Production of jasmonates in filamentous fungi

Abstract

The present invention relates to an improved method for the production of jasmonates such as jasmonic acid and methyl jasmonate in filamentous fungi under shaking conditions, thus enabling the scale-up of the production process using conventional fermenters. In particular, during cultivation of the filamentous fungi, one or more fungal quorum-sensing molecules and / or a jasmonate production inducer can be added to the nutrient medium to induce favorable morphogenesis and jasmonate production.

Description

[0001] Related applications This application claims the benefit of U.S. Provisional Application No. 63 / 019,429, filed May 4, 2020, entitled “Production of jasmonic acids in filamentous fungi,” based on 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Invention Field

[0002] This invention relates to filamentous fungi such as *Trichoderma iridis* (… Lasiodiplodia iranensis Jasmonic acid is produced in ). Background of the Invention Jasmonic acids, including jasmonic acid (JA), methyl jasmonic acid (MeJA), and other precursors and derivatives in the jasmonic acid biosynthesis pathway, are α-linolenic acid-derived compounds of great economic importance. They are a class of plant hormones that play a crucial role in plant defense against carrion-producing pathogens and herbivorous insects. They are also powerful inducers of the biosynthesis of numerous secondary metabolites, such as caffeoyl putrescine, in tomato leaves. See, for example, Chen et al., Proc. Natl. Acad. Sci. USA, 102:19237-19242 (2005); Vijayan et al., Proc. Natl. Acad. Sci. USA, 95:7209-7214 (1998); and Chen et al., FEBS Lett. 580: 2540-2546 (2006).

[0004] Methyl jasmonate has a scent reminiscent of the heart of a jasmine flower and is used as a floral note in peach, apricot, grape, and other spices. Since 1973, the Flavor Extract Manufacturers Association has classified it as Generally Recognized As Safe (GRAS). Furthermore, methyl jasmonate has shown great potential as a novel class of anticancer drugs. Specifically, by inducing the release of cytochrome C in the mitochondria of cancer cells, methyl jasmonate can kill cancer cells without harming normal cells. See Rotem et al., Cancer Res., 65: 1984–1993 (2005).

[0005] Due to the importance of jasmonic acids in agriculture, the fragrance and aroma industry, and potential pharmaceutical applications, there is considerable interest in their large-scale production. Although jasmonic acids can be synthesized through organic chemistry, consumer demand for “natural” fragrances has created a market for jasmonic acids produced via bio-based methods. More importantly, chemically synthesized jasmonic acids are a mixture of bioactive and inactive isomers, while bio-based jasmonic acids are primarily bioactive isomers. Unfortunately, similar to other plant hormones, jasmonic acid and methyl jasmonic acid are present in higher plants only in trace amounts (e.g., less than 10 μg in 1 kg of induced fresh tomato leaves), thus preventing the utilization of higher plants as a commercial source of jasmonic acids. See Chen et al., FEBS Lett. 580: 2540-2546 (2006).

[0006] Conversely, filamentous fungi such as *Dioscorea cakopifolia* (…) are used. Lasiodiplodia theobromae (Synonyms include Coccobacillus dispora) Botryodiplodia theobromae ) and cottony color dispora ( Diplodia gossypina Fusarium oxysporum and Fusarium oxysporum ( )), Fusarium oxysporum and Fu Gibberella fujikuroi Large quantities of jasmonic acid (e.g., 1-1.5 g / L) can be synthesized using biological production methods. See U.S. Patent No. 6,333,180 and Eng et al., PLoS One, 11: e0167627. In fact, jasmonic acid as a natural product was first isolated from cultures of the fungus *Dioscorea opposita* in 1971. See Aldridge et al., J. Chem. Soc. C, pp. 1623-1627 (1971).

[0007] However, jasmonic acid was also found to be produced only in static flask cultures or static shallow dish cultures. On the other hand, large-scale fermentation manufacturing uses fermenters that operate under shaking or orbital oscillation to maximize production.

