Novel terpenoid cyclase as well as coding gene and application thereof
By providing terpene cyclases and their encoding genes, heterologous synthesis of harzianol I and wickerol A was achieved, solving the technical challenge of resistant spore production in Trichoderma fungi, enhancing the fungi's resistance in extreme environments, and prolonging their viability in soil.
Patent Information
- Application Number
- CN202511157266.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-11-14
AI Technical Summary
In the existing technology, the key enzymes of biosynthesis in Trichoderma fungi and their biological functions have not been elucidated, resulting in the cyclization enzymes of Trichoderma harzianum and Trichoderma-like compounds not being identified, and thus failing to effectively enhance the production of fungal resistant spores.
Terpenoid cyclases and their encoding genes are provided, including amino acid sequences such as Tri4155 to Tarm302 as shown in SEQ ID NO.1 to 8, and nucleotide sequences such as encoding genes shown in SEQ ID NO.9 to 16, for the synthesis of harazianol I and wickerol A, and their heterologous synthesis is achieved through recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria, thereby enhancing the production of fungal resistant spores.
The efficient biosynthesis of harzianol I and wickerol A was achieved, which enhanced the production of chlamydospores and sclerotia of Trichoderma fungi, improved the fungal resistance to extreme environments, and prolonged their viability in soil.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product biosynthesis technology, specifically relating to terpenoid cyclases and their encoding genes for the biosynthesis of Trichoderma diterpenes and their enhancement of the biological function of fungal resistant spores, as well as the application of terpenoid cyclases, their encoding genes, and their product harzianol I in enhancing the production of fungal resistant spores. Background Technology
[0002] Trichoderma fungi are important biocontrol fungi, widely used in agriculture and forestry. Commercially available products include *Trichoderma harzianum*, *Trichoderma echinosporum*, *Trichoderma viride*, *Trichoderma reesei*, *Trichoderma longifolia*, and multi-component *Trichoderma* fungi. *Trichoderma* fungi produce two types of novel and bioactive diterpenoids: harziananes and trichomycins. However, the key enzymes involved in their biosynthesis and their biological functions have long remained unclear.
[0003] Currently, 40 harzianol diterpenoids have been isolated and identified from the *Trichoderma* genus, including harzianol I, harzianone, and harziamdione. Harzianol I is an upstream compound in the biosynthesis of harzianol diterpenoids. Harzianol diterpenoids possess a wide range of biological activities, including antifungal, anti-HIV, anti-inflammatory, and cytotoxic effects. Eight wikeryl diterpenoids have also been identified from the *Trichoderma* genus, including wickerol A and wickerol B. Wikerol A is an upstream compound in the biosynthesis of wikeryl diterpenoids. Harzianol diterpenoids exhibit significant antiviral activity against the H1N1 influenza virus, with an IC50 value of [missing information]. 50 = 0.07 μg / mL. Harziram and trichomycin-like natural products are derived exclusively from the genus *Trichoderma* and are specific secondary metabolites of this genus. Isotope feeding experiments indicate that the skeletons of both harziram and trichomycin-like products originate from a one-step cyclization of GGPP, but the enzyme responsible for cyclization has not yet been identified.
[0004] Trichoderma fungi comprise three asexual propagation forms: hyphae, conidia, and chlamydospores. Chlamydospores are resistant spores with thickened cell walls, exhibiting stronger resistance to extreme environments such as high temperatures, drought, and ultraviolet radiation. Trichoderma products, primarily composed of chlamydospores, show significantly longer viability in soil than conidia and hyphae; therefore, enhancing chlamydospore production is considered to enhance the biocontrol function of Trichoderma. Aspergillus oryzae, a fungus in the Aspergillus genus, is widely used in the production of fermented foods such as soy sauce and rice wine. Furthermore, it is a widely used engineered strain in biosynthetic research. The asexual propagation forms of Aspergillus oryzae include hyphae, conidia, and sclerotia. Similarly, sclerotia are resistant spores of Aspergillus oryzae, consisting of spherical or elliptical granules formed by dense hyphae encased in a hard, brown shell. Enhancing the production of Aspergillus oryzae sclerotia can prolong the viability of the strain during long-term storage. Currently, there are no reports or applications of natural products regulating the production of chlamydospores and sclerotia. Summary of the Invention
[0005] The purpose of this invention is to provide terpenoid cyclases and their encoding genes for the biosynthesis of Trichoderma diterpenes and their enhancement of the biological function of fungal resistant spores, as well as the application of terpenoid cyclases, their encoding genes, and their product harzianol I in enhancing and regulating the production of fungal resistant spores.
