Mechanistic Insight into the Biosynthesis and Detoxification of

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Mechanistic insight into the biosynthesis and detoxification of fumonisin mycotoxins Kevin M N Burgess, Justin B Renaud, Tim McDowell, and Mark W Sumarah ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00438 • Publication Date (Web): 22 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Mechanistic insight into the biosynthesis and detoxification of fumonisin mycotoxins

Kevin M. N. Burgess, Justin B. Renaud, Tim McDowell, and Mark W. Sumarah*

*Author to whom correspondence is to be addressed London Research and Development Centre Agriculture and Agri-Food Canada 1391 Sandford St London, Ontario, Canada N5V 4T3 Tel.: +1 519 953 6723; fax: +1 519 457 3997 e-mail: [email protected]


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Abstract: Fumonisins, notably FB1, FB2, FB3, and FB4 are economically important mycotoxins produced by a number Fusarium sp. that occur on corn, rice, and sorghum as well as by Aspergillus sp. on grapes. The fumonisin scaffold is comprised of a C18 polyketide backbone functionalized with two tricarballylic esters and an alanine derived amine. These functional groups contribute to fumonisin’s ability to inhibit sphingolipid biosynthesis in animals, plants and yeasts. We report for the first time the isolation and structure elucidation of two classes of non-aminated fumonisins (FPy and FLa) produced by Aspergillus welwitschiae. Using a Lemna minor (duckweed) bioassay, these new compounds were significantly less toxic in comparison to the fumonisin B mycotoxins, providing new insight into the mechanism of fumonisin toxicity. Time course fermentations monitoring the production of FB4, FPy4, and FLa4, as well as 13C and 15

N stable isotope incorporation, suggest a novel post-biosynthetic oxidative deamination process

for fumonisins. This pathway was further supported by a feeding study with FB1, a fumonisins not produced by Aspergillus sp., which resulted in its transformation to FPy1. This study demonstrates that Aspergillus have the ability to produce enzymes that could be used for fumonisin detoxification.


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Fumonisins are a group of sphingolipid analogue mycotoxins1 produced primarily by Fusarium verticillioides, F. proliferatum and more recently by Aspergillus species.2 Fumonisin exposure results in pulmonary edema in pigs, leukoencephalomalacia in horses, and has been associated with an increased risk for developing neural tube birth defects in humans.3,4,5 In addition, the International Agency for Research on Cancer (IARC) has classified fumonisins as group 2B; possibly carcinogenic to humans. Fumonisin B1 (FB1) and B2 (FB2) were first isolated and structurally characterized from F. verticillioides6 and confirmed by total syntheses.7,8 Since their characterization, additional fumonisins have been reported, which mainly differ by the number and position of hydroxyl groups on the polyketide backbone.9,10 Other, albeit minor, fumonisins have also been identified, including N-acetyl analogues (FA series)6 as well as those containing a shorter carbon backbone (FC series).11 The FB series of fumonisins possess two tricarballylic ester (TCE) functional groups and a primary amine, which is derived by the condensation of L-alanine to the polyketide backbone during biosynthesis.12,13,14 Fumonisins are competitive inhibitors of ceramide synthase,15,16,17 an enzyme responsible for the biosynthesis of sphingolipids. The aminated head group of FB1 is believed to interact with the binding site of sphinganine, while a TCE group interacts with the fatty acyl-coenzyme A binding site.18,19 Thus, the amine functionality is believed to be essential for toxicity of intact fumonisins. This was partially demonstrated with N-acetyl fumonisins, which are less toxic and do not interfere with ceramide synthase.20 However, since the primary amine at the C2 position is α-hydroxylated at C3 in fumonisins, spontaneous rearrangement of the acetyl group can occur yielding an O-acetyl fumonisin derivative. This analogue was reported to be as potent as FB1 in elevating sphinganine levels in rat liver slices.21 Hydrolyzed FB1 (HFB1), where one or both of the TCE-derived side chains are cleaved off, exhibit a