[0008] Therefore, there is still a need in the art for improved methods for the bioproduction of jasmonic acid, especially those methods that can be scaled up and achieve high production titers under stirred conditions. Summary of the Invention

[0009] This invention addresses the aforementioned problem by inducing jasmonic acid production in filamentous fungi using quorum-sensing molecules and / or inducers. A correlation was found between the mycelial morphology of these filamentous fungi and the level of jasmonic acid production. Specifically, high levels of jasmonic acid production are observed only when the filamentous fungi are able to form mycelial clusters, while no high levels of jasmonic acid production are observed when the filamentous fungi are in the form of free-floating spheres or clustered into mycelial particles. The use of quorum-sensing molecules enables the formation of mycelial clusters even when the filamentous fungi are grown under oscillating conditions (e.g., by a stirring system). Jasmonic acid production inducers are small molecules capable of inducing or activating cryptic biosynthetic pathways. In this case, jasmonic acid production inducers are chosen because they can induce or activate the cryptic biosynthetic pathways involved in the production of jasmonic acids by filamentous fungi.

[0010] Therefore, in one aspect, the present invention provides a method for producing one or more jasmonic acids (e.g., jasmonic acid and / or methyl jasmonicate), wherein the method comprises culturing a strain of filamentous fungal organisms in a nutrient medium under stirring, and isolating the jasmonic acid product from the nutrient medium. Representative genera of filamentous fungi include *Trichoderma*, *Fusarium*, and *Gibberella*. In various embodiments, the filamentous fungal organisms may be selected from *Trichoderma iridis*, *Trichoderma cocovenenans*, *Fusarium oxysporum*, and *Gibberella fujikuro*. In a representative embodiment, the filamentous fungal organism is *Trichoderma iridis* DWH-2, deposited under CCTCC accession number M2017288.

[0011] In various embodiments, the nutrient medium may contain at least one fungal quorum sensing molecule. In some embodiments, the nutrient medium may contain at least one jasmonic acid production inducer. In some embodiments, the nutrient medium may contain at least one fungal quorum sensing molecule and at least one jasmonic acid production inducer. In some embodiments, the nutrient medium may contain two or more fungal quorum sensing molecules. In some embodiments, the nutrient medium may contain two or more jasmonic acid production inducers. Using two or more fungal quorum sensing molecules, or using two or more jasmonic acid production inducers, or using a combination of at least one fungal quorum sensing molecule and at least one jasmonic acid production inducer, can produce a synergistic effect on jasmonic acid production at higher titers.

[0012] Examples of fungal quorum sensing molecules suitable for use in this invention include, but are not limited to, farnesol, tyrosol, tryptol, γ-heptanol, farnesic acid, 1-phenylethanol, 2-phenylethanol, multicolanic acid, multicolosic acid, multicolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, factor alpha pheromone, α-factor pheromone, 3-octanone, 3-octanol, and 1-octen-3-ol. Preferred fungal quorum sensing molecules include farnesol, tyrosol, tryptol, and γ-heptanol. In some embodiments, the nutrient medium may include farnesol, tyrosol, or both. In typical embodiments, the fungal quorum sensing molecule may be present in the nutrient medium at a concentration of about 10-500 mg / L.

[0013] According to this teaching, examples of suitable jasmonic acid inducers include, but are not limited to, various plant hormones, oxidative stressors, and histone deacetylase inhibitors. Representative plant hormones include, but are not limited to, ethylene (ET), indole-3-acetic acid (IAA), salicylic acid (SA), and acetylsalicylic acid (ASA). Also included are various auxins, such as 4-chloroindole-3-acetic acid (4-Cl-IAA), 2-phenylacetic acid (PAA), indole-3-butyric acid (IBA), and indole-3-propionic acid (IPA), as well as gibberellins such as gibberellin A1 (GA1), gibberellic acid (Ga3), ent-gibberellane, and ent-kaurene ( ent -kaurene). In some preferred embodiments, the jasmonic acid production inducer is a plant defense hormone such as abscisic acid (ABA).

[0014] In some implementations, the oxidative stressor can be a reactive oxygen species (ROS) added to the nutrient medium. Typical ROS species include hydrogen peroxide, peroxide salts, peroxyacids, and superoxide salts. Oxidative stress can also be induced by adding organic compounds known to have redox activity. Viologen is a well-known family of redox-active heterocyclic compounds, and viologen-paraquat (methyl viologen or MV) is widely used to induce oxidative stress. Its mechanism is believed to be acting as a superoxide generator, inducing ROS through interaction with complex I within the mitochondrial matrix.

