, the target band was recovered using the Gene JET Gel Extraction Kit according to the instructions.

[0087] (2) Construction of Golden Gate Carrier

[0088] 1) Add 0.1 pmol (pmol = mass ng / (fragment length bp × 0.65)) to each of the above purified DNA fragments (promoter, terminator, and pathway gene) and perform a Golden Gate reaction; the reaction system is as follows:

[0089]

[0090] PCR reaction conditions: 37℃ for 3 min, 16℃ for 4 min, 25 cycles; 50℃ for 5 min; 80℃ for 5 min; 4℃ maintenance.

[0091] 2) Transformation of ligation products into clonal competent cells

[0092] The ligation product was added to 50 μL of Trans1-T1 competent cells and sequenced for verification. The sequencing results were compared with the original sequence using Seqman software. The expression vector plasmid with correct sequencing was stored at -20℃ for later use, thus obtaining the double transcription module.

[0093] 3) Obtaining electrotransgenic module fragments

[0094] Design a dual transcription module 'L1-T' TPI1 -TwCPS3-P TDH3 -P ADH1 -TwKSL2-T PGI -L2'、'L2-T ADH1 -TwCPS3-P PGK1 -P TEF2 -TwKSL2-

[0095] T CYC1 -L3'、'L3-T FBA1 -TwKO-P FBA -P HXT7 -TwCPR3-T PDC1 The cloning primers for -L4' (to enhance precursor supply capacity, express two terpene synthase modules TwCPS3 and TwKSL2, and one redox module TwKO and TwCPR3) are as follows:

[0096]

[0097] Using the constructed Golden Gate vector as a template, PCR amplification was performed to obtain the PCR amplification product. PCR reaction conditions: 98℃ for 30s; 98℃ for 20s, 55℃ for 20s, 72℃ for 3min, 35 cycles; 72℃ for 7min, hold at 4℃; after the reaction, 6×DNA Loading Buffer was added to the system, mixed well, and then 1.5% agarose gel electrophoresis was performed (160V, 15min) to detect the presence of the target band; if the target band appeared, it was recovered using the Gene JET Gel Extraction Kit according to the instructions.

[0098] (3) Electroporation of brewer's yeast cells

[0099] The information on the BY-HZ16 Saccharomyces cerevisiae cell strain retained in the laboratory is as follows:

[0100] BY4742,rox1Δ,erg9::Δ-220-176,yjl064wΔ,ypl062wΔ,ΔTrp1,Trp1::HIS3-PPGK1-BTS1 / ERG20-TADH1-PTDH3-SaGGPS-TTP11-PTEF1-tHMG1-TCYC1. (T.Hu,J.Zhou,Y.Tong,P.Su,L.Huang.Engineering chimeric diterpene synthases and isoprenoidbiosynthetic pathways enables high-level production of miltiradiene inyeast.Metabolic Engineering. 2020; 60:87-96.)

[0101] 1) Take BY-HZ16 Saccharomyces cerevisiae cells retained from the laboratory and streak them onto an SD-His plate. Incubate in the dark at 30°C for 2-3 days until single colonies appear. 2) Pick a single colony and place it in 5ml LYP (1% Yeast Extract, 2% Peptone) + 2% Gal liquid medium. Incubate at 30°C with shaking at 200 rpm until OD (October Expiratory Time) is reached. 600 =0.6-1.0;

[0102] 3) Take the electric rotary cup (0.2cm) soaked in ethanol, rinse with ultrapure water, place it upside down on absorbent filter paper, and sterilize it in a laminar flow hood;

[0103] 4) Take 1-2 mL of bacterial culture into a sterile 1.5 mL EP tube, centrifuge at 10000×g at room temperature for 1 min, and discard the supernatant;

[0104] 5) Add 1 mL of pre-cooled sterile water to resuspend, centrifuge at 10000×g at room temperature for 1 min, and repeat once;

[0105] 6) Add an equal volume of pre-cooled buffer (10 mM LiAc, 10 mM DTT, 0.6 M sorbitol, 10 mM pH 7.5 Tris–HCl).