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reduction of fumonisin’s toxicological effects.20,22,23,24,25 While assessing the risk of fumonisin and ochratoxin A contamination on grapes grown in wine producing regions in Canada, two new series of non-aminated fumonisins, FPy and FLa, were putatively identified by liquid chromatography mass spectrometry (LC-MS) from cultures of A. welwitschiae.26,27 A limited number of studies have examined the toxicological effect of removing the amine group either chemically28,29 or with a microbial enzyme, suggesting that the amine is critical for fumonisins toxicity.30 We report the isolation and NMR structural characterization of the naturally occurring fumonisins, FPy4 and FLa4, from the large-scale fermentation of a strain of A. welwitschiae on table grapes. As fumonisins are known phytotoxins, the toxicity of FPy4 and FLa4 with respect to their amine-bearing counterpart, FB4, as well as with FB1 was assessed in a Lemna minor (duckweed) assay by measuring conductivity and plant biomass as endpoints.31,32 The duckweed assay allowed for the determination of the structure-activity relationship between non-aminated and aminated fumonisins, thereby providing new insight into the function of the amine group for fumonisin toxicity. Additionally, over an eight day fermentation, we monitored the production of FPy4, FLa4, and FB4. This led to the hypothesis that FPy and FLa fumonisins can be biosynthesized either from (i) an enzyme that catalyzes the decarboxylative condensation of pyruvic or L-lactic acid rather than L-alanine onto the fumonisin backbone or (ii) an oxidative deamination of fumonisin post-biosynthesis. These biosynthetic routes are explored using LCMS, stable-isotope labelling, and FB1 feeding experiments.


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Results and Discussion Fermentation, isolation and characterization of FPy4 and FLa4 From our recent survey of black Aspergillus species in Canadian vineyards, A. welwitschiae DAOMC 250207 was identified as the optimal producer of the new fumonisins.27 The highest concentrations of FPy4 and FLa4 were obtained when DAOMC 250207 was inoculated on red table grapes in the laboratory. Additionally, since a standard of FB4, was not commercially available, it was isolated from a culture of F. verticillioides MRC 826 (DAOMC 250206) fermented on corn. Upon extraction of either grapes (5kg) or corn (0.5 kg) with 100% MeOH, the desired fumonisins were purified from the extract initially by C18 reversed phase flash column chromatography. The fumonisin-containing fractions were then pooled and pure standards of FPy4, FLa4, and FB4 were obtained using semi-preparative high performance liquid chromatography (HPLC). Since fumonisins are not UV active, their presence was monitored in the fractions using LC-MS.

Figure 1: Structures of FB4, FPy4, and FLa4 with the numbering scheme used for NMR assignments in Table 1 and Table S1 (see Supporting Information). The 1H and

13

C resonance assignments for C1 to C4 of FB4, FPy4, and FLa4 are

summarized in Table 1, whereas the complete assignments are reported in Table S1 of the


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Supporting Information. For positions C1 to C4, Gelderblom et al.33 reported 1H and

13

C

assignments for FB4 and 3-epi-FB4 in dimethylsulfoxide (DMSO)-d6, which were in agreement with our NMR assignments for FB4 reported in methanol-d4. Fumonisin FPy4 (C34H56O14) and FLa4 (C34H58O14) were isolated as yellow oils (1.2 and 0.9 mg respectively) as confirmed by high resolution mass spectrometry (HRMS). The 1H and

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C assignments for FPy4 and FLa4 are

consistent with a fumonisin scaffold when compared with those of the well characterized FB1, FB2, and FB3 compounds.6 As the fumonisin backbone is fully hydrogenated with the exception of C2 for FPy4 (vide infra), positional assignments by NMR were straightforward using both 1H– 13

C H2BC (or 1H–1H DQFCOSY) and 1H–13C HMBC correlations. The numbering system for

the resonance assignments can be found in Figure 1 and key correlations for FPy4 and FLa4 are illustrated in Figure S13 of the Supporting Information. For both FPy4 and FLa4, the C11 to C20 and TCE side chain assignments (see Table S1) were in agreement with those observed for FB1 and FB2.6 As for the remainder of the CH2 resonances, significant overlap between each of them in the 1H NMR spectrum hampered their assignment to the fumonisin backbone, thus the remaining 13C methylene resonances were assigned to the C5–C10 positions. The major differences between the NMR spectra for FB4 compared to FPy4 and FLa4 stem from the nature of the substituent at the C2 position (see Figure 1). For FB4 and FLa4, a cross peak was observed between the expected doublet for the H1 methyl protons and the H2 proton. A large difference in the 13C chemical shift value was observed at C2 for FB4 (53.5 ppm) compared to FLa4 (71.8 ppm). Both of these δC values are consistent with either an amine, in the case of FB4, or a hydroxyl substituent in FLa4. In addition, the H3 resonance can be assigned given it’s correlation to H2 in the COSY spectra of both FB4 and FLa4. The H1 methyl protons for FPy4 appear as a singlet at 2.16 ppm, which has a strong HMBC cross peak to a deshielded