[0015] Representative histone deacetylase inhibitors include, but are not limited to, valproic acid (VA) and sodium butyrate. Typically, jasmonic acid inducers are present in nutrient media at concentrations of approximately 10–500 mg / L.

[0016] In various embodiments, the nutrient medium may include at least one carbon source and at least one nitrogen source. Examples of suitable carbon sources include, but are not limited to, sucrose, starch, maltose, glucose, and fructose. The nitrogen source may be an organic nitrogen source, an inorganic nitrogen source, or both. Examples of organic nitrogen sources include, but are not limited to, beef extract, peptone, corn steep liquor, yeast extract, and malt extract. Examples of inorganic nitrogen sources include, but are not limited to, sodium nitrate, potassium nitrate, urea, and ammonium nitrate.

[0017] While this disclosure is readily adaptable to various modifications and alternatives, specific embodiments thereof are illustrated by way of example in the accompanying drawings and will be described in detail herein. However, it should be understood that the drawings and detailed description presented herein are not intended to limit this disclosure to the specific embodiments disclosed, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure as defined by the appended claims.

[0018] Other features and advantages of the invention will become apparent in the following detailed description of preferred embodiments of the invention with reference to the accompanying drawings. Brief description of the attached diagram Figure 1 Showing the chemical structures of jasmonic acid (JA) and its methyl ester, methyl jasmonic acid (MeJA).

[0020] Figure 2 The morphology of the fungal strain *Trichoderma iridis* grown under different culture conditions is shown: (A) with shaking at 250 rpm; (B) static. The temperature under both conditions was 30 °C.

[0021] Figure 3 The chemical structures of exemplary fungal quorum sensing molecules that can be used according to the teachings of the present invention are shown, specifically: farnesol, tyrosol, croterol, and γ-heptyl lactone.

[0022] Figure 4 The images show HPLC (top left) and UV (top right) spectra of Sigma-Aldrich's JA standard (MO, USA), as well as those of the ethyl acetate extract of the fungal culture that produces JA (bottom left and bottom right, respectively). The JA standard was prepared in methanol at a concentration of 1 g / L.

[0023] Figure 5This image shows the effects of farnesol on the morphology and JA production levels of fungal cultures under shaking conditions, 9 days post-inoculation. CK represents the control culture, in which 0.1% ethanol was added to the medium; "FAR" indicates the culture supplemented with 100 mg / L farnesol. The image on the left shows the different morphologies between the control and farnesol-induced cultures. The image on the right shows that the farnesol-induced cultures achieved JA production titers exceeding 300 mg / L.

[0024] Figure 6 This image shows the effect of tyrosol on the morphology and JA production levels of fungal cultures under shaking conditions, 7 days post-inoculation. CK3 represents the control culture, in which 0.1% ethanol was added to the medium; "Tyr3" represents the culture with 100 mg / L tyrosol added. The image on the left shows the different morphologies between the control and tyrosol-induced cultures. The image on the right shows that the tyrosol-induced cultures achieved JA production titers exceeding 300 mg / L.

[0025] Figure 7 The chemical structures of various jasmonic acid-derived inducers that can be used according to the teachings of this invention are shown, including representative plant hormones, oxidative stressors, and histone deacetylase inhibitors. Invention Details This teaching relates to a method for producing one or more jasmonic acids. The method generally involves culturing a strain of filamentous fungal organism in a nutrient medium under stirring, and isolating the jasmonic acid product from the nutrient medium. The jasmonic acids may be selected from jasmonic acid, methyl jasmonate, 7-isojasmonic acid, 9,10-dihydrojasmonic acid, 2,3-didehydrojasmonic acid, 3,4-didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5-didehydrojasmonic acid, 4,5-didehydro-7-isojasmonic acid, cucurbitacin, 6-epicucurbitacin, 6-epicucurbitacin lactone, 12-hydroxyjasmonic acid, 12-hydroxyjasmonicin lactone, 11-hydroxyjasmonic acid, 8... 1-Hydroxyjasmonic acid, homojasmonic acid, dihomojasmonic acid, 11-hydroxydihomojasmonic acid, 8-hydroxydihomojasmonic acid, tuberose acid, tuberose acid-O-β-glucopyranoside, cucurbitacin-O-β-glucopyranoside, 5,6-didehydrojasmonic acid, 6,7-didehydrojasmonic acid, 7,8-didehydrojasmonic acid, methyl dihydroisojasmonic acid, amino acid conjugates of jasmonic acid, and their lower alkyl esters, salts and stereoisomers.