[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:
[0007] This invention provides terpenoid cyclases for the synthesis of hazianol I and wickerol A, including but not limited to one or more of the terpenoid cyclases in 1) to 9):
[0008] 1) The amino acid sequence is Tri4155 as shown in SEQ ID NO.1;
[0009] 2) The amino acid sequence is Tcie612 as shown in SEQ ID NO.2;
[0010] 3) The amino acid sequence is Trei084 as shown in SEQ ID NO.3;
[0011] 4) The amino acid sequence is Tass213 as shown in SEQ ID NO.4;
[0012] 5) The amino acid sequence is Tasm819 as shown in SEQ ID NO.5;
[0013] 6) The amino acid sequence is Tham208 as shown in SEQ ID NO.6;
[0014] 7) The amino acid sequence is Tgai159 as shown in SEQ ID NO.7;
[0015] 8) The amino acid sequence is as shown in SEQ ID NO.8, Tarm302;
[0016] 9) Derivative proteins with the same function but with the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 substituted and / or deleted and / or added one or more amino acid residues.
[0017] This invention also provides the encoding genes for the terpene cyclases described in the above scheme, including one or more of the encoding genes in 1) to 9):
[0018] 1) The gene encoding Tri4155 with a nucleotide sequence as shown in SEQ ID NO.9;
[0019] 2) The gene encoding Tcie612, whose nucleotide sequence is shown in SEQ ID NO.10;
[0020] 3) The gene encoding Trei084, whose nucleotide sequence is shown in SEQ ID NO.11;
[0021] 4) The gene encoding Tass213 with a nucleotide sequence as shown in SEQ ID NO.12;
[0022] 5) The gene encoding Tasm819 with a nucleotide sequence as shown in SEQ ID NO.13;
[0023] 6) The gene encoding Tham208 with a nucleotide sequence as shown in SEQ ID NO.14;
[0024] 7) The gene encoding Tgai159 with a nucleotide sequence as shown in SEQ ID NO.15;
[0025] 8) The gene encoding Tarm302 with the nucleotide sequence shown in SEQ ID NO.16;
[0026] 9) Nucleotide sequences that are substituted and / or deleted and / or added with one or more nucleotides according to the nucleotide sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16 and express the same functional protein.
[0027] The present invention also provides recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the coding genes described above.
[0028] The present invention also provides the application of the terpene cyclase described in the above-described scheme, or the encoding gene, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in the synthesis of harazianol I and wickerol A.
[0029] The application of the terpene cyclase, the encoding gene, the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in the synthesis of Trichoderma diterpenoid compounds.
[0030] According to the application described, the diterpenoid compound comprises chemical structural formulas as shown in the formulas shown below. Harzianol I and wickerol A, as shown in Formula II, and downstream products derived from Harzianol I and wickerol A,
[0031] Mode Mode .
[0032] In addition, the present invention also provides trichoderma diterpenoid compounds and the application of harzianol I in enhancing the production of fungal resistant spores.
[0033] The application of the terpene cyclase, the encoding gene, the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in enhancing the production of fungal resistant spores.
[0034] According to the above applications, the fungal resistant spores include chlamydospores of the Trichoderma genus, sclerotia of Aspergillus oryzae, and resistant spores of other fungi.
[0035] The terpenoid cyclases TriDTCs of this invention catalyze the synthesis of upstream compounds, harzianol I and wickerol A, from the GGPP pathway for the biosynthesis of the Trichoderma diterpenoid harzianol and trichomycin. Expression of TriDTCs in Trichoderma and Aspergillus oryzae enhances the production of Trichoderma chlamydospores and Aspergillus oryzae sclerotia. Similarly, the addition of harzianol I to Trichoderma and Aspergillus oryzae culture media also enhances the production of these sclerotia. This invention provides an efficient biosynthetic method for the heterologous synthesis of harzianol I and wickerol A, and offers biological and chemical pathways for enhancing the production of resistant spores in Trichoderma and Aspergillus oryzae. Attached Figure Description
[0036] Figure 1Schematic diagram of the catalytic and biological functions of TriDTCs.
[0037] Figure 2 This study investigated the production and quantification of harazianol I and wickerol A by triDTCs in recombinant prokaryotic bacteria. A) GC-MS chromatograms of harazianol I and wickerol A by each triDTC in the E. coli BL21 system, with results shown as extracted ion EIC: m / z = 272, compared with the identified product peaks of harazianol I and wickerol A standards. B) MS / MS chromatograms of harazianol I and wickerol A standards. C) Quantitative results of harazianol I and wickerol A products by each triDTC in the E. coli BL21 system, with yields shown as mean ± SD (n=3 biological replicates).
[0038] Figure 3 The production and quantification of harazianol I and wickerol A by triDTCs in eukaryotic recombinant bacteria. A, GC-MS chromatograms of harazianol I and wickerol A by each triDTC in the *A. oryzae* NSAR1 system, results shown as extracted ion EIC: m / z = 272. B, Quantitative results of harazianol I and wickerol A products by each triDTC in the *A. oryzae* NSAR1 system, yields shown as mean ± SD (n=3 biological replicates).
[0039] Figure 4 Purity and state of purified proteins were determined using Tcie612 and Tri4155. The gel column type was Superdex 200Increase 10 / 300 GL.