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carbonyl resonance at 213.5 ppm. This δC value for a carbonyl resonance assignment at C2, along with an additional HMBC cross peak between H1 and C3 (78.3 ppm), is consistent with an α-hydroxy ketone moiety in FPy4. In all three fumonisins, H4/C4 was assigned via an H2BC cross peak between H3 and C4. Table 1: Select 1H (600 MHz, CD3OD) and 13C (151 MHz, CD3OD) NMR assignments for FB4, FPy4 and Fla4 position 1 2 3 4

FB4

FPy4

FLa4

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

16.02 53.5 73.1 34.7

1.27, d (6.7) 3.10, dq (6.7, 6.7) 3.45, m 1.56, m; 1.42, m

25.6 213.5 78.3 34.4

2.16, s 4.06, dd (8.2, 4.2) 1.72, m; 1.54, m

18.4 71.8 76.7 33.8

δH (J in Hz) 1.15, d (6.4) 3.56, dq (6.3, 6.3) 3.36, m 1.57, m; 1.34, m

Fumonisin B4 and FLa4 contain 8 stereocentres whereas FPy4 has 7. Given that the multiplicities as well as the measured J-coupling constants from H14 (for FPy4, ddd, J = 11.0, 3.0, 3.0 Hz) and H15 (for FPy4, dd, J = 8.4, 3.5 Hz) were nearly identical to those reported for fumonisin B1,6 we concluded that the configurations at C14 and C15 are 14S and 15R.34 The 25R and 31R configurations35 were assigned based on the similarities of the J-coupling constants and 1

H chemical shift values for the TCE methylene protons (i.e., H24/H26 and H30/H32) in

comparison to those reported for FB1 and to the recently reported fumonisin B6.36 As for the configurations at C16 and C12 bearing the C22 and C21 methyl substituents respectively, these were assigned as 16R and 12S in FB4, FPy4, and FLa4 as their δH, δC, and J-coupling constants were consistent with the values reported for FB1.6 For position C2 of FB4 and FLa4, the H2 resonances in both cases have a doublet of quartet multiplicity with similar J-coupling constants (6.7 and 6.7 Hz for FB4 and 6.3 and 6.3 Hz for FLa4), thus the relative configuration between C2 and C3 is identical to the FB1 molecule. As for the absolute stereochemical configurations of C2 and C3, Larsen and co-workers36 showed that FB2 produced by A. niger was identical to FB2


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produced by Fusarium species. In addition, our FB4 assignments for C2 and C3 are consistent with Gelderblom et al.’s assignments for 2S,3S-FB4.33 Given that the stereochemistry of FB1 is well understood37 and, combined with the aforementioned information, we are confident in the 2S configuration in FLa4 and 3S configurational assignment for FPy4 and FLa4. Fumonisin toxicity Fumonisins are well understood to be phytotoxic and as such, duckweed has previously been used to assay fumonisins and AAL-toxins.31 Figure 2b summarizes the phytotoxic effects of FPy4 and FLa4 in comparison to FB4 and FB1 in regards to their ability to inhibit the growth of duckweed as determined by dry weight biomass after 7 days. Since it has been previously reported that FB2 and FB1 have similar phytotoxic effects to duckweed in the low µM range,38 identical concentrations were used for FB1 and FB4 ranging between 0.3 and 5.0 µM. Preliminary data indicated that much higher concentrations up to 40.0 µM of FPy4 and FLa4 were necessary to observe similar phytotoxic effects. The highest fumonisin concentrations (40.0 µM for FPy4 and FLa4 or 5.0 µM for FB4 and FB1) resulted in inhibition of plant growth as well as in frond discoloration in comparison to control wells (Figure 2a). For each compound, the minimum inhibitory concentration (MIC) and the measured plant biomass data were analyzed by ANOVA followed by Fisher’s least significant difference (LSD). Growth inhibition was observed at a FB1 concentration of 0.6 µM, which is in agreement with previous studies.31 For FB4, significant growth inhibition was observed at a concentration of 5.0 µM. The MICs for the non-aminated fumonisins were an order of magnitude greater than FB1 (i.e. 40.0 µM for FLa4 and 20.0 µM for FPy4 based on statistical analysis of data used for Figure 2b).


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Figure 2: (a) An example 24-well plate of the L. minor growth inhibition assay as a function of fumonisin concentration in µM. The dried duckweed biomass data is summarized in (b) where the error bars represent standard error in the mass measurements between 3 replicates. The italicized letters above each bar represