An allene oxide and 12-oxophytodienoic acid are key intermediates in jasmonic acid biosynthesis by Fusarium oxysporum[S]

Fungi can produce jasmonic acid (JA) and its isoleucine conjugate in large quantities, but little is known about the biosynthesis. Plants form JA from 18:3n-3 by 13S-lipoxygenase (LOX), allene oxide synthase, and allene oxide cyclase. Shaking cultures of Fusarium oxysporum f. sp. tulipae released over 200 mg of jasmonates per liter. Nitrogen powder of the mycelia expressed 10R-dioxygenase-epoxy alcohol synthase activities, which was confirmed by comparison with the recombinant enzyme. The 13S-LOX of F. oxysporum could not be detected in the cell-free preparations. Incubation of mycelia in phosphate buffer with [17,17,18,18,18-2H5]18:3n-3 led to biosynthesis of a [2H5]12-oxo-13-hydroxy-9Z,15Z-octadecadienoic acid (α-ketol), [2H5]12-oxo-10,15Z-phytodienoic acid (12-OPDA), and [2H5]13-keto- and [2H5]13S-hydroxyoctadecatrienoic acids. The α-ketol consisted of 90% of the 13R stereoisomer, suggesting its formation by nonenzymatic hydrolysis of an allene oxide with 13S configuration. Labeled and unlabeled 12-OPDA were observed following incubation with 0.1 mM [2H5]18:3n-3 in a ratio from 0.4:1 up to 47:1 by mycelia of liquid cultures of different ages, whereas 10 times higher concentration of [2H5]13S-hydroperoxyoctadecatrienoic acid was required to detect biosynthesis of [2H5]12-OPDA. The allene oxide is likely formed by a cytochrome P450 or catalase-related hydroperoxidase. We conclude that F. oxysporum, like plants, forms jasmonates with an allene oxide and 12-OPDA as intermediates.

The fungal biosynthesis of (+)-JA is intriguing in many aspects. First, fungi can form remarkably large amounts of (+)-JA in the laboratory, as discussed below. Second, in spite of the large production of (+)-JA, the mechanism of the fungal biosynthesis is not firmly established. Third, (+)-JA produced by fungi can augment plant disease (15,16); yet many plants form jasmonates as defense reactions to fungal infections (11,12). Fourth, the initial steps of (+)-JA biosynthesis in plants occur in plastids, which are absent in fungi.
The present investigation had three goals. The first goal was to optimize the growth conditions of F. oxysporum with respect to biosynthesis of jasmonates, and we chose to investigate Fot (15). The second goal was to determine the transformation of unsaturated C 18 fatty acids by nitrogen powder of these mycelia. This led us to investigate the oxidation of 18:3n-3 and 18:1n-9 by recombinant 10R-DOX-EAS of F. oxysporum. The third goal was to detect possible intermediates in jasmonate biosynthesis, which could be formed by the intact mycelium.
()-JA-Ile-MO} was prepared as follows. N-[()-jasmonoyl]-(S)-isoleucine (95 mg), prepared as described in (28), was added to 2 H 2 O (10 ml) and stirred with 300 mg of K 2 CO 3 at 23°C for 46 h. A solution of 30 mM O-methyl-hydroxylamine hydrochloride in methanol was added, and the mixture was kept at 23°C for 18 h. Extraction with ethyl acetate gave a residue that was subjected to RP-HPLC using a 250 × 10 mm column of Nucleosil 100-7 C 18 eluted with methanol/water/acetic acid (60:40:0.015, v/v/v) at a flow rate of 4 ml/min. This afforded pure [ 2 H 3 ]()-JA-Ile-MO (68 mg) as a white solid. An aliquot was treated with diazomethane and analyzed by GC-MS. Two peaks (ratio, 6:1), due to the MO syn-anti isomers, appeared. The mass spectrum of the major isomer showed the following prominent ions [deuterium isotope shifts relative to the corresponding unlabeled derivative (30)