[0040] Figure 5 Characterization of the in vitro enzymatic properties of TriDTCs. A. In vitro enzyme activity results of Tcie612 and Tri4155. B. Substrate specificity results of Tcie612. C. Metal ion dependence results of Tcie612. D. Michaelis constant K of Tcie612. M Measurement results. E, F, Tcie612 Mg without GGPP and with GGPP. 2+ Titration experiment results.
[0041] Figure 6Trichoderma diterpenoids are associated with the production of chlamydospores. Among them: 1 is harzianol I, 2 is wickerol A, and 3 and 4 are harzianol compounds.
[0042] Figure 7 The effects of triDTC gene knockout on chlamydospore production: A. Gene knockout verification and diterpenoid product analysis of tri4155, where 1 is harzianol I, 2 is wickerol A, and 3 and 4 are harzianol compounds, the same below. B. Knockout verification and diterpenoid product analysis of triDTCs in *T. asperellum* and *T. gamsii*. C. Comparison of hyphae and chlamydospore production in the three wild-type (WT) and knockout strains of *Trichoderma*, scale bar = 50 μm.
[0043] Figure 8 Effect of harzianol I replenishment to the culture medium on chlamydospore production in Trichoderma strains. Where: 1 represents harzianol I, scale bar = 50 μm.
[0044] Figure 9 Effects of TriDTCs and harazianol I on Aspergillus oryzae sclerotium formation. The figure shows a comparison of sclerotium formation in wild-type Aspergillus oryzae strain (A. oryzae), heterologously expressed Tri4155 Aspergillus oryzae strain (A. oryzae-tri4155), complement compound harazianol I (A. oryzae-S1), and complement compound wickerol A (A. oryzae-S2). Detailed Implementation
[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0046] Example 1: Obtaining the triDTCs gene.
[0047] The tri4155 gene was obtained by PCR reaction using the PCR primers described in Table 1 with the cDNA of *Trichoderma atroviride* as a template. Using the amino acid sequence of Tri4155 as a blueprint, a BLAST search was performed in the NCBI database to obtain the amino acid sequences of other TriDTCs. Then, gene synthesis was performed to optimize the codons for the *E. coli* expression system, resulting in plasmids containing the gene sequences Tcie612, Trei084, Tass213, Tasm819, Tham208, Tgai159, and Tarm302.
[0048] Table 1: PCR amplification primers used for constructing triDTCs gene expression vectors and cassettes
[0049]
[0050] Example 2: Construction of prokaryotic and eukaryotic recombinant vectors, expression cassettes, and recombinant strains of triDTCs.
[0051] Recombinant vectors for the triDTCs gene were constructed using homologous recombination ligation. The preferred prokaryotic vector was pCold-TF, which was double-digested with SalI and KpnI, and the 20bp sequences at both ends of the restriction sites were selected as homologous arms (sequences shown in Table 1). The eukaryotic vector was pTAex3, containing the arginine synthesis gene, used for Aspergillus oryzae strains with arginine deficiency. NdeI and SmaI restriction sites were used, and 20bp homologous arms were selected (sequences shown in Table 1). Homologous arms of the vectors were added to both ends of the gene amplification primers listed in Table 1 to synthesize homologous recombination primers. Using Trichoderma viride cDNA or plasmids containing gene sequences obtained through gene synthesis as templates, PCR amplification was performed to obtain each triDTCs gene fragment containing homologous arms. Homologous recombination ligation reaction was performed using a seamless cloning kit (50℃, 20 min). After the reaction, the fragments were transformed into E. coli DH5α for screening and replication. Subsequently, plasmid extraction was performed to obtain the recombinant vectors pCold-TF-tri4155, pTAex3-tri4155, pCold-TF-Tcie612, pCold-TF-Trei084, pCold-TF-Tass213, pCold-TF-Tasm819, pCold-TF-Tham08, pCold-TF-Tgai159, and pCold-TF-Tarm302.