[0106] Incubate at 25℃ for 20 min; centrifuge at 10000×g at room temperature for 1 min, and discard the supernatant.

[0107] 7) Resuspend in 1 mL of pre-cooled sorbitol (1 M) solution, centrifuge at 10000 × g for 1 min at room temperature, repeat once, and discard the supernatant.

[0108] 8) Resuspend the cells in 100 μL of pre-cooled sorbitol (1 M) solution to prepare competent cells;

[0109] 9) Mix all DNA fragments in equal molar ratios, with a total mass of 500 ng (total volume not exceeding 1 / 10 of the competent cell volume), add to the competent cells, mix well and transfer to an electroporation cuvette (0.2 cm), incubate on ice for 2-5 min; electroporate under the conditions of 2.7 kV, 25 μF, 200 Ω (Bio-Rad, Hercules, CA);

[0110] 10) Immediately after the electric shock, add 1 mL of sorbitol (1 M) solution and transfer to a sterile 1.5 mL EP tube, and incubate at 30°C for 1–2 h;

[0111] 11) Centrifuge at 10000×g at room temperature for 1 min, discard the supernatant, resuspend the bacterial cells in the remaining 100μL solution, drop it onto the center of a defective SD-Ura-His solid plate, spread it evenly with a spreader until all the bacterial solution is completely absorbed, and incubate upside down in a 30℃ incubator for 2-3 days.

[0112] (4) Detection of precursor compounds during fermentation

[0113] 1) Pick 5 single colonies and place them in 50 mL of SD-Ura-His liquid medium, and incubate at 30 °C and 200 rpm for 3 days with shaking.

[0114] 2) Collect the fermentation broth, add an equal volume of ethyl acetate, and sonicate at low temperature for 1 hour;

[0115] 3) Centrifuge at 3000×g for 2 min to collect the upper extract, shake the lower fermentation broth well and add an equal volume of ethyl acetate for ultrasonic extraction once more;

[0116] 4) Combine the two upper extracts, remove the ethyl acetate solvent by vacuum evaporation using a rotary evaporator (water temperature set to 30℃), wash the inner wall of the rotary evaporator flask with 500μL of ethyl acetate three times, and combine the reconstituted solutions;

[0117] 5) The reconstituted solution was dried by blowing with high-purity nitrogen, 100 μL of chromatographic grade methanol was added, the solution was vortexed for 30 s to reconstitute, and centrifuged at 12000×g for 10 min. The supernatant was then subjected to UPLC / Q-TOF MS for product analysis. The strain that could detect the final product 16α-hydroxyl-ent-kaurenoic acid indicated that the pathway genes (TwCPS3, TwKSL2, TwCPR3, TwKO) were successfully integrated. This strain was named BY-HZ16-16OH strain. An equal volume of 50% glycerol was added, and the strain was stored at -80℃ for later use.

[0118] 2. The expression vector TW19G00978 was transformed into BY-HZ16-16OH

[0119] (1) The expression vector TW19G00978 was transformed into BY-HZ16-16OH

[0120] BY-HZ16-16OH yeast competent cells were prepared using the Zymo Research Frozen-EZ Yeast Transformation Kit II. 1 μg of expression vector plasmid pESC-Leu::(TwCPR3+TW19G00978) was transformed into the yeast competent cells, plated on SD-His-Ura-Leu plates, and cultured at 30℃ for 2-3 days until single colonies appeared. Single colonies were picked and cultured in 5 mL of SD-Ura-His-Leu + 2% Glc liquid medium overnight at 30℃ and 200 rpm for activation.

[0121] (2) Fermentation induced by bacterial culture

[0122] 1) The activated bacterial solution was prepared with an initial OD value. 600 0.05 mg was inoculated into 20 mL of SD-Ura-His-Leu + 2% Glc liquid medium and incubated at 30 °C with shaking at 200 rpm for 24 h until OD reached. 600 It reaches approximately 0.8-1.0;

[0123] 2) Collect bacterial cells by centrifugation at 3000×g for 3 min;

[0124] 3) Resuspend the bacterial cells in 20mLYP (1% Yeast extract, 2% Peptone) + 2% Gal, and continue to induce fermentation at 30℃ and 200rpm for 72h.

[0125] (3) Extraction and detection of fermentation products

[0126] 1) Collect the fermentation broth, add an equal volume of 20 mL of ethyl acetate, and sonicate at low temperature for 1 h;

[0127] 2) Centrifuge at 3000×g for 2 min to collect the upper extract, shake the lower fermentation broth well and add an equal volume of ethyl acetate for ultrasonic extraction once more;

[0128] 3) Combine the two upper extracts, remove the ethyl acetate solvent by vacuum evaporation using a rotary evaporator (water temperature set to 30℃), wash the inner wall of the rotary evaporator flask with 500μL of ethyl acetate three times, and combine the reconstituted solutions;

[0129] 4) Dry the reconstituted solution with high-purity nitrogen, add 100 μL of chromatographic grade methanol, vortex for 30 s to reconstitute, centrifuge at 12000 × g for 10 min, and take the supernatant for UPLC / Q-TOF MS analysis. The results are as follows: Figure 5 As shown,

[0130] The results showed that yeast containing the TW19G00978 gene produced four new product peaks containing triptolide and neotriptolide compared to the empty vector control group. In positive ion mode, product a (Peak a) had a retention time of 6.31 min and a molecular weight of 319.2290, consistent with the peak of the standard triptolide; product b (Peak b) had a retention time of 7.64 min and a molecular weight of 319.2290, also consistent with the peak of the standard neotriptolide; in negative ion mode, product c (Peak c) had a retention time of 4.68 min and a molecular weight of 335.2173; and product d (Peak d) had a retention time of 4.93 min and a molecular weight of 335.2253 (it is speculated that products c and d are transition products before lactone formation).

[0131] The yeast strain that can produce Tripterygium wilfordii and neo-Tripterygium wilfordii (BY-HZ16-16OH yeast strain with introduced plasmid pESC-Leu::(TwCPR3+TW19G00978) and can be backfilled with His-Ura-Leu) was named MW-1.

Claims

1. Application of the enzyme with the amino acid sequence shown in SEQ ID NO: 2 in catalyzing the hydroxylation of 16-hydroxy-enantiomeric kaurene acid C-20.

2. The use of the enzyme with the amino acid sequence shown in SEQ ID NO: 2 in the synthesis of triptolide and / or neotriptolide, wherein the use is to catalyze the hydroxylation of 16-hydroxy-enantio-kauridine C-20 in the synthetic pathway of triptolide and / or neotriptolide.

3. The use of a recombinant yeast expressing an enzyme having the amino acid sequence shown in SEQ ID NO: 2 in catalyzing the hydroxylation of 16-hydroxy-enantiomeric kaurene acid C-20, said yeast comprising a polynucleotide encoding the enzyme shown in SEQ ID NO:

2.

4. The use of a recombinant yeast expressing an enzyme having the amino acid sequence shown in SEQ ID NO: 2 in the synthesis of triptolide and / or neotriptolide, wherein the use is to catalyze the hydroxylation of 16-hydroxy-enantioretinic acid C-20 in the synthetic pathway of triptolide and / or neotriptolide, said yeast comprising a polynucleotide encoding the enzyme shown in SEQ ID NO:

2.

5. The application according to claim 3 or 4, wherein the polynucleotide is the nucleotide molecule shown in SEQ ID NO:

1.

6. The application according to claim 3 or 4, wherein the yeast is a BY series yeast.

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