Indole diterpenoid biosynthesis genes and their applications

Abstract

The present invention discloses an indole diterpene biosynthesis gene and its application. tsnG、tsnC、tsnM and tsnB , expanding the gene and enzyme library for the biosynthesis of indole diterpenoid natural products. It can express and produce enzymes TsnG, TsnC, TsnM and TsnB, which can efficiently play the enzyme catalytic function in the biosynthesis of paspaline, realizing the efficient biosynthesis of paspaline, a key precursor of indole diterpenes, and can meet the needs of cell factories for the efficient synthesis of active indole diterpene compounds such as penicillin and shearinines.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an indole diterpene biosynthesis gene and application thereof in the preparation of paspalin and other compounds. Background Art

[0002] Indole diterpenes are a class of fungal natural products with diverse chemical structures and biological activities. The main producers are Penicillium, Aspergillus, and Claviceps. This class of compounds has more than 200 family members, many of which show significant insecticidal activity or insect resistance and are low or non-toxic to mammals, such as polyarthrosporic acid, penicillin, epoxy-janthitrem, shearinines, and nominine. Among them, polyarthrosporic acid has shown high insecticidal activity and no obvious adverse reactions to mammals, making it an important candidate compound for the development of new insecticides. In addition, indole diterpenoids also exhibit antibacterial, antiviral and anticancer effects. For example, tolypocladins have significant inhibitory effects on plant pathogenic fungi, Staphylococcus aureus and other pathogenic bacteria, Oxalerpene A and B have antiviral activity against H1N1 and respiratory syncytial virus, and penicillin exhibits significant anticancer activity against human A-549 and HL-60 cancer cell lines. They are important candidate compounds for antibacterial, antiviral and anticancer drugs.

[0003] Most indole diterpenoids have a paspaline (compound 3)-type basic skeleton, which is an angular hexanuclear ring formed by the fusion of a tetracyclic diterpene with a 5-6-6-6 structure and indole ( Figure 1 ). Paspaline is further modified by prenylation, oxidation, reduction, cyclization, etc. to form indole diterpenoids with different structures, such as penicillin, penicillin, shearinines, etc. The yield of indole diterpenoids in the original strain is low, such as polysporic acid A in the wild type H . pulicicidum The yield in the experiment is only 10 mg / L. People have also tried to obtain indole diterpenes through chemical synthesis, but the chemical synthesis process is long, the conditions are harsh, and the yield is low. These problems make it difficult to optimize the structure of indole diterpenes and the production cost is high, which seriously restricts its development and application potential in pesticides and medicines. Mining the indole diterpenoid biosynthetic genes, reconstructing the biosynthetic pathway in chassis cells such as Saccharomyces cerevisiae with the help of synthetic biology, and efficiently synthesizing the key precursor paspalin and modified active indole diterpenes are effective means to solve the above problems. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the lack of genetically engineered bacteria for efficiently synthesizing the key precursor of indole diterpenoid paspaline in the prior art, and to provide a paspaline biosynthesis gene and its application. The paspaline biosynthesis gene of the present invention and its encoded protein can convert farnesyl pyrophosphate and isopentenyl pyrophosphate into geranylgeranyl pyrophosphate, and then efficiently synthesize paspaline through a series of enzyme catalytic reactions.

[0005] An indole diterpene biosynthesis gene selected from one or more of the following genes:

[0006] (1) Gene encoding geranylgeranyl synthase tq , tq The nucleotide sequence is shown in SEQ ID NO.1;

[0007] (2) Gene encoding geranylgeranyltransferase tqCy , tqCy The nucleotide sequence is shown in SEQ ID NO.2;

[0008] (3) Genes encoding flavin-dependent oxidases tsn , tsn The nucleotide sequence is shown in SEQ ID NO.3;

[0009] (4) Gene encoding indole diterpene cyclase tnB , tnB The nucleotide sequence is shown in SEQ ID NO.4.

[0010] The nucleotide sequence or at least part of the nucleotide sequence may be modified or mutated. These approaches include insertion, substitution or deletion, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutation, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical agents, etc.