[0027] In the broadest sense, the nutrient medium according to the invention comprises at least one fungal quorum sensing molecule or at least one jasmonic acid production inducer, at least one carbon source, and at least one nitrogen source. As demonstrated by the experimental results included herein, the inventors have unexpectedly discovered that the addition of one or more fungal quorum sensing molecules and / or jasmonic acid production inducers to the nutrient medium allows for the formation of favorable fungal morphologies even under stirred conditions. The formation of variable fungal morphologies (mycelial clusters) shows enhanced jasmonic acid production in filamentous fungi such as *Trichoderma iliforme*.

[0028] Quorum sensing (QS) is a communication mechanism among microorganisms that coordinates group-based behavior based on quorum density, relying on the production and release of small, diffusible chemical signaling molecules in the extracellular environment (Mehmood et al., Molecules 2019 May; 24(10): 1950). This study first reported the presence of the marine bacterium *Vibrio fischeri* (QS). Alivibrio fischeri Group sensing in (Nealson et al., (1970)) J. Bacteriol . 104, 313-322). Farnesol is produced by the dimorphic fungus Candida albicans ( Candida albicans The first fungal quorum sensing molecule discovered in [the study] (Hornby et al., (2001) Appl. Environ. Microbiol. 67, 2982–2992).

[0029] To date, numerous fungal quorum-sensing molecules have been identified, including farnesol, tyrosol, tryptol, and γ-heptylactin. Docolic acid, another fungal quorum-sensing molecule, has been reported to increase sclerotinum protein production in *Penicillium sclerotiorum*. J Biotechnol 2010 Jul 20;148(2-3):91-8). γ-Hemprolactone has been shown to regulate growth and secondary metabolite production in Aspergillus nidulans (Williams et al., Appl. Microbiol Biotechnol. 2012 Nov; 96(3):773-81). Furthermore, farnesol has been shown to regulate growth and secondary metabolite production in the dimorphic fungus *Picea longifolia* (Williams et al., Appl. Microbiol Biotechnol. 2012 Nov; 96(3):773-81). Ophiostoma piceae It induces morphological transformation and higher extracellular esterase activity in cells (De Salas et al., Appl Environ Microbiol. 2015 Jul;81(13):4351-7).

[0030] However, the biological activities of quorum sensing molecules can be quite diverse. For example, farnesol blocks the filamentous transition of yeast at high cell densities and promotes yeast cell dispersion by inhibiting germ tube / hyphae formation, while tyrosol stimulates hyphal production and promotes germ tube formation in the early stages of biofilm development (Padder et al., Microbiol Res. 2018 May;210:51-58). Despite their different biological activities, farnesol and tyrosol were surprisingly found to have similar effects on morphological changes and jasmonic acid production in JA-producing filamentous fungi.

[0031] Fungal quorum sensing molecules that can be used according to the teachings of the present invention include, but are not limited to, farnesol, tyrosol, tryptol, γ-heptanol, farnesic acid, 1-phenylethanol, 2-phenylethanol, docoracic acid, docosolic acid, docosolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, factor alpha pheromone, α-factor pheromone, 3-octanone, 3-octanol, and 1-octen-3-ol, or more thereof. Preferred fungal quorum sensing molecules include farnesol, tyrosol, tryptol, and γ-heptanol. In some embodiments, the nutrient medium may include farnesol, tyrosol, or both. Typically, fungal quorum sensing molecules may be present in the nutrient medium at a concentration of about 10-500 mg / L.