[0052] An expression cassette was constructed using the promoter, terminator, restriction enzyme sites, and His-tag sequence of tri4155 for the expression and purification of TriDTCs in Aspergillus oryzae, aiming to improve protein expression levels and product yield. PCR primers for Ptri4155 and Ttri4155 (as shown in Table 1) were synthesized, and the Ptri4155 and Ttri4155 sequences were amplified using Trichoderma viride genomic DNA as a template. PCR primers for tri4155-C-His (as shown in Table 1) were synthesized, with NotI and KpnI restriction enzyme sites introduced at the F and R ends, respectively, to replace other triDTC genes; an 8 × His-tag sequence was introduced outside the C-terminal restriction enzyme site for protein purification of each TriDTC. The Ptri4155, Tri4155, and tri4155-C-His fragments were ligated with the pTAex3 vector, which had been double-digested with NdeI and SmaI, using a seamless cloning kit for homologous recombination ligation (50℃, 30 min). After the reaction, the vector was transformed into E. coli DH5α for selection and replication. Plasmid extraction yielded the tri4155 recombinant vector pTAex3-Ptri4155-tri4155 containing the expression cassette. The -F / -R homologous arms of pTAex3-Ptri4155 from Table 1 were added to both ends of other triDTCs PCR primers to synthesize the required homologous recombination primers. PCR amplification was performed using a plasmid containing the triDTCs gene as a template to obtain the homologous recombination gene fragment. The recombinant vector was double-digested with NotI and KpnI and ligated with the triDTCs homologous recombination fragment for homologous recombination ligation (50℃, 20 min). After the reaction, the vector was transformed into E. coli. DH5α was screened and replicated, and plasmids were extracted to obtain triDTCs recombinant vectors containing expression cassettes: pTAex3-Ptri4155-Tcie612, pTAex3-Ptri4155-Trei084, pTAex3-Ptri4155-Tass213, pTAex3-Ptri4155-Tasm819, pTAex3-Ptri4155-Tham08, pTAex3-Ptri4155-Tgai159, and pTAex3-Ptri4155-Tarm302.
[0053] The engineered strain *E. coli* BL21, which produces high-yield precursor GGPP, was selected as the prokaryotic chassis strain. This strain contains two plasmids: pBbA5c-MevT-MBIS (for high-yield FPP) and pET28a-crtE (for expressing GGPP synthase). The prokaryotic expression recombinant vector was transformed into this chassis strain to obtain the triDTCs prokaryotic expression recombinant strain. *Aspergillus oryzae* NSAR1 (Arg...) was selected... - / Ade- / Met - The constructed recombinant vectors pTAex3-tri4155, pTAex3-Ptri4155-tri4155, pTAex3-Ptri4155-Tcie612, pTAex3-Ptri4155-Trei084, pTAex3-Ptri4155-Tass213, pTAex3-Ptri4155-Tasm819, pTAex3-Ptri4155-Tham08, pTAex3-Ptri4155-Tgai159, and pTAex3-Ptri4155-Tarm302 were transformed into Aspergillus oryzae NSAR1 protoplasts and expressed in Arg. - The culture medium was used for screening and culture to obtain triDTCs eukaryotic recombinant strains.
[0054] Example 3: Production and quantification of harzianol I and wickerol A by triDTCs in prokaryotic recombinant bacteria.
[0055] Fermentation of prokaryotic recombinant strains: Recombinant *E. coli* strains were inoculated into 6 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing antibiotics (100 mg / L ampicillin, 50 mg / L chloramphenicol, 50 mg / L kanamycin) and cultured overnight at 37°C with shaking at 180 rpm. 1 mL of the culture was then inoculated into 100 mL of TB medium (12 g / L tryptone, 24 g / L yeast extract, 25 mL / L glycerol, 2.31 g / L KH₂PO₄, 12.54 g / L K₂HPO₄) and cultured until OD₀. 600 The values reached 0.6-0.8. Subsequently, 0.3 mM IPTG was added to induce protein expression, and the induction was carried out at 16℃ for 18 h, followed by incubation at 30℃ for 72 h. The culture was extracted with 150 mL ethyl acetate using ultrasound-assisted extraction for 20 min. The organic phase was concentrated by rotary evaporation, and the extract was transferred to a 10 mL glass vial with petroleum ether. After drying the solvent under nitrogen, it was reconstituted in 800 μL of n-hexane for GC-MS analysis. All experiments were performed in triplicate.
[0056] GC-MS analysis was performed using an Agilent 7890A gas chromatograph system equipped with an Agilent 5795C inert triple quadrupole mass spectrometer detector. The column dimensions were 30 m × 250 μm × 0.25 μm, and high-purity helium was used as the carrier gas. The temperature program was set as follows: initial temperature 80 °C, increased to 220 °C at a rate of 15 °C / min, then increased to 270 °C at a rate of 4 °C / min and held for 2 minutes. The carrier gas flow rate was kept constant at 1.2 mL / min, and a splitless injection mode was used with an injection volume of 5 μL. The injector and detector temperatures were set to 250 °C and 300 °C, respectively.
[0057] The analytical results were compared with the retention times of harzianol I and wickerol A standards using secondary mass spectrometry to identify the product peaks. Standard curves were constructed and the products were quantified under the same conditions as the harzianol I and wickerol A standards. Chromatographic, mass spectrometric, and quantitative results are shown below. Figure 2 As shown in the figure, the results indicate that harazianol I is the major product of TriDTCs, while wickerol A is a minor product. Tri4155 produced 3.55 ± 0.85 mg / L of harazianol I and 0.20 ± 0.05 mg / L of wickerol A; Tricie612 produced 33.34 ± 4.71 mg / L of harazianol I and no wickerol A; Trei084 produced 1.43 ± 0.40 mg / L of harazianol I and no wickerol A. The remaining TriDTCs produced only trace amounts of the product.
[0058] Example 4: Production of harazianol I and wickerol A by triDTCs in eukaryotic recombinant bacteria and their quantification.
[0059] Fermentation of recombinant eukaryotic strains: Spores of the recombinant Aspergillus oryzae strain were inoculated onto fresh DPY plates (20 g / L dextrin, 10 g / L polypeptone, 5 g / L yeast extract, 2.31 g / L KH2PO4, 0.5 g / L MgSO4, 2% agar) and incubated statically at 30°C for 3 days. Mycelia were then transferred to 100 mL of DPY liquid medium and cultured at 30°C with shaking at 180 rpm for 7 days. The culture was extracted with 150 mL of ethyl acetate using ultrasonic-assisted extraction for 30 min. The organic phase was concentrated by rotary evaporation, and the extract was transferred to 10 mL glass vials with petroleum ether. After drying the solvent under nitrogen, the extract was reconstituted in 800 μL of n-hexane for GC-MS analysis. All experiments were performed in triplicate.
[0060] The GC-MS analysis conditions were the same as in Example 3.
[0061] The analytical quantification of products harzianol I and wickerol A was consistent with that in Example 3. Results are attached. Figure 3 As shown, all eight TriDTCs exhibited functionality in the Aspergillus oryzae system, producing either hazianol I or both hazianol I and wickerol A. Specifically, Tri4155 produced 10.09 ± 2.08 mg / L of hazianol I and 1.55 ± 0.39 mg / L of wickerol A; Tass213 and Tgai159 also produced both products. Tcie612 produced 10.80 ± 1.54 mg / L of hazianol I but did not produce wickerol A. Trei084, Tham208, and Tarm302 also produced only hazianol I and no wickerol A.
[0062] The results show that all eight triDTCs catalyze the production of harazianol I and wickerol A in both prokaryotic and eukaryotic systems, with consistent product patterns. However, the relative yields vary depending on the system, which may be due to the different stability of the proteins in different systems.
[0063] Example 5: Protein expression and purification of TriDTCs.
[0064] Construction of recombinant vectors and expression strains: pCold-II was selected as the protein expression vector, and recombinant vectors were constructed using homologous recombination ligation. The vectors were double-digested with SalI and KpnI, and the 20bp sequences at both ends of the digestion sites were selected as homologous arms (sequences shown in Table 1) and added to both ends of the tri4155 and Tcie612 cloning primers to synthesize homologous recombination primers. PCR amplification was performed using *Trichoderma viride* B7 cDNA and the Tcie612 plasmid as templates to obtain the homologous recombination gene fragment. This fragment was then ligated with the digested vector using a seamless cloning kit (50℃, 20 min). After the reaction, the fragment was transformed into *E. coli* DH5α for selection and replication, followed by plasmid extraction to obtain the recombinant vectors. The recombinant vectors pCold-II-tri4155 and pCold-II-Tcie612 were transformed into *E. coli* BL21 to obtain the protein expression strain.
[0065] Protein expression condition optimization: The target protein exhibits instability in the *E. coli* expression system, necessitating optimized protein expression conditions. Key points include: 1. Using TB medium containing glycerol and buffer salts to increase protein stability; 2. Using a low concentration of inducer for slow induction to reduce protein aggregation and precipitation; 3. Using low-temperature induction to avoid protein denaturation and precipitation. The specific protein expression procedure is as follows: The protein expression strain is inoculated into 100 mL of TB medium containing ampicillin (100 mg / L) and cultured at 37°C with shaking at 180 rpm until OD... 600 Once the OD value reached 0.6-0.8, it was transferred to 3L of TB medium containing antibiotics and cultured at 37℃ with shaking at 180 rpm until the OD value reached 0.6-0.8. 600 The value was approximately 0.5, and then 0.03 mM IPTG was added to induce protein expression. The expression was induced at 16°C and 180 rpm for 16 h.
[0066] Protein purification condition optimization: The target protein exhibits instability in the *E. coli* expression system, necessitating optimized purification conditions. Key points include: 1. Employing mild methods such as freeze-thaw cycles for cell disruption. Ultrasonic disruption should be performed at low power and slow speed to minimize protein aggregation and precipitation. 2. Continuously adding a high concentration of NaCl to the purification buffer to increase protein solubility and stability. 3. Maintaining the entire process at low temperatures to prevent protein denaturation and precipitation. The specific protein purification procedure is as follows: Collect bacterial cells by centrifugation at 4℃ and 4000 rpm. Resuspend the cells once in disruption buffer (50mM Tris, 1M NaCl, 2mM DTT, 10% glycerol, pH adjusted to 8.0 with hydrochloric acid). Centrifuge to remove the supernatant. Freeze-thaw the cells once at -80℃, then resuspend them in disruption buffer. Disrupt the cells on ice using an ultrasonic cell disruptor (2% power, 1.5s sonication time, 5.5s pause time, total time 1h). Centrifuge at 12000 rpm for 30 min at 4℃. Add elution buffer (50 mM Tris, 1 M NaCl, 2 mM DTT, 10% glycerol, 500 mM imidazole, pH adjusted to 8.0 with hydrochloric acid) to adjust the imidazole concentration to 20 mM. The mixture binds to Ni-NTA at low temperature, followed by elution with 40 mM and 300 mM imidazole buffers. Collect the 300 mM eluent and concentrate it to 500 μL using a 10 kDa ultrafiltration tube. Perform molecular sieve column chromatography using a ӒKTA pure M protein purification system equipped with a Superdex200 Increase 10 / 300 GL gel column. The mobile phase is 50 mM Tris, 1 M NaCl, 2 mM DTT, pH adjusted to 8.0 with hydrochloric acid; the flow rate is 0.5 mL / min; and the target protein is detected by SDS-PAGE gel chromatography. Further purification was performed using a Capto HiResQ 5 / 50 gel column. Mobile phase A consisted of 50 mM Tris, 50 mM NaCl, and 2 mM DTT, adjusted to pH 8.0 with hydrochloric acid. Mobile phase B consisted of 50 mM Tris, 1 M NaCl, and 2 mM DTT, adjusted to pH 8.0 with hydrochloric acid. The elution program was 50 min, mobile phase B 10%–100%. The target protein was detected by SDS-PAGE gel chromatography. Finally, the purity and state of the protein were assessed using a Superdex 200 Increase 10 / 300 GL gel column. The protein purification results are shown below. Figure 4 As shown, the theoretical molecular weights of Tri4155 and Tcie612 are 71.6 and 72.6 kDa, respectively. Based on the exit point position, both proteins are monomers.
[0067] Example 6: Characterization of the in vitro enzymatic properties of TriDTCs.
[0068] In vitro enzyme activity assay: The 200 μL enzyme activity reaction system contained: 100 μL reaction buffer (50 mM Tris-HCl, 25 mM MgCl2, 100 mM KCl, 5 mM DTT, 10% glycerol, 5 mM β-hydroxypropyl cyclodextrin, pH 7.5), 50 μL enzyme solution, 10 μL GGPP, and 40 μL ddH2O. After reacting in a 30℃ water bath for 30 min, the mixture was extracted with 400 μL n-hexane. This process was repeated once. The extracts were combined, dried under nitrogen, and reconstituted with 50 μL n-hexane. GC-MS analysis was performed. The analytical results are as follows: Figure 5 As shown: Tri4155 produces harazianol I and wickerol A, while Tcie612 only produces harazianol I and does not produce wickerol A, consistent with the in vivo enzyme activity products.
[0069] Substrate specificity: The substrate specificity of Tcie612 was tested. When the substrate for the enzyme activity reaction was changed to FPP or GFPP, no related cyclization products were detected, indicating that Tcie612 can only utilize GGPP as a substrate. When the substrate was changed to DMAPP + 3 IPP or FPP + IPP, no diterpene cyclization products were detected, indicating that Tcie612 does not possess isopentenyltransferase function.
[0070] Metal ion dependence: The utilization efficiency of different metal ions was tested using Tcie612. Mg in the reaction buffer was... 2+ Replaced by other divalent metal ions, including Co 2+ Mn 2+ Fe 2+ Ca 2+ Zn 2+ The production of harazianol I was detected under the same reaction conditions, and the results showed (see attached). Figure 5 Tcie612's dependence on metal ions is: Mg 2+ Co 2+ > Mn 2+ = Fe 2+ Ca cannot be used 2+ and Zn 2+ .
[0071] Michaelis constant K MDetermination of kinetics: Kinetic experiments were conducted using a 100 μL reaction system with different concentrations of GGPP (4, 8, 12, 16, 20, 30, 60, 80, and 200 μM). The reaction mixture was incubated at 30 °C for 30 min, and then immediately terminated by rapid freezing with liquid nitrogen. The reaction products were subsequently extracted with n-hexane and analyzed by GC-MS. Each experiment included three biological replicates. Based on the Michaelis-Menten equation, nonlinear regression analysis was performed on the data from the three replicate experiments using Origin 2021 software to determine the kinetic parameters (see attached). Figure 5 ).
[0072] Mg 2+ Determination of binding number: The number of Mg²⁺ molecules bound to each enzyme molecule was determined using an isothermal titration calorimeter (ITC) (MicroCal PEAQ-ITC). Tcie612 was prepared in Tris-HCl buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) at a concentration of 20 μM. Before titration, the enzyme solution was pre-incubated with GGPP (40 μM, i.e., two equivalents) or without GGPP as a control. Subsequently, 280 μL of protein solution was added to the sample cell, and 60 μL of MgCl₂ solution (1.2 mM) was added to the syringe. The titration conditions were as follows: stirring speed 750 rpm, sample cell temperature 25 °C, initial injection of 0.4 μL (3.0 s), followed by 18 injections of 2 μL (3.0 s / injection), with each injection spaced 150 s apart. Data were processed using MicroCal PEAQ-ITC analysis software (Malvern Panalytical), and curve fitting was performed using a "Two Sets of Sites" binding model. Results are as follows: Figure 5 As shown, without the addition of substrate GGPP, Mg 2+ It does not bind to enzymes; when incubated with enzymes after the prior addition of GGPP, Mg 2+ It binds to enzymes, with a binding number of 2.
[0073] Example 7: Experimental characterization of TriDTCs and harzianol I enhancing the formation of chlamydospores in the genus Trichoderma.
[0074] Trichoderma diterpenoids were associated with the production of chlamydospores: Five Trichoderma species (T. atroviride, T. asperellum, T. gamsii, T. reesei, and T. harzianum) were inoculated into 100 mL PDB (200 g / L potato, 20 g / L glucose) medium and cultured at 30 °C with shaking at 180 rpm for 3 days. Mycelia were then collected for microscopic observation of chlamydospore formation. The fermentation broth was extracted with 150 mL ethyl acetate using ultrasonic-assisted extraction for 30 min. The organic phase was concentrated by rotary evaporation, and the extract was transferred to a 10 mL glass vial with petroleum ether. After drying the solvent under nitrogen, the extract was redissolved in 200 μL of n-hexane, and the diterpenoid products were analyzed by GC-MS. The results showed (…) Figure 6 T. atroviride, T. asperellum, and T. gamsii produce diterpenoid products and chlamydospores; T. reesei and T. harzianum produce trace amounts of diterpenoid products and do not produce chlamydospores.
[0075] Knockout of triDTCs genes in *Trichoderma* strains: A split-marker strategy was used to knock out triDTCs in the genomes of *T. atroviride*, *T. asperellum*, and *T. gamsii*. The hygromycin resistance gene hygB was used as a resistance marker. Using the primer sequences shown in Table 2, and with *T. atroviride*, *T. asperellum*, and *T. gamsii* genomic DNA and plasmid pUC-hygB as templates, PCR amplification was performed to obtain the resistance cassette fragments tri4155-up, tri4155-down, TasDTC-up, TasDTC-down, TgaDTC-up, TgaDTC-down, and hygB. The hygB resistance cassette fragment was split into two parts, 969 bp and 1336 bp, namely split1 and split2 (containing a 174 bp overlap region). Using the hygB-up-R / hygB-down-F primer sequences shown in Table 2, overlap PCR was used to ligate the fragments with each upstream and downstream homologous arm to obtain the upstream and downstream homologous arm sequences containing split1 and split2. For example, tri4155-up-split1 and tri4155-down-split2 were used as a pair of gene knockout fragments for the knockout of tri4155. Trichoderma protoplasts were prepared using the Yatalase enzymatic digestion method. The gene knockout fragment was introduced into the protoplasts using the PEG transformation method, specifically as follows: 6 μg of the upstream knockout fragment and 6 μg of the downstream knockout fragment were mixed with 200 μL of protoplasts and 50 μL of PEG buffer (25% PEG6000, 10 mM Tris-HCl, 50 mM CaCl2, pH 7.5) and incubated on ice for 30 min. Then, 2 mL of PEG buffer was added, and the mixture was allowed to stand at room temperature for 20 min. After adding 5 mL of stabilization buffer (1.2 M sorbitol, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5), the mixture was stirred and centrifuged at 2500 rpm at room temperature to collect the cells. The cells were resuspended in upper culture medium (M medium containing 100 mg / L hygromycin B and 0.8% agar) at 40℃ and then plated onto lower culture medium (M medium containing 1.0% agar). Positive transformants were verified by PCR using primers flanking the deleted region (Table 2). Figure 7 ).
[0076] Table 2: Primers used in the gene knockout experiment of Trichoderma strains triDTCs
[0077]
[0078] Fermentation and chlamydospore production of the knockout strain: The gene knockout strain and the wild-type strain were inoculated into PDB medium and cultured at 30℃ and 180 rpm with shaking. After one day, mycelia were collected and observed under a microscope for chlamydospore formation. After 3 days of continued fermentation, the fermentation broth was extracted with 150 mL of ethyl acetate using ultrasonic-assisted extraction for 30 min. The organic phase was concentrated by rotary evaporation, and the extract was transferred to a 10 mL glass vial with petroleum ether. After drying the solvent with nitrogen, it was redissolved in 200 μL of n-hexane, and the diterpenoid products were analyzed by GC-MS. The results are attached. Figure 7 As shown, the gene knockout strain no longer produces Trichoderma diterpenoids; compared with the wild-type strain, the gene knockout strain no longer produces chlamydospores, and the hyphae exhibit hydrophobic aggregation in liquid culture medium.