Fungal cultures
In initial experiments, 50-100 ml CDB or PDB in 250-500 ml flasks were inoculated from PDA plates (0.5-1 cm 2 ) and grown under various conditions [dark or light, 22°C or 28°C, with or without moderate shaking (100 rpm)]. These incubations were terminated after 18-21 days.
In subsequent studies, PDB was inoculated with growth medium (2.5-50 ml PDB) from a growing culture and incubated at 28°C (100 rpm) in the dark (shaking incubator SI-300R; Jeio Tech). After 4-5 days, the culture was colored deep red due to bikaverin, and they were harvested after 10-15 days. The reddish mycelia were collected by filtration, washed with saline, frozen in liquid N 2 , and ground to a fine powder in a mortar with liquid N 2 . The powder was stored at 80°C. An aliquot was added to 0.1 M KHPO 4 buffer (pH 7.4)/2 mM EDTA/0.04% Tween-20 and homogenized with a Potter-Elvehjem glass homogenizer with Teflon pestle (10 passes) and then incubated with fatty acids.
The growth media were centrifuged (3,100 g) and the supernatant was then assayed for jasmonates. An aliquot (7-10 ml) was extracted on a cartridge of C 18 silica (SepPak/C 18 ), washed with water (2 ml), and lipids were eluted with ethyl acetate (4 ml) (31). The organic extract was evaporated under a stream of N 2 , dissolved in ethanol (1 ml), and 1-10 l were then analyzed for JA-conjugates by LC-MS/MS. For detection of JA, the organic extract of 10 ml medium was evaporated to dryness and purified by semi-preparative RP-HPLC (65% methanol) with subsequent analysis of the evaporated polar fractions by RP-HPLC-MS/MS (60% methanol).
For studies of oxidation of fatty acid by mycelia and release of intermediates in JA biosynthesis in small scale, 2.5 ml of the F. oxysporum liquid culture was centrifuged (3,100 g; 20 min, +4°C) and the supernatant was discarded. The mycelia were suspended in 2.5 ml 0.1 NaBO 3 buffer (pH 8.

GC-and LC-MS/MS analysis
An Agilent mass selective detector model 5977E connected to an Agilent model 7820A gas chromatograph were used for GC-MS. A capillary column of 5% phenylmethylsiloxane (12 m, 0.33 m film thickness) with helium as the carrier gas was used. The temperature was raised from 80°C to 300°C at a rate or 10°C/min. The scan and selected ion monitoring modes were used for data acquisition. RP-HPLC with MS/MS analysis was performed with a Surveyor MS pump (ThermoFisher) and an analytical or semi preparative octadecyl silica column (5 m; 2.0 × 150 mm, Phenomenex; 5 m; 4.6 × 150 mm, Dr. Maisch). The columns were eluted at 0.25-0.3 ml/min or 1 ml/min, respectively, with methanol/water/acetic acid, 600:400:0.05, 650:350:0.05, 700:300:0.05, 750:250:0.05, or 800:200:0.05 (v/v/v). The effluent was subject to ESI in a linear ion trap mass spectrometer (LTQ; ThermoFisher). The heated transfer capillary was set at 315°C, the ion isolation width at 1.5 amu (5 amu for analysis of hydroperoxides), the collision energy at 35 (arbitrary scale), and the tube lens at 112 V. Samples were injected manually (Rheodyne 7510) or by an autosampler (Surveyor Autosampler Plus; ThermoFisher).

Analysis of jasmonates in the growth medium
Cultures were inoculated from agar plates. We first examined the secretion of JA and JA-conjugates by cultures for 3 weeks (22°C) in flasks with CDB with and without shaking (100 rpm) and in the dark or under fluorescent light. Cultures in CDB, which were grown with shaking in the dark, released the largest amounts of ()-JA-Ile and (+)-JA-Ile, as shown in Fig. 3A. Cultures grown in PDB in the dark (or in subdued light) at 28°C with shaking (100 rpm) released the highest amounts of jasmonates after 3 weeks (Fig. 3A). The amount of (+)-and ()-JA was only 0.2% of the amount of (+)-JA-Ile in this sample, which is lower than in other strains of F. oxysporum (15,20). Growth in PDB with shaking at 28°C and in the dark yielded the highest levels and these conditions were, therefore, used routinely.
GC-MS analysis showed that the secretion of jasmonates to the PDB was low for the first 4 days, but caught momentum after about a week and reached about 4 mg l 1 (+)-JA-Ile (Fig. 3B). The yield varied between incubations (15). Inoculation of PDB with an aliquot of a liquid culture led to accumulation of over 0.23 g l 1 (+)-JA-Ile after 15 days (Fig. 3C). The largest amounts obtained were 0.31 g l 1 (+)-JA-Ile (almost 1 mM).
The elution of jasmonates on RP-HPLC is shown in Fig. 4A. JA-Ile was the main product along with variable relative amounts of 9,10-dihydro-JA-Ile. In addition, small amounts of Ile-Val and 9,10-dihydro-JA-Val were also detected. The MS 2 spectrum of JA-Ile is shown in Fig. 4B, and dominated by the signal at m/z 130 (Ile carboxylate anion; compare inset in Fig. 4B). The MS 2 spectra of 9,10-dihydro-JA-Ile, JA-Val, and 9,10-dihydro-JA-Val showed a similar fragmentation pattern (supplemental Fig. S1).
A minor metabolite eluted before 16-KOTrE (marked by arrow in the chromatogram, Fig. 6A). The MS 2 spectrum and the retention time of this product were both identical to that of authentic 12-OPDA. We next examined its formation with [ 2 H 5 ]18:3n-3 as a substrate.
The mass spectra of 12-OPDA and [ 2 H 5 ]12-OPDA are shown in Fig. 7B, C. The signal at m/z 165 in the spectrum of 12-OPDA is due to charge-directed fragmentation by the  carboxyl group and the fragment is illustrated in the insert in Fig. 7C (35 (Fig. 8A). The [ 2 H 5 ]11-HOTrE could not be detected, which is a major product of Fo-MnLOX (22). Steric analysis by CP-HPLC showed that over 95% of [ 2 H 5 ]13-HOTrE coeluted with 13S-HOTrE (Fig. 8B).
Products formed in a large-scale incubation of mycelia with the KHPO 4 buffer were extracted and purified by semi-preparative RP-HPLC, and 10 l of 1 ml fractions were analyzed by direct injection into the mass spectrometer. The fraction with 12-OPDA was reduced to dryness and then analyzed by GC-MS, which separated the cis and trans side chain isomers of 12-OPDA (supplemental Fig. S4A). The electron impact mass spectrum is shown in supplemental Fig. S4B. We conclude that 12-OPDA was identified by both LC-and GC-MS analyses.
We expected exogenous 13S-HPOTrE to be transformed to 12-OPDA. Incubation of mycelia with 100 M [ 2 H 5 ]13S-HPOTrE for 1 h did not lead to significant amounts of

Metabolism of 18:3n-3-Ile by mycelia of Fot
Standards were prepared with flaxseed AOS. The 13S-HPOTrE-Ile was obtained by oxidation with sLOX-1 and further transformed by acetone powder of flaxseed (AOS) 3 BLAST analysis at NCBI showed that AOS (GenBank AAF00225) and 12-OPDA reductase (OPR3; GenBank OAP09565) of A. thaliana could be aligned with putative CYP (27% identity; 24% query cover) and with a putative NADPH-dependent dehydrogenase (45% identity; 97% query cover) of F. oxyporum Fo47. 4 BLAST analysis at NCBI showed no significant homology between proteins of F. oxysporum and AOC (GenBank CAC83764) and JAR1 (GenBank OAP073631) of A. thaliana.
Mycelia of Fot did not release detectable amounts of 12-OPDA-Ile or the -ketol-Ile conjugate. We conclude that conjugation of (+)-JA with Ile likely occurs as a final step.

DISCUSSION
We report a prominent biosynthesis of the JA conjugate (+)-JA-Ile by the fungus Fot, about 0.2 g l 1 . This is of the same magnitude as that reported for the biosynthesis of (+)-JA by L. theobromae, which commonly produces 0.3-0.5 g l 1 (17,19). The prominent (+)-JA biosynthesis by Fot enabled our main finding, the detection of two key intermediates in plant biosynthesis of (+)-JA. 13S-HPOTrE was also converted to 12-OPDA, albeit in low yields. This suggests that the initial steps of the fungal biosynthetic pathway to jasmonates are analogous to those operating in plants (Fig. 10).
The first step is likely oxidation of 18:3n-3 to 13S-HPOTrE. As far as is known, the fungal DOXs of the cyclooxygenase gene family do not oxidize 18:3n-3 at C-13, but there are two possible LOX candidates (Fig. 2). These LOXs have only been studied by recombinant expression and expressed sequence tags have not yet been detected in F. oxysporum. 2 Fo-MnLOX is likely secreted and forms 11-HPOTrE as a major metabolite (22). The latter could not be detected. The 13S-LOX with catalytic iron is therefore the obvious candidate (26).
We have little information on the other fungal enzymes leading to biosynthesis of (+)-JA-Ile. AOS, AOC, 12-OPDA reductase (OPR3), and JAR1 proteins of Arabidopsis thaliana are well-characterized (39)(40)(41)(42). AOS can be aligned with putative CYP of F. oxysporum at the C-terminal end with 27% identity, and OPR3 with NADPH-dependent reductases with 46% identity. 3 We could not detect homologs of AOC or JAR1 in the genome of F. oxysporum. 4  plant enzymes lack catalytic metals. The active site of JAR1 relies on an adaptable three-dimensional scaffold (8) and the crystal structure of AOC indicates an active site inside a barrel cavity (10). It seems likely that fungal AOC and acyl amino acid synthase could be examples of parallel evolution of active sites.
-Ketols are formed by nonenzymatic hydrolysis of allene oxides taking place with predominant inversion of configuration (43). The -ketol of the present work was mainly 13R, suggesting its formation from 12,13S-EOT. The latter is likely formed from 13S-HPOTrE by a 13S-AOS. AOSs belong to two families of heme proteins, CYP of plants and fungi and catalases of corals and cyanobacteria (36,44,45). Fungal AOSs have so far only been detected in DOX-CYP fusion proteins, e.g., 8R-, 8S-, 9R-, and 9S-DOX-AOS and not in fungal catalases (24,(46)(47)(48). In contrast to plant CYP74, which contains a characteristic insertion sequence of seven to nine amino acids in the Cys pocket, the fungal AOSs of DOX-AOS enzymes do not share common characteristic sequences (9,47,49). It will be a challenging task to identify the putative 13S-AOS. The efficient synthesis of jasmonates in fungi suggests a tight coupling between the AOS and cyclase activities and even the possibility that they may reside in the same protein (compare Ref. 50).
The 12-OPDA is formed in plants by AOS and AOC in plastids. The location of the fungal AOS and AOC is unknown. Eukaryotic CYP enzymes are typically membrane-bound and found in the endoplasmic reticulum. -Oxidation in fungi only occurs in peroxisomes (51). In analogy with plants, 12-OPDA could be transferred to peroxisomes where the ring double bond could be reduced and the side chain shortened by -oxidation.
We expected N 2 powder of mycelia to oxidize 18:3n-3 to 13S-HPOTrE, but this was not detected with certainty. This may explain why the fungal biosynthesis of JA has been enigmatic. Biosynthesis of (+)-JA-Ile in shaking cultures in PDB was associated with prominent expression of 10R-DOX-EAS and bikaverin, which colored the mycelium dark red. Bikaverin biosynthesis in Fusarium and JA biosynthesis by L. theobromae start when glucose and/or nitrogen have been partly consumed and can be optimized in different ways (17,18). It is not unlikely that the biosynthesis of jasmonates by Fot also can be optimized.
How can the enzymes in the fungal cascade to (+)-JA-Ile be identified? The classical method to purify enzymes from cell-free preparations does not appear to be feasible due to undetectable enzyme activities. This is in striking similarity to the undetectable prostaglandin biosynthesis by subcellular fractions of the coral, Plexaura homomalla (52). This enigma was solved by recombinant expression of the coral cyclooxygenase (52). The 13S-LOX of F. oxysporum has also been expressed (26) and we detected biosynthesis of 13S-HOTrE by the mycelium. The enzymes further down the biosynthetic pathway to (+)-JA-Ile are unknown. It is possible that comparison of mRNA expression under growth conditions with little and augmented JA biosynthesis might generate hypotheses for subsequent recombinant enzyme expression and analysis.
Access to strains of L. theobromae with a high capacity to form (+)-JA is restricted by environmental and commercial considerations. In contrast, Fot is generally available for future studies from the ARS culture collection. Our study raises many questions, which could be investigated in mycelia of Fot, e.g., the effect of gene deletion of 13S-LOX, the subcellular location of the biosynthesis of 12-OPDA, the reduction of the ring double bond, and conditions for optimal biosynthesis of (+)-JA-Ile in liquid cultures.

CONCLUSIONS
We have identified 12-OPDA as an intermediate, which was formed from 18:3n-3 and 13S-HPOTrE, in the biosynthesis of jasmonates in F. oxysporum. An -ketol was also detected, which provides evidence for an allene oxide serving as the immediate precursor of 12-OPDA. It seems likely that further conversions of 12-OPDA take place as established for higher plants, i.e., by reduction of the ring double bond, -oxidation, and conjugation with Ile.