[0011] An indole diterpene biosynthesis protein selected from one or more of the following proteins:

[0012] (1) geranylgeranyl synthase TsnG, the amino acid sequence of TsnG is shown in SEQ ID NO.5;

[0013] (2) geranylgeranyltransferase TsnC, the amino acid sequence of TsnC is shown in SEQ ID NO.6;

[0014] (3) flavin-dependent oxidase TsnM, the amino acid sequence of TsnM is shown in SEQ ID NO.7;

[0015] (4) Indole diterpene cyclase TsnB, the amino acid sequence of TsnB is shown in SEQ ID NO.8.

[0016] The polypeptide of the amino acid sequence or at least a part of the sequence may still have biological activity or even new biological activity after removing or replacing certain amino acids, or improve the yield or optimize the protein kinetic characteristics or other properties that are intended to be obtained.

[0017] The indole diterpene is 3-geranylgeranyl indole, 19,20-epoxy-GGI or paspalin.

[0018] A recombinant expression vector comprises the indole diterpene biosynthesis gene.

[0019] A genetically engineered bacterium for producing indole diterpenes, the genetically engineered bacterium comprising the recombinant expression vector.

[0020] The starting strain of the genetically engineered bacteria is yeast, preferably Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0021] A method for preparing indole diterpenoids, the method comprising fermenting and culturing the genetically engineered bacteria in a fermentation medium.

[0022] Beneficial effects of the present invention: The indole diterpene biosynthesis gene and its encoded protein provided by the present invention can efficiently synthesize penicillin in genetically engineered strains 、 Paspalin is a key precursor of active indole diterpene compounds such as shearinines. Therefore, the biocatalytic method provided by the present invention can realize the efficient synthesis of active indole diterpene compounds such as penicillin and shearinines in cell factories, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chemical structural formula of compound 1-3.

[0024] Figure 2 This is the plasmid map of the recombinant vector pXW06F-tsnGC.

[0025] Figure 3 This is the plasmid map of the recombinant vector pXW06F-tsnGCM.

[0026] Figure 4 This is the plasmid map of the recombinant vector pXW06F-tsnGCB.

[0027] Figure 5 LC-MS spectrum of the fermentation extract of Saccharomyces cerevisiae expressing tsnG, tsnC, tsnM and tsnB in heterologous combination.

[0028] Figure 6 is the ESIMS spectrum of compound 1.

[0029] Figure 7 is the hydrogen NMR spectrum of compound 2.

[0030] Figure 8 is the carbon NMR spectrum of compound 2.

[0031] Fig. 9 This is the ESIMS spectrum of compound 2.

[0032] Fig.10 is the H NMR spectrum of compound 3.

[0033] Fig.11 is the carbon NMR spectrum of compound 3.

[0034] Fig.12 This is the ESIMS spectrum of compound 3. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0036] The experimental methods without specifying the specific conditions in the examples were carried out according to conventional methods, and molecular cloning was carried out according to the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the manufacturer.

[0037] Enzymes and kits: Restriction endonucleases and T4 DNA ligase were purchased from NEB. DNA polymerase (PhantaMaster Mix) and seamless cloning kit (ClonExpress Multis One Step Cloning Kit) were purchased from Nanjing Novozyme Biotechnology Co., Ltd. Escherichia coli DH5a competent cells, SuperStain DNA Marker and 6×SuperStain Loading Buffer were purchased from Comverse Century. DNA gel extraction kit (GeneJETTM GelExtraction Kit) was purchased from Thermo Fisher Scientific, USA. Rapid plasmid mini-extraction kit and ampicillin were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., with a storage concentration of 100 mg / mL and a working concentration of 100 µg / mL. Reverse transcription kit (PrimeScript TM RT reagent kit with gDNA Eraser) was purchased from Takara, Japan. RNase removal solution was purchased from Shanghai Beibo Biotechnology Co., Ltd. PCR primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. Frozen yeast transformation kit was purchased from ZYMO RESEARCH Biological Company.

[0038] Chemical reagents and consumables: Analytical grade methanol and ethanol were produced by Sinopharm Chemical Reagent Co., Ltd. Chromatographic grade methanol, acetonitrile, and formic acid were produced by Fisher Chemical. CHROMATOREX C18 MB 100-40 / 75 reversed-phase ODS packing was produced by Fuji Silicon Chemical Co., Ltd., Japan. Deuterated reagents used in nuclear magnetic resonance spectroscopy tests were produced by Cambridge Incorporated (CIL), USA. Poroshell 120 EC-C18 (2.1 mm × 100 mm, 1.5 µm) and ZORBOX RX-C18 reversed-phase semi-preparative HPLC columns (9.4 mm ´ 250 mm, 5 µm) were purchased from Agilent Technologies, USA.

[0039] Main instruments and equipment: BUCHI R-300 rotary evaporator, BUCHI I-300 touch central control unit, BUCHI V-300 PTFE membrane vacuum pump, BUCHI B-300 electric constant temperature water bath, all produced by BUCHI, Switzerland. Low temperature coolant circulation pump produced by Shanghai Zhixin Experimental Instrument Technology Co., Ltd. 1290 Infinity-G6125B ultra-high performance liquid chromatography-single quadrupole mass spectrometer, 1260 Infinity II semi-preparative high performance liquid chromatography, 600MHz nuclear magnetic resonance spectrometer Agilent DD2 produced by Agilent Technologies, USA. 400 MHz nuclear magnetic resonance spectrometer AVANCE produced by Bruker, Germany.

[0040] Culture medium: E. coli culture medium is LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0. SC-Trp medium (g / L): 6.7 g amino acid-free yeast nitrogen source, 0.72 g Trp-DO Supplement, 10 g glucose. 1% YPD medium (g / L): 10 g yeast extract, 10 g glucose, 20 g peptone. 2% YPD medium (g / L): 10 g yeast extract, 20 g glucose, 20 g peptone. Add 2% agar powder when preparing solid culture medium.

[0041] Strain culture conditions and plasmids: Penicillium thiersii NRRL 28147 was purchased from ARS Culture Collection. The genetically engineered strains and plasmids used are shown in Table 1. Escherichia coli was grown in LB medium at 37°C, supplemented with ampicillin when necessary. Saccharomyces cerevisiae recombinant strains were grown in SC-Trp medium at 30°C.

[0042] Table 1 Strains and plasmids used or produced in the present invention

[0043]

[0044] Example 1 Penicillium thiersii NRRL 28147 genome sequencing and assembly

[0045] The Novasek 6000 (PE150) high-throughput sequencing platform was used to Penicillium thiersiiThe NRRL28147 genome was sequenced. After removing low-quality data, a total of 14.17 Gb of high-quality clean data was obtained, with an average insert length of 150 bp. SOAPdenovo was used to assemble the genome, resulting in a genome containing 160 scaffolds and 165 contigs, with a total genome length of 30.73 Mb, a Scaffold N50 length of 2.84 Mb, and a Contig N50 length of 2.19 Mb. A total of 10,280 genes were predicted using bioinformatics analysis tools such as GeneWise_v2.20, SNAP_2010-07-28, Augustus_v3.2.1, GeneMark-ES_v4.21, and EVM_v1.1.1.

[0046] Example 2 Analysis of the Paspalin Biosynthetic Gene Cluster in Penicillium thiersii NRRL 28147

[0047] The antiSMASH online software (https: / / fungismash.secondarymetabolites.org) was used for analysis Penicillium thiersii NRRL 28147 genome sequencing data, a total of 59 biosynthetic gene clusters were predicted, including an indole diterpenoid gene cluster tsn , which may be responsible for the biosynthesis of paspalin. tsn The gene cluster is about 3.4 kb in length and contains 13 genes. NCBI-BLASTp analysis results show that these genes encode transcription factor Tsn5410, hypothetical protein Tsn5411, PLP-dependent aminotransferase Tsn5412, geranylgeranyltransferase (GGT) TsnC, indole diterpene cyclase TsnB, flavin-dependent oxidase (FMO) TsnM, acetyltransferase TsnV, three cytochrome P450 oxidases TsnP, TsnQ, TsnJ, geranylgeranyl synthase (GGPPS) TsnG, short-chain dehydrogenase TsnH, and methyltransferase 5421. Among them, TsnG, TsnC, TsnM and TsnB are closely related to Penicillium mycoides ( Penicillium paxilli ) have homology with the paspaline biosynthetic enzymes PaxG, PaxC, PaxM and PaxB in the wild, with amino acid sequence identities of 58%, 69%, 62% and 69%, respectively (Table 2).

[0048] Table 2 Indole diterpene biosynthesis gene clusters tsn Gene function prediction

[0049]

[0050] Example 3 Heterologous Expression of Paspalin Biosynthesis Genes

[0051] To further verify the functions of TsnG, TsnC, TsnM and TsnB, extract Penicillium thiersii The RNA of NRRL 28147 was reverse transcribed to obtain cDNA, and primers were designed to amplify the genes encoding TsnG, TsnC, TsnM, and TsnB, respectively. The Saccharomyces cerevisiae expression vectors pXW06F-tsnGC, pXW06F-tsnGCM, and pXW06F-tsnGCMB for combined expression of the four genes were constructed. Figure 2-Figure 4 ). The vector was sent to a sequencing company for sequencing and comparison, and the sequences shown in SEQ ID NO. 1 to NO. 4 were obtained. tq , tqCy , tsn and tnB .

[0052] Saccharomyces cerevisiae RC01 was inoculated into 2% YPD medium and cultured at 30°C and 200 r / min until the OD value was 0.8-1.0. Saccharomyces cerevisiae competent cells were prepared and the recombinant plasmids were transformed according to the instructions provided by ZYMO. The recombinant plasmids pXW06F-tsnGC, pXW06F-tsnGCM and pXW06F-tsnGCMB were transformed into Saccharomyces cerevisiae, respectively, and spread on SC solid defective medium (SC-Trp) lacking tryptophan, and cultured at 30°C for about 3 days. Positive yeast transformants were selected, streaked and cultured on new SC-Trp defective medium, and cultured in a 30°C incubator for about 2 days. In the same way, yeast transformants transferred into the empty plasmid pXW06F were obtained as blank controls.

[0053] A two-step fermentation technique was adopted. First, an appropriate amount of Saccharomyces cerevisiae transformants were inoculated into 25 mL SC-Trp liquid defective medium, cultured at 30°C and 200 rpm for 16 hours, and then 25 ml of 1% YPD medium was added for 48 hours. After the fermentation was completed, the fermentation product was extracted with 50 ml ethyl acetate. The ethyl acetate concentrated extract was recovered by rotary evaporator, re-dissolved in 1 ml methanol, filtered and used for LC-MS detection.

[0054] Example 4 Detection of Paspalin in Recombinant Yeast Fermentation Extract

[0055] LC-MS was used to detect indole diterpenoid products in the fermentation extract of recombinant yeast. The liquid chromatography detection conditions were as follows: the chromatographic column was Poroshell 120 EC-C18 (2.1 mm × 100 mm, 1.5 µm), the UV detection wavelength was 280 nm, the mobile phase was acetonitrile / water containing 0.1% formic acid, the flow rate was 0.4 mL / min, and the elution program was: 0-15 min, 30-100%; 15.1-18 min, 100%; 18.1-20 min, 30%. The main MS test conditions were: positive ion mode, ion source was ESI source, mass spectrometry mass-to-charge ratio scanning range was 100-800 amu, drying gas temperature was 350℃, dryer flow rate was 10 L / min, nebulizer pressure was 35 psi, and capillary voltage was 3.5 kV.