[0079] Effect of harzianol I supplementation in Trichoderma culture medium on chlamydospores: Harzianol I dissolved in DMSO and added to the PDA medium of the Trichoderma tri4155 knockout strain. A final concentration of 10 μM promoted conidial production; increasing the final concentration to 400 μM restored chlamydospore production in the knockout strain. *T. reesei* and *T. harzianum* produced only small amounts of trichoderma diterpenes and no chlamydospores. Supplementation of 400 μM harzianol I to the PDA medium of *T. harzianum* promoted the production of a large number of chlamydospores. Supplementation of 400 μM harzianol I to the PDB medium of *T. reesei* promoted chlamydospore production. The above experimental results are shown in the appendix. Figure 8 The results showed that the addition of harzianol I restored the biological function of Tri4155, and that the addition of harzianol I could promote the production of chlamydospores by Trichoderma strains that do not produce chlamydospores.
[0080] Example 8: Experimental characterization of TriDTCs and harzianol I enhancing sclerotium formation in Aspergillus oryzae.
[0081] The *Aspergillus oryzae* NSAR1 strain began producing conidia 1-2 days after culture on PDA plates, and white sclerotia formed around the conidial clusters on day 5. These structures gradually matured, forming mature sclerotia with thick, brownish-brown walls. In contrast, the strain heterologously expressing tri4155 produced significantly denser white sclerotia on day 5, which covered the conidial clusters, while the area of the conidial clusters was significantly reduced. Figure 9Similar to the phenotype of heterologous tri4155 expression, the addition of 10 μM harzianol I to the culture medium also promoted sclerotium formation in Aspergillus oryzae, while 10 μM wickerol A had no such effect. These results indicate that tri4155 can promote the formation of host-resistant sclerotia through heterologous synthesis of harzianol I.
Claims
1. Terpenoid cyclases TriDTCs, including Tri4155 and its homologous proteins with a protein sequence similarity greater than 57%, including but not limited to one or more of the terpenoid cyclases in 1) to 9): 1) The amino acid sequence is Tri4155 as shown in SEQ ID NO.1; 2) The amino acid sequence is Tcie612 as shown in SEQ ID NO.2; 3) The amino acid sequence is Trei084 as shown in SEQ ID NO.3; 4) The amino acid sequence is Tass213 as shown in SEQ ID NO.4; 5) The amino acid sequence is Tasm819 as shown in SEQ ID NO.5; 6) The amino acid sequence is Tham208 as shown in SEQ ID NO.6; 7) The amino acid sequence is Tgai159 as shown in SEQ ID NO.7; 8) The amino acid sequence is as shown in SEQ ID NO.8, Tarm302; 9) Derivative proteins with the same function but with the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 substituted and / or deleted and / or added one or more amino acid residues.
2. The encoding gene of the terpene cyclase according to claim 1, including but not limited to one or more of the encoding genes in 1) to 9): 1) The gene encoding Tri4155 with a nucleotide sequence as shown in SEQ ID NO.9; 2) The gene encoding Tcie612, whose nucleotide sequence is shown in SEQ ID NO.10; 3) The gene encoding Trei084, whose nucleotide sequence is shown in SEQ ID NO.11; 4) The gene encoding Tass213 with a nucleotide sequence as shown in SEQ ID NO.12; 5) The gene encoding Tasm819 with a nucleotide sequence as shown in SEQ ID NO.13; 6) The gene encoding Tham208 with a nucleotide sequence as shown in SEQ ID NO.14; 7) The gene encoding Tgai159 with a nucleotide sequence as shown in SEQ ID NO.15; 8) The gene encoding Tarm302 with the nucleotide sequence shown in SEQ ID NO.16; 9) Nucleotide sequences of the nucleotide sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16 that have been substituted and / or have had one or more nucleotides deleted and / or added and express the same functional protein.
3. A recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium containing the encoding gene of claim 2.
4. The use of the terpene cyclase of claim 1, the encoding gene of claim 2, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria of claim 3 in the synthesis of Trichoderma diterpenoid compounds.
5. The application according to claim 4, characterized in that, The diterpenoid compounds include chemical structural formulas as shown in the formulas shown below. Harzianol I and wickerol A, as shown in Formula II, and downstream products derived from Harzianol I and wickerol A, Mode , Mode .
6. The use of Trichoderma diterpenoid compounds synthesized from the terpene cyclase of claim 1, the encoding gene of claim 2, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria of claim 3 in enhancing the production of fungal resistant spores.
7. The use of harzianol I synthesized from the terpene cyclase of claim 1, the encoding gene of claim 2, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria of claim 3 in enhancing the production of fungal resistant spores.
8. The use of the terpene cyclase of claim 1, the encoding gene of claim 2, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria of claim 3 in enhancing the production of fungal resistant spores.
9. The application according to claim 6, 7, or 8, characterized in that, The resistant fungal spores include chlamydospores of the Trichoderma genus, sclerotia of Aspergillus oryzae, and resistant spores of other fungi.