[0032] Jasmonic acid production inducers are small molecules capable of inducing or activating cryptic biosynthetic pathways; in this case, JA production using filamentous fungi such as *Trichoderma hainanensis* is involved. According to this teaching, examples of suitable jasmonic acid production inducers include, but are not limited to, various plant hormones, oxidative stressors, and histone deacetylase inhibitors. Representative plant hormones include, but are not limited to, ethylene (ET), indole-3-acetic acid (IAA), salicylic acid (SA), and acetylsalicylic acid (ASA). Also included are various auxins, such as 4-chloroindole-3-acetic acid (4-Cl-IAA), 2-phenylacetic acid (PAA), indole-3-butyric acid (IBA), and indole-3-propionic acid (IPA), and gibberellins such as gibberellin A1 (GA1), gibberellic acid (Ga3), ent-gibberellane, and ent-kaurene ( ent -kaurene). In some preferred embodiments, the jasmonic acid production inducer is a plant defense hormone such as abscisic acid (ABA).

[0033] In an exemplary embodiment, the oxidative stressor can be a reactive oxygen species (ROS) added to the nutrient medium. Typical ROS species include hydrogen peroxide, peroxide salts, peroxyacids, and superoxide salts. Oxidative stress can also be induced by adding organic compounds known to have redox activity. Viologen is a well-known family of redox-active heterocyclic compounds, and viologen paraquat (methyl viologen or MV) is widely used to induce oxidative stress. Its mechanism is believed to be acting as a superoxide generator, inducing ROS through interaction with complex I within the mitochondrial matrix.

[0034] Representative histone deacetylase inhibitors include, but are not limited to, valproic acid (VA) and sodium butyrate. Typically, jasmonic acid inducers are present in nutrient media at concentrations of approximately 10–500 mg / L.

[0035] In various embodiments, the method of the present invention can be carried out in batch or continuous operation modes. In batch fermentation, the nutrient medium, culture medium, and substrate are combined and fermented until the jasmonic acid product becomes constant. In the continuous method, the substrate in the nutrient medium can be continuously recycled via the fermentation reactor, provided that the substrate and product are added separately and removed from the recycled medium.

[0036] In carrying out the method of this invention, in addition to producing one or more fungal quorum sensing molecules and / or jasmonic acid-based inducers, the fungal strain is cultured and fermented in an aqueous culture medium in the presence of conventional nutrients (carbon source, nitrogen source, inorganic salts, and growth factors). Examples of inorganic salts that may be included in the nutrient medium include, but are not limited to, phosphates and / or sulfates of sodium, calcium, magnesium, and potassium. Other nutrients may also be added, such as one or more B vitamins, one or more trace minerals such as iron, manganese, cobalt, copper, zinc, etc., as known to those skilled in the art. Fungal growth hormones such as 10-oxo-trans-8-decenoic acid and histidine betaine may also be included in the nutrient medium.

[0037] In a typical method, filamentous fungal organisms are first cultured at an inoculum size to produce a mature culture in a nutrient medium. The culture is then inoculated into a fermenter nutrient medium and allowed to establish itself. Substrate is then added, and fermentation continues until a stable concentration of jasmonic acid products is achieved.

[0038] The culture and fermentation incubation of filamentous fungal organisms can be carried out with stirring at a speed of approximately 150 rpm to approximately 1500 rpm. The culture temperature can be between approximately 20°C and approximately 35°C. Culture and incubation can be carried out under aerobic conditions at a pH range of approximately 4.5 to approximately 9, preferably 6. Jasmonic acid products can be isolated after incubation for at least 2 days following substrate addition.

[0039] In various embodiments, jasmonic acid products can be separated from the nutrient medium by extraction with an extraction solvent such as ethyl acetate to form a jasmonic acid extract. The extraction solvent can be vapor stripped to provide a concentrated jasmonic acid extract. Jasmonic acid present in the jasmonic acid extract can be converted to methyl jasmonic acid by esterification using methanol. The resulting methyl jasmonic acid can be further concentrated using techniques known to those skilled in the art. For example, fractionation can be performed, for instance, with silica gel to separate different isomers.

[0040] Jasmonic acids, such as jasmonic acid and methyl jasmonic acid, produced according to the teachings of this invention, can be used in a variety of applications in agriculture, food, fragrance, and medicine. For example, jasmonic acid has been tested as a natural pest control tool for crops against herbivores. Methyl jasmonic acid can be used as a food and flavoring ingredient in products such as perfumes, personal care products, home care products, and oral care products. Furthermore, given its reported antidepressant, anti-inflammatory, and anti-inflammatory effects, methyl jasmonic acid also has great potential for development for pharmaceutical uses.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred materials and methods are described below.

[0042] This disclosure will be more fully understood after considering the following non-limiting embodiments. It should be understood that these embodiments, while indicating preferred embodiments of the subject matter, are given by way of illustration only. From the foregoing discussion and these embodiments, those skilled in the art can determine the essential features of the subject matter, and various changes and modifications can be made to the subject matter to adapt it to various uses and conditions without departing from its spirit and scope. Example

[0043] Example 1: Morphology of *Trichoderma iridis* under different culture conditions To produce jasmonic acid (JA), cultures of *Trichoderma iridis* DWH-2 (deposited under CCTCC accession number M2017288) were grown under two different conditions. One culture was grown in a shaker under stirring conditions (250 rpm). The other culture was cultured in an incubator for static growth.

[0044] like Figure 2 As shown, under oscillating conditions, fungal mycelia are freely dispersed throughout the culture with a porridge-like consistency (i.e., they form mycelial clusters). Conversely, under static conditions, fungal mycelia aggregate into mats.

[0045] Furthermore, HPLC analysis showed that little or no JA was produced under oscillating conditions, while under static conditions, the culture produced a JA titer of approximately 1 g / L, which is similar to that reported in Chinese patent CN107227264B.

[0046] Based on the above results, especially regarding fungal morphology, it is demonstrated that the generation of JA is related to hyphal aggregation in filamentous fungi such as Trichoderma iridis.

[0047] Example 2: Effect of adding farnesol on JA of Trichoderma iridis Cultures of *Trichoderma iridis* DWH-2 (accessed by CCTCC accession number M2017288) were kept on potato dextrose agar (PDA, from Millipore Sigma, MO, USA) plates in an incubator at 30°C.

[0048] Cut a square slice (~1 cm x 1 cm) from a PDA plate containing a culture of Trichoderma iridis and inoculate it with 50 ml of nutrient medium containing (Far) or without (CK) farnesol.

[0049] Specifically, farnesol (purchased from Sigma-Aldrich, MO, USA) was dissolved in 70% ethanol to prepare a stock solution with a concentration of 100 g / L. For samples containing farnesol, the final working concentration in the culture medium was 100 mg / L (1,000-fold dilution). The nutrient medium contained the following components: glucose (50 g / L); KNO3 (8.9 g / L); KH2PO4 2.0 (g / L); KCl 0.3 (g / L); MgSO4·7H2O 0.6 (g / L); FeSO4·7H2O (0.6 g / L); ZnSO4·7H2O (0.03 g / L); MnSO4·7H2O (0.003 g / L); CuSO4·7H2O (0.003 g / L); Na2MoO4·2H2O 0.003 (g / L); and yeast extract (1.0 g / L).

[0050] The flask was placed in a shaker set at 250 rpm and 30°C. After nine days of incubation, the extract was collected, and its JA content was analyzed by HPLC.

[0051] For porridge-like cultures, 0.5 ml of the entire culture was used as a sample for further analysis. For cushion-like cultures, 0.5 ml of supernatant was used. Each sample was acidified with 10 μl of 2N HCl, followed by JA extraction with 0.5 ml of ethyl acetate. After shaking at room temperature for 30 min, the sample was centrifuged at 15,000 rpm for 15 min. The ethyl acetate phase was used for HPLC analysis.

[0052] HPLC was performed on a Thermo Scientific Dionex Ultimate 3000 using an Acclaim™ 120, C18 column (3 µm 120A, 3 x 150 mm). The mobile phases were: A, 0.1% TFA (trifluoroacetic acid) and B, acetonitrile, with the following gradients: 0–5 min, 5% B; 5–9 min, 5–80% B; 9–13 min, 80% B; 13–14 min, 80–5% B; 14–17 min, 5% B. The detector wavelength for JA was 200 nm. Figure 4 It was confirmed that JA from fungal cultures had the same retention time and UV spectrum as JA standards from Sigma-Aldrich (MO, USA).

[0053] Figure 5 The effect of adding farnesol on JA production under shaking conditions was verified. As shown, the extract without farnesol supplementation exhibited a porridge-like morphology and produced very little jasmonic acid (approximately 19 mg / L) under shaking conditions. In contrast, farnesol induced mycelial aggregation in *Trichoderma iridis*, resulting in the formation of cushion-like microbial communities even under shaking conditions. The sample with added farnesol was able to produce jasmonic acid at titers of ~327 mg / L.

[0054] Therefore, these results confirm that the addition of farnesol can be used to control the fungal morphology of filamentous fungi such as *Trichoderma iridis*. Specifically, when farnesol is added, *Trichoderma iridis* is able to form mycelial clusters under shaking conditions. Considering that the cushion-like morphology appears to be crucial for jasmonic acid biosynthesis in filamentous fungi, the addition of farnesol results in the production of JA at higher titers under shaking conditions.

[0055] Example 3: Effect of adding tyrosol on JA production of Trichoderma iridis Repeat the steps described in Example 2 using casein instead of farnesol. Specifically, cut a square slice (approximately 1 cm × 1 cm in area) from a PDA plate containing a culture of *Trichoderma iridis* and inoculate it with 30 ml of the same nutrient medium containing (Tyr3) or without (CK3) casein. Dissolve casein (purchased from Sigma-Aldrich, MO, USA) in 70% ethanol to prepare a stock solution with a concentration of 100 g / L. For samples containing casein, the final working concentration in the medium is 100 mg / L (1,000-fold dilution).

[0056] The flask was placed in a shaker set at 250 rpm and 30°C. After seven days of incubation, the extract was removed, and its JA content was analyzed by HPLC.

[0057] Figure 6 The effect of adding casein on JA production under shaking conditions was verified. As shown, the extract without casein supplementation exhibited a porridge-like morphology and produced very little jasmonic acid (approximately 81 mg / L) under shaking conditions. In contrast, casein induced mycelial aggregation in *Trichoderma iridis*, resulting in the formation of cushion-like microbial communities even under shaking conditions. The sample with added casein was able to produce jasmonic acid at titers of ~314 mg / L.

[0058] Therefore, these results confirm that the addition of tyrosol can be used to control the fungal morphology of filamentous fungi such as *Trichoderma iridis*. Specifically, when tyrosol is added, *Trichoderma iridis* is able to form mycelial clusters under shaking conditions. Considering that the cushion-like morphology appears to be crucial for jasmonic acid biosynthesis in filamentous fungi, the addition of tyrosol results in the production of JA at higher titers under shaking conditions.

[0059] Example 4: Inducing JA production of *Trichoderma iridis* by adding one or more jasmonic acid inducers Effect Replacing farnesol with a jasmonic acid production inducer, repeat the steps described in Example 2. Specifically, cut a square slice (approximately 1 cm × 1 cm in area) from a PDA plate containing a culture of *Trichoderma iridis* and inoculate it with 50 ml of the same nutrient medium, with or without a jasmonic acid production inducer. A jasmonic acid production inducer is a small molecule capable of inducing or activating a cryptic biosynthetic pathway; in this case, it involves production using JA from filamentous fungi such as *Trichoderma iridis*. Typical jasmonic acid production inducers can be plant hormones, oxidative stressors, histone deacetylase inhibitors, or antibiotics. Figure 7 The chemical structures of various jasmonic acids that can be used in accordance with this teaching to produce inducers are shown, including representative plant hormones such as indole-3-acetic acid (IAA), salicylic acid (SA), and acetylsalicylic acid (ASA); representative oxidative stressors such as methyl viologen (MV) and hydrogen peroxide (H2O2); and representative histone deacetylase inhibitors such as valproic acid (VA) and sodium butyrate.

[0060] Sodium indole-3-acetate (IAA), sodium salicylate (SA), acetylsalicylic acid (ASA), sodium butyrate, sodium valproate (VA), and hydrogen peroxide were purchased from Sigma-Aldrich (MO, USA). Stock solutions of the sodium salts and H2O2 were prepared in water, while the stock solution of ASA was prepared in 70% ethanol. The final working concentrations in the culture medium were 200 mg / L for IAA and sodium butyrate; 100 mg / L for SA and ASA; 10 mg / L for VA; and 2 mM for H2O2.

[0061] The flask was placed in a shaker set at 250 rpm and 30°C. After eight days of incubation, the extract was removed, and its JA content was analyzed by HPLC.

[0062] In the control group (i.e., without jasmonic acid production inducers), less than 50 mg / L of JA was produced. As summarized in Table 1 below, cultures with added jasmonic acid production inducers produced JA at significantly higher titers.

[0063] Table 1. JA production with or without jasmonic acid-induced agents. Comparison 50 IAA 400 SA 150 ASA 100 Sodium butyrate 175 VA 350 <![CDATA[H2O2]]> 160

Claims

1. A method for producing jasmonic acids, the method comprising: *Trichoderma iridis* DWH-2, deposited under CCTCC accession number M2017288, was cultured with stirring in a nutrient medium containing at least one fungal quorum sensing molecule, at least one carbon source, and at least one nitrogen source; and Jasmonic acid products were isolated from the nutrient culture medium. The nutrient culture medium contains at least one of farnesol or tyrosol.

2. The method according to claim 1, wherein the nutrient culture medium further comprises one or more fungal quorum sensing molecules selected from tryptophan, γ-heptanol, farnesic acid, 1-phenylethanol, 2-phenylethanol, docolactic acid, docosolic acid, docolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, α-factor pheromone, α-factor pheromone, 3-octanone, 3-octanol and 1-octen-3-ol.

3. The method according to claim 1, wherein the nutrient culture medium comprises farnesol and tyrosol.

4. The method according to claim 1, wherein the nutrient culture medium further comprises at least one jasmonic acid-based inducing agent.

5. The method according to claim 4, wherein the jasmonic acid production inducer is selected from plant hormones, oxidative stressors, and histone deacetylase inhibitors.

6. The method according to claim 4, wherein the jasmonic acid generating inducer is selected from indole-3-acetic acid, salicylic acid, acetylsalicylic acid, methyl viologen, hydrogen peroxide, valproic acid, and sodium butyrate.

7. The method according to claim 4, wherein the nutrient culture medium comprises two or more jasmonic acid production inducers selected from indole-3-acetic acid, salicylic acid, acetylsalicylic acid, methyl viologen, hydrogen peroxide, valproic acid and sodium butyrate.

8. The method according to any one of claims 1-7, wherein the carbon source is selected from sucrose, starch, maltose, glucose and fructose.

9. The method according to any one of claims 1-7, wherein the nitrogen source is an organic nitrogen source selected from beef extract, peptone, corn steep liquor, yeast extract and malt extract.

10. The method according to any one of claims 1-7, wherein the nitrogen source is an inorganic nitrogen source selected from sodium nitrate, potassium nitrate, urea and ammonium nitrate.

11. The method according to any one of claims 1-7, wherein the stirring is performed between 150 rpm and 1500 rpm.

12. The method according to any one of claims 1-7, wherein the culture step is performed at a temperature between 20°C and 35°C.

13. The method according to any one of claims 1-7, wherein the culture step is performed for at least 2 days.

14. The method according to any one of claims 1-7, wherein the at least one fungal quorum sensing molecule is present in the nutrient culture medium at a concentration of 10-500 mg / L.

15. The method according to any one of claims 4-7, wherein if the jasmonic acid production inducer is a solid, the at least one jasmonic acid production inducer is present in the nutrient culture medium at a concentration of 10-500 mg / L.

16. The method according to any one of claims 1-7, wherein the nutrient culture medium further comprises a fungal growth hormone selected from 10-oxo-trans-8-decenoic acid and histidine betaine.

17. The method according to any one of claims 1-7, wherein the jasmonic acid product comprises jasmonic acid.

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