Production of Jasmonates in Filamentous Fungi

Abstract

The present invention relates to an improved method for producing jasmonates, such as jasmonic acid and methyl jasmonate, in filamentous fungi under shaking conditions, thereby enabling the scale-up of the production process using conventional fermentors. Specifically, one or more fungal quorum-sensing molecules and / or jasmonate-producing elicitors can be added to the nutrient medium during the cultivation of the filamentous fungi to induce the formation of desired morphologies and the production of jasmonates.

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 019,429, filed May 4, 2020, entitled "PRODUCTION OF JASMONATES IN FILAMENTOUS FUNGI", the entire content of which is incorporated herein by reference.

[0002] The field of the invention relates to the production of jasmonates in filamentous fungi such as Lasiodiplodia iranensis.

Background Art

[0003] Jasmonates, including jasmonic acid (JA), methyl jasmonate (MeJA), and other precursors and derivatives in the jasmonic acid biosynthetic pathway, are economically very important α-linolenic acid-derived compounds. They are a class of plant hormones that play a central role in plant defense against necrotrophic pathogens and herbivorous insects. They are also powerful elicitors that induce the biosynthesis of numerous secondary metabolites such as caffeoylputrescine 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, which imparts an odor reminiscent of the floral heart of jasmine, is used in its floral notes for peach, apricot, grape and other flavors. Since 1973, it has been classified as generally recognized as safe (GRAS) by the Flavor Extract Manufacturers Association. Furthermore, methyl jasmonate has also been shown to have great potential as a new class of anti-cancer agents. Specifically, by inducing cytochrome C release 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 jasmonates in agriculture, flavor and fragrance industries, and potentially in medicine, there has been considerable interest in large-scale production of jasmonates. Jasmonates can be synthesized by organic chemistry, but consumer demand for "natural" flavors has created a market for jasmonates produced by biobased processes. More importantly, chemically synthesized jasmonates are a mixture of biologically active and inactive isomers, while biobased jasmonates have a predominance of biologically active isomers. Unfortunately, like other plant hormones, both jasmonic acid and methyl jasmonate are present only in trace amounts in higher plants (e.g., less than 10 μg in 1 kg of induced fresh tomato leaves), which hinders the development of higher plants as commercial sources of jasmonates. See Chen et al., FEBS Lett. 580:2540-2546 (2006).

[0006] In contrast, in a bioproduction process using filamentous fungi such as Lasiodiplodia theobromae (synonyms include Botryodiplodia theobromae and Diplodia gossypina), Fusarium oxysporum, and Gibberella fujikuroi, a large amount of jasmonic acid can be synthesized (e.g., 1 - 1.5 g / L). 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 a culture of the fungus Lasiodiplodia theobromae in 1971. See Aldridge et al., J.Chem.Soc.C, pp.1623 - 1627 (1971).

[0007] However, it has also been found that jasmonic acid is produced only in static flask culture or static tray culture. On the other hand, large - scale fermentation production uses fermenters that operate with rocking or orbital shaking to maximize production.

[0008] Therefore, in the art, there is still a need for an improved method for the bioproduction of jasmonic acids, particularly one that is scalable and can achieve high productivity values under agitation conditions.

Summary of the Invention

Means for Solving the Problems

[0009] The present invention addresses the above problems by inducing jasmonic acid production in filamentous fungi using quorum sensing molecules and / or elicitors. A correlation was found between the mycelial morphology of such filamentous fungi and the level of jasmonic acid production. Specifically, high levels of jasmonic acid production were observed only when the filamentous fungi were able to form a mycelial mat, but not when the filamentous fungi were in a floating pellet form or aggregated into mycelial particles. The use of quorum sensing molecules enables the formation of a mycelial mat even when the filamentous fungi grow under shaking conditions (e.g., by a stirring system). A jasmonic acid production elicitor is a small molecule that can induce or awaken a potential biosynthetic pathway. In this case, the jasmonic acid production elicitor is selected for its ability to induce or awaken a potential biosynthetic pathway involved in jasmonic acid production by filamentous fungi.

[0010] Accordingly, in one aspect, the present invention provides a method for producing one or more jasmonic acids (e.g., jasmonic acid and / or methyl jasmonate), the method comprising culturing a strain of a filamentous fungal organism in a nutrient medium under agitation and isolating the jasmonic acid product from the nutrient medium. Representative genera of filamentous fungi include Lasiodiplodia, Fusarium, and Gibberella. In various embodiments, the filamentous fungal organism can be selected from the group consisting of Lasiodiplodia iranensis, Lasiodiplodia theobromae, Fusarium oxysporum, and Gibberella fujikuroi. In a representative embodiment, the filamentous fungal organism is Lasiodiplodia iranensis DWH-2 deposited under CCTCC deposit number M2107288.

[0011] In various embodiments, the nutrient medium can include at least one fungal quorum-sensing molecule. In some embodiments, the nutrient medium can include at least one jasmonate-producing elicitor. In certain embodiments, the nutrient medium can include at least one fungal quorum-sensing molecule and at least one jasmonate-producing elicitor. In some embodiments, the nutrient medium can include two or more fungal quorum-sensing molecules. In some embodiments, the nutrient medium can include two or more jasmonate-producing elicitors. The use of two or more fungal quorum-sensing molecules, or two or more jasmonate-producing elicitors, or the combined use of at least one fungal quorum-sensing molecule and at least one jasmonate-producing elicitor can produce a synergistic effect on jasmonate production at a higher titer.

[0012] Examples of fungal quorum-sensing molecules suitable for use according to the present teachings include, but are not limited to, farnesol, tyrosol, tryptofol, γ-heptalactone, farnesoic acid, 1-phenyl-ethanol, 2-phenylethanol, multicolanic acid, multicolosic acid, multicolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, a-factor pheromone, α-factor pheromone, 3-octanone, 3-octanol, and 1-octen-3-ol. Preferred fungal quorum-sensing molecules include farnesol, tyrosol, tryptofol, and γ-heptalactone. In certain embodiments, the nutrient medium can include farnesol, tyrosol, or both. In exemplary embodiments, the fungal quorum-sensing molecule(s) can be present in the nutrient medium at a concentration of about 10 - 500 mg / L.

[0013] Examples of jasmonic acid-producing elicitors suitable for use according to the present teachings include, but are not limited to, various plant hormones, oxidative stress factors, 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). 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, are also included. In some preferred embodiments, the jasmonic acid-producing elicitor is a plant defense hormone such as abscisic acid (ABA).

[0014] In some embodiments, the oxidative stress factor may be reactive oxygen species (ROS) added to the nutrient medium. Typical reactive oxygen species include hydrogen peroxide, peroxide salts, peroxy acids, and superoxide salts. Oxidative stress can also be induced by the addition of organic compounds known to be redox-active. Biologens are a well-known family of redox-active heterocycles, and biologen paraquat (methyl viologen, or MV) is widely used to induce oxidative stress by a mechanism thought to act as a superoxide generator and generates ROS through interaction with complex I in the inner mitochondrial matrix.

[0015] Representative histone deacetylase inhibitors include, but are not limited to, valproic acid (VA) and sodium butyrate. Typically, the jasmonic acid-producing elicitor(s) are present in the nutrient medium at a concentration of about 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 either 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 pulp, 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] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. However, the drawings and detailed description presented herein are not intended to limit the present disclosure to the particular embodiments disclosed, and on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0018] Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0026] The present disclosure relates to a method for producing one or more jasmonates. The method generally includes culturing a strain of filamentous fungal organism in a nutrient medium under agitation and isolating the jasmonate product from the nutrient medium. The jasmonate(s) can be selected from jasmonic acid, methyl jasmonate, 7-iso-jasmonic 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-iso-jasmonic acid, cucurbitic acid, 6-epi-cucurbitic acid, 6-epi-cucurbitic acid-lactone, 12-hydroxy-jasmonic acid, 12-hydroxy-jasmonic acid-lactone, 11-hydroxy-jasmonic acid, 8-hydroxy-jasmonic acid, homojasmonic acid, dihomojasmonic acid, 11-hydroxy-dihomojasmonic acid, 8-hydroxy-dihomojasmonic acid, tuberonic acid, tuberonic acid-O-β-glucopyranoside, cucurbitic acid-O-β-glucopyranoside, 5,6-didehydrojasmonic acid, 6,7-didehydrojasmonic acid, 7,8-didehydrojasmonic acid, methyldihydroisojasmonate, 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 present disclosure contains at least one fungal quorum sensing molecule or at least one jasmonate elicitor, at least one carbon source, and at least one nitrogen source. As demonstrated by the experimental results included herein, the inventors have unexpectedly found that by adding one or more fungal quorum sensing molecules and / or jasmonate elicitors to the nutrient medium, a preferred fungal morphology can be formed even under agitation conditions. The formation of variable fungal morphology (mycelial mat) has been shown to enhance the production of jasmonates in filamentous fungi such as Lasiodiplodia iranensis.

[0028] Quorum sensing (QS) is a method of microbial communication that enables the coordination of group-based behaviors based on population density, which depends on the production and release of small diffusible chemical signaling molecules in the extracellular environment (Mehmood et al., Molecules 2019 May;24(10):1950). Quorum sensing was first reported in the marine bacterium Alivibrio fischeri (Nealson et al. (1970) J. Bacteriol. 104, 313 - 322). Farnesol was the first fungal quorum sensing molecule discovered in the dimorphic fungus Candida albicans (Hornby, et al. (2001) Appl. Environ. Microbiol. 67, 2982 - 2992).

[0029] To date, many fungal quorum sensing molecules, including farnesol, tyrosol, tryptofol, and γ-heptalactone, have been identified. Another fungal quorum sensing molecule, multicolic acid, has been reported to improve sclerotiorin production in Penicillium sclerotiorum (J Biotechnol. 2010 Jul 20;148(2 - 3):91 - 8). γ-Heptalactone 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 induce morphological transition and higher extracellular esterase activity in the dimorphic fungus Ophiostoma piceae (De Salas et al., Appl Environ Microbiol. 2015 Jul;81(13):4351 - 7).

[0030] However, the biological activities of quorum sensing molecules can be highly diverse. For example, farnesol promotes the dispersion of yeast cells by blocking the transition from yeast form to filamentous form at high cell density and inhibiting germ tube / hypha formation, while tyrosol stimulates hyphal growth and promotes germ tube formation at the initial stage of biofilm development (Padder et al., Microbiol Res. 2018 May;210:51-58). Despite their different biological activities, both farnesol and tyrosol were unexpectedly found to show 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 present teachings include, but are not limited to, one or more of farnesol, tyrosol, tryptofol, γ-heptalactone, farnesenic acid, 1-phenyl-ethanol, 2-phenylethanol, multicolanic acid, multicolosic acid, multicolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, a-factor pheromone, α-factor pheromone, 3-octanone, 3-octanol, and 1-octen-3-ol. Preferred fungal quorum sensing molecules include farnesol, tyrosol, tryptofol, and γ-heptalactone. In certain embodiments, the nutrient medium may contain farnesol, tyrosol, or both. Typically, the fungal quorum sensing molecule(s) can be present in the nutrient medium at a concentration of about 10 - 500 mg / L.

[0032] Jasmonate-producing elicitors are small molecules that can induce or awaken potential biosynthetic pathways and, in this example, are involved in JA production by filamentous fungi such as Lasiodiplodia iranensis. Examples of jasmonate-producing elicitors suitable for use according to the present teachings include, but are not limited to, various plant hormones, oxidative stress factors, and histone deacetylase inhibitors. Representative plant hormones include, but are not limited to, ethylene (ET), indole-3-acetic acid (IAA), salicylic acid (SA), acetylsalicylic acid (ASA). 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 are also included. In some preferred embodiments, the jasmonate-producing elicitor is a plant defense hormone such as abscisic acid (ABA).

[0033] In an exemplary embodiment, the oxidative stress factor can be reactive oxygen species (ROS) added to the nutrient medium. Typical reactive oxygen species include hydrogen peroxide, peroxide salts, peroxy acids, and superoxide salts. Oxidative stress can also be induced by the addition of organic compounds known to be redox-active. Biologens are a well-known family of redox-active heterocycles, and biologen paraquat (methylviologen, or MV) is widely used to induce oxidative stress by a mechanism thought to act as a superoxide generator and generates ROS through interaction with complex I inside the mitochondrial matrix.

[0034] Representative histone deacetylase inhibitors include, but are not limited to, valproic acid (VA) and sodium butyrate. Typically, the jasmonate-producing elicitor(s) is present in the nutrient medium at a concentration of about 10 - 500 mg / L.

[0035] In various embodiments, the method can be carried out in batch or continuous operation modes. In batch fermentation, a nutrient medium, a culture, and a substrate are combined and fermented until the jasmonates product becomes constant. In a continuous process, the substrate in the nutrient medium can be continuously recycled through the fermentation reactor under conditions where the substrate and the product are each added and removed from the recycled medium.

[0036] When carrying out this process, the cultivation and fermentation incubation of the fungal strain are achieved in an aqueous medium in the presence of normal nutrients (carbon source, nitrogen source, inorganic salts, and growth factors) in addition to one or more fungal quorum sensing molecules and / or jasmonates production elicitors. Examples of inorganic salts that can be included in the nutrient medium include, but are not limited to, phosphate and / or sulfate salts of sodium, calcium, magnesium, and potassium. As is known to those skilled in the art, additional nutrients such as one or more vitamins B, one or more trace minerals such as iron, manganese, cobalt, copper, zinc, etc. can also be added. Fungal growth hormones such as 10-oxo-trans-8-decenoic acid and hercynine can also be included in the nutrient medium.

[0037] In a typical process, the filamentous fungal organism is first cultured at an inoculum level to produce a mature culture in a nutrient medium. The culture is inoculated into the fermenter nutrient medium and allowed to establish itself. Then, the substrate is added and fermentation is continued until a certain concentration of jasmonates product is present.

[0038] The cultivation and fermentation incubation of the filamentous fungal organism can be carried out under agitation at about 150 rpm to about 1500 rpm. The culture temperature is from about 20 °C to about 35 °C. The cultivation and incubation can proceed under aerobic conditions in a pH range of about 4.5 to about 9, preferably 6. The jasmonates product can be isolated at least 2 days after cultivation following the addition of the substrate.

[0039] In various embodiments, the jasmonates product can be isolated from the nutrient medium by extracting with an extraction solvent such as ethyl acetate to form a jasmonates extract. The extraction solvent can be removed to obtain a concentrated jasmonates extract. Jasmonic acid present in the jasmonates extract can be converted to methyl jasmonate by esterification using methyl alcohol. The obtained methyl jasmonate can be further concentrated using techniques known to those skilled in the art. For example, fractionation can be performed using, for example, silica gel to separate different isomers.

[0040] Jasmonates such as jasmonic acid and methyl jasmonate produced according to the present teachings can be used in various applications in agriculture, food, fragrance, and pharmaceuticals. For example, jasmonic acid has been tested as a natural pest control tool for crop plants against herbivores. Methyl jasmonate can be used as a food and flavor ingredient in products such as fragrances, personal care products, household care products, and oral consumables. Furthermore, methyl jasmonate also has great potential for development for pharmaceutical use considering its reported antidepressant, anti-aggression, and anti-inflammatory effects.

[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 belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred materials and methods are described below.

[0042] The present disclosure will be more fully understood by considering the following non-limiting examples. It should be understood that these examples illustrate preferred embodiments of the subject technology but are provided by way of illustration only. From the above description and these examples, one of ordinary skill in the art can identify the essential features of the subject technology and make various changes and modifications to the subject technology to adapt it to various applications and conditions without departing from the spirit and scope thereof.

[0043] Example Example 1: Morphology of Lasiodiplodia iranensis under different culture conditions. Cultures of Lasiodiplodia iranensis DWH-2 (deposited under CCTCC deposit number M2107288) were grown under two different conditions for jasmonic acid (JA) production. One culture was grown in a shaker under stirring conditions (250 rpm). Another culture was grown in an incubator for static growth.

[0044] As shown in Figure 2, under shaking conditions, the fungal mycelia were freely dispersed with a porridge-like consistency throughout the culture (i.e., they did not form mycelial clumps). In contrast, under static conditions, the fungal mycelia aggregated into a mat.

[0045] Furthermore, HPLC analysis demonstrated that under shaking conditions, little or no JA was produced, while under static conditions, the culture produced JA at a titer of approximately 1 g / L similar to that reported in Chinese Patent No. 107227264.

[0046] In light of the above results, it was demonstrated that JA production correlates with mycelial aggregation in filamentous fungi such as Lasiodiplodia iranensis, especially with regard to the morphology of the fungus.

[0047] Example 2: Effect of addition of farnesol on JA production by Lasiodiplodia iranensis Cultures of Lasiodiplodia iranensis DWH-2 (deposited under CCTCC deposit number M2107288) were maintained on potato dextrose agar (PDA, manufactured by MilliporeSigma, MO, USA) plates in an incubator at 30 °C.

[0048] Square pieces (approx. 1 cm x 1 cm) of PDA plates containing L. iranensis cultures were cut and used to inoculate 50 ml of nutrient medium containing farnesol (Far) or no farnesol (CK).

[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. The final working concentration in the culture medium was 100 mg / L (1,000 - fold dilution) for samples containing farnesol. The nutrient medium contained the following: 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 flasks were placed in a shaker set at 250 rpm and 30 °C. After 9 days of culture, the extracts were collected and their JA content was analyzed by HPLC.

[0051] For the broth - like cultures, 0.5 ml of the whole culture was taken as a sample for further analysis. For the mat - like cultures, 0.5 ml of the supernatant was used. To each sample, 10 μl of 2N HCl was added for acidification, followed by 0.5 ml of ethyl acetate for JA extraction. After shaking at room temperature for 30 minutes, the samples were centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate phase was used for HPLC analysis.

[0052] HPLC was performed using a Thermo Scientific Dionex Ultimate 3000 with an Acclaim™ 120, C18 column (3 μm 120 Å, 3 x 150 mm). The mobile phase was A, 0.1% TFA (trifluoroacetic acid) and B, acetonitrile, gradient: 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 confirms that JA from the fungal culture has the same retention time and UV spectrum as the JA standard from Sigma - Aldrich (MO, USA).

[0053] The effect of the addition of farnesol on JA production under shaking conditions was demonstrated in Figure 5. As shown, cultures without farnesol supplementation showed a porridge - like morphology and produced little jasmonic acid (about 19 mg / L) under shaking conditions. In contrast, farnesol induced hyphal aggregation in L. iranensis and led to the formation of a mat - like microbiota despite the shaking conditions. Samples supplemented with farnesol were able to produce jasmonic acid at a titer of about 327 mg / L.

[0054] Therefore, these results confirmed that the addition of farnesol can be used to control the fungal morphology of filamentous fungi such as Lasiodiplodia iranensis. Specifically, when farnesol was added, Lasiodiplodia iranensis was able to form a mycelial mat under shaking conditions. Considering that the mat - like morphology seems to be important for jasmonic acid biosynthesis by filamentous fungi, the addition of farnesol led to JA production at a higher titer under shaking conditions.

[0055] Example 3: Effect of the addition of tyrosol on JA production by Lasiodiplodia iranensis Using tyrosol instead of farnesol, the procedure described in Example 2 was repeated. Specifically, square pieces (area approximately 1 cm x 1 cm) of a PDA plate containing a culture of L. iranensis were cut and used to inoculate 30 ml of the same nutrient medium with (Tyr3) or without (CK3) tyrosol. Tyrosol (purchased from Sigma-Aldrich, MO, USA) was dissolved in 70% ethanol to prepare a stock solution with a concentration of 100 g / L. The final working concentration in the culture medium was 100 mg / L (1,000-fold dilution) for the samples containing tyrosol.

[0056] The flasks were placed in a shaker set at 250 rpm and 30 °C. After 7 days of culture, the extracts were collected and their JA content was analyzed by HPLC.

[0057] The effect of the addition of tyrosol on JA production under shaking conditions was demonstrated in Figure 6. As shown, the cultures without tyrosol supplementation showed a porridge-like morphology and produced little jasmonic acid (about 81 mg / L) under shaking conditions. In contrast, tyrosol induced hyphal aggregation in L. iranensis and led to the formation of a mat-like microbiota despite the shaking conditions. The samples with added tyrosol were able to produce jasmonic acid at a titer of about 314 mg / L.

[0058] Therefore, these results confirmed that the addition of tyrosol can be used to control the fungal morphology of filamentous fungi such as Lasiodiplodia iranensis. Specifically, when tyrosol was added, Lasiodiplodia iranensis was able to form a mycelial mat under shaking conditions. Considering that the mat-like morphology seems to be important for jasmonic acid biosynthesis by filamentous fungi, the addition of tyrosol led to JA production at a higher titer under shaking conditions.

[0059] Example 4: Effect of addition of one or more jasmonate elicitors on JA production by Lasiodiplodia iranensis The procedure described in Example 2 was repeated using a jasmonate elicitor instead of farnesol. Specifically, square pieces (area approximately 1 cm x 1 cm) of PDA plates containing L. iranensis cultures were cut and used to inoculate 50 ml of the same nutrient medium with or without the jasmonate elicitor. Jasmonate elicitors are small molecules that can induce or awaken potential biosynthetic pathways and, in this case, are involved in JA production by filamentous fungi such as Lasiodiplodia iranensis. Typical jasmonate elicitors can be plant hormones, oxidative stress factors, histone deacetylase inhibitors, or antibiotics. Figure 7 shows the chemical structures of various jasmonate elicitors that can be used according to the present teachings, including representative plant hormones such as indole-3-acetic acid (IAA), salicylic acid (SA), acetylsalicylic acid (ASA); representative oxidative stress factors 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, and 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 8 days of culture, the extracts were collected and their JA contents were analyzed by HPLC.

[0062] In the control (i.e., without jasmonate elicitor), less than 50 mg / L of JA was produced. Each culture supplemented with jasmonate elicitor produced JA at significantly higher titers, as summarized in Table 1 below.

Table 1

Claims

**Claim 1** A method for producing jasmonates, comprising: culturing a strain of filamentous fungal organisms in a nutrient medium containing at least one fungal quorum sensing molecule, at least one carbon source, and at least one nitrogen source with stirring; isolating a jasmonate product from the nutrient medium; wherein the filamentous fungal organisms belong to the genus Lasiodiplodia; the fungal quorum sensing molecule is selected from the group consisting of farnesol and tyrosol; a method for producing jasmonates. **Claim 2** The method according to claim 1, wherein the filamentous fungal organisms are the Lasiodiplodia iranensis strain. **Claim 3** The method according to claim 1 or 2, wherein the nutrient medium further comprises one or more fungal quorum sensing molecules selected from the group consisting of tryptofol, γ-heptalactone, farnesenic acid, 1-phenyl-ethanol, 2-phenylethanol, multicolanic acid, multicolosic acid, multicolic acid, butyrolactone-I, γ-butyrolactone, α-(1,3)-glucan, a-factor pheromone, 3-octanone, 3-octanol, and 1-octen-3-ol. **Claim 4** The method according to any one of claims 1 to 3, wherein the nutrient medium contains farnesol and tyrosol. **Claim 5** The method according to any one of claims 1 to 4, wherein the carbon source is selected from the group consisting of sucrose, starch, maltose, glucose, and fructose. **Claim 6** The method according to any one of claims 1 to 5, wherein the nitrogen source is an organic nitrogen source selected from the group consisting of beef extract, peptone, corn pulp, yeast extract, and malt extract. **Claim 7** The method according to any one of claims 1 to 5, wherein the nitrogen source is an inorganic nitrogen source selected from the group consisting of sodium nitrate, potassium nitrate, urea, and ammonium nitrate. **Claim 8** The method according to any one of claims 1 to 7, wherein the stirring is carried out at 150 rpm to 1500 rpm. **Claim 9** The method according to any one of claims 1 to 8, wherein the culturing is carried out at a temperature of 20°C to 35°C. **Claim 10** The method according to any one of claims 1 to 9, wherein the cultivation is carried out for at least 2 days. **Claim 11** The method according to any one of claims 1 to 10, wherein the at least one fungal quorum sensing molecule is present in the nutrient medium at 10 to 500 mg / L. **Claim 12** The method according to any one of claims 1 to 11, wherein the nutrient medium further comprises a fungal growth hormone selected from the group consisting of 10-oxo-trans-8-decenoic acid and helsinin. **Claim 13** The method according to any one of claims 1 to 12, wherein the jasmonic acid product comprises jasmonic acid.

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