[0056] LC-MS analysis revealed that compared with yeast transformants transformed with the empty plasmid pXW06F (blank control), yeast transformants transformed with the expression plasmid pXW06F-tsnGC were able to specifically synthesize compound 1. The positive ion mode mass spectrum of compound 1 showed [M+H] + The peak mass-to-charge ratio is 390 ( Figure 6 ), corresponding to a molecular weight of 389, which is consistent with the molecular weight of 3-geranylgeranyl indole (GGI). Yeast transformants transformed with the expression plasmid pXW06F-tsnGCM were able to specifically synthesize compound 2. The positive ion mode mass spectrum showed that the [M+H] + The peak mass-to-charge ratio is 406 ( Fig. 9 ), corresponding to a molecular weight of 405, which is consistent with the molecular weight of 19,20-epoxy-GGI. Yeast transformants transformed with pXW06F-tsnGCMB were able to specifically synthesize compound 3. The positive ion mode mass spectrum of compound 3 showed [M+H] + The peak mass-to-charge ratio is 422 ( Fig.12 ), corresponding to a molecular weight of 421, which is consistent with the molecular weight of paspalin ( Figure 5 ).

[0057] Example 5 Isolation and purification of paspalin from recombinant yeast fermentation extract

[0058] The yeast transformants transformed with pXW06F-tsnGCM and pXW06F-tsnGCMB were fermented on a large scale (4 L), and the fermentation extracts were separated by ODS medium-pressure reverse phase chromatography. Five fractions Fr. A-Fr. E were obtained after gradient elution with methanol / water, v / v, 50:50→100:0. After LC-MS detection of the above components, it was found that compound 2 was enriched in the fraction Fr. C eluted with 80% methanol from the fermentation extract of the pXW06F-tsnGCM yeast transformant. It was separated and purified by semi-preparative high-performance liquid chromatography with UV detection wavelength at 280 nm and isocratic elution (v / v, 80:20) using acetonitrile-water (containing 0.1% formic acid) as the mobile phase at a flow rate of 2.5 mL / min to obtain compound 2 (21 mg) monomer ( Figure 7-9 Compound 3 was enriched in the fraction Fr. D of the fermentation extract of the pXW06F-tsnGCMB yeast transformant. It was separated and purified by semi-preparative HPLC with UV detection at 280 nm and isocratic elution (v / v, 85:15) using acetonitrile-water (containing 0.1% formic acid) as the mobile phase at a flow rate of 2.5 mL / min to obtain compound 3 (45 mg) monomer ( Figure 10-12 ). By analyzing the nuclear magnetic resonance hydrogen spectrum and carbon spectrum, it was determined that compound 2 was 19,20-epoxy-GGI and compound 3 was paspaline ( Figure 1 ), the structure identification data are as follows:

[0059] Compound 2: white powder, 1 H NMR (600 MHz, CDCl3) δ8.07 (1H, brs, H-1), 7.59(1H, d, 7.8, H-5), 7.35 (1H, d, 8.2, H-8), 7.18 (1H, t, 7.6, H-7), 7.11 (1H,t, 7.4, H-6), 6.94 (1H, s, H-2), 5.46 (1H, t, 7.1, H-11), 5.19 (1H, t, 6.6,H-15), 5.09 (1H, t, 6.9, H-23), 3.46 (1H, d, 7.2, H-10), 2.71 (1H, t, 6.2, H-19), 2.14 (1H, m, H-14a), 2.13 (1H, m, H-17a), 2.08 (1H, m, H-14b) , 2.06(1H, m, H-17b), 2.05 (2H, m, H2-13) , 2.05 (2H, m, H2-22), 1.76 (3H, s, H3-27), 1.69 (3H, s, H3-26), 1.63 (1H, m, H-21a), 1.61 (3H, s, H3-28), 1.60 (3H,s, H3-25), 1.39 (1H, m, H-21b), 1.25 (3H, s, H3-29); 13 C NMR (150 MHz, CDCl3) δ 136.7 (C-9), 135.5 (C-12), 134.3 (C-16), 132.0 (C-24), 127.6 (C-4), 124.9 (C-15), 123.8 (C-23), 123.3 (C-11), 122.0 (C-7), 121.4 (C-2), 119.2 (C-6), 119.1(C-5), 116.2 (C-3), 111.2 (C-8), 63.6 (C-19), 61.1 (C-20), 39.7 (C-13), 39.0(C-21), 36.4 (C-17), 27.5 (C-18), 26.5 (C-14), 25.8 (C-26), 24.1 (C-22), 24.0(C-10), 17.8 (C-25), 16.6 (C-29), 16.2 (C-27), 16.1 (C-28);ESIMS m / z 406.4 [M+H] + 。

[0060] Compound 3: white powder, 1 H NMR (400 MHz, CDCl3) δ 7.72 (1H, brs, H-1), 7.42(1H, m, H-2), 7.29 (1H, m, H-8), 7.07 (2H, s, H-6, H-7), 3.21 (1H, dd, 11.8,3.1, H-18), 3.03 (1H, dd, 11.7, 3.9, H-19), 2.76 (1H, m, H-11), 2.68 (1H, dd,13.2, 6.3, H-10a), 2.33 (1H, dd, 13.2, 10.6, H-10b), 1.96 (1H, m, H-21a), 1.83 (1H, m, H-16a), 1.79 (1H, m, H-12a), 1.69 (1H, m, H-17a), 1.68 (2H, m,H2-20), 1.64 (1H, m, H-13a), 1.62 (1H, m, H-21b), 1.59 (1H, m, H-12b), 1.46(2H, m, H2-14), 1.41 (1H, m, H-13b), 1.38 (1H, m, H-17b), 1.11 (1H, m, H-16b), 1.19 (3H, s, H3-26), 1.18 (3H, s, H3-25), 1.14 (3H, s, H3-28), 1.03 (3H,s, H3-27), 0.89 (3H, s, H3-29); 13 C NMR (100 MHz, CDCl3) δ151.0 (C-2), 140.1(C-9), 125.3 (C-4), 120.6 (C-7), 119.7 (C-6), 118.5 (C-5), 118.4 (C-3), 111.6(C-8), 85.8 (C-19), 84.8 (C-18), 72.1 (C-24), 53.1 (C-23), 48.9 (C-11), 46.6(C-14), 40.2 (C-22), 37.8 (C-16), 36.7 (C-15), 34.1 (C-21), 27.7 (C-10), 26.2(C-26), 25.4(C-12), 24.8(C-20), 23.9 (C-25), 22.1 (C-17), 22.1 (C-13), 20.1(C-28), 14.7 (C-27), 12.8 (C-29); ESIMS m / z 422.4 [M+H] + .

[0061] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An indole diterpene biosynthesis gene, characterized in that: It is selected from one or more of the following genes: (1) Gene encoding geranylgeranyl synthase tq , tq The nucleotide sequence is shown in SEQ ID NO.1; (2) Gene encoding geranylgeranyltransferase tqCy , tqCy The nucleotide sequence is shown in SEQ ID NO.2; (3) Genes encoding flavin-dependent oxidases tsn , tsn The nucleotide sequence is shown in SEQ ID NO.3; (4) Gene encoding indole diterpene cyclase tnB , tnB The nucleotide sequence is shown in SEQ ID NO.

4.

2. An indole diterpene biosynthesis protein, characterized in that It is selected from one or more of the following proteins: (1) geranylgeranyl synthase TsnG, the amino acid sequence of TsnG is shown in SEQ ID NO.5; (2) geranylgeranyltransferase TsnC, the amino acid sequence of TsnC is shown in SEQ ID NO.6; (3) flavin-dependent oxidase TsnM, the amino acid sequence of TsnM is shown in SEQ ID NO.7; (4) Indole diterpene cyclase TsnB, the amino acid sequence of TsnB is shown in SEQ ID NO.

8.

3. The indole diterpene biosynthesis gene according to claim 1 or the indole diterpene biosynthesis protein according to claim 2, characterized in that: The indole diterpene is 3-geranylgeranyl indole, 19,20-epoxy-GGI or paspalin.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the indole diterpene biosynthesis gene according to claim 1.

5. A genetically engineered bacterium for producing indole diterpenes, characterized in that: The genetically engineered bacteria comprises the recombinant expression vector according to claim 4.

6. The genetically engineered bacteria for producing indole diterpenes according to claim 5, characterized in that: The starting strain of the genetically engineered bacteria is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

7. A method for preparing indole diterpenes, characterized in that: The method comprises fermenting the genetically engineered bacteria according to claim 5 or 6 in a fermentation medium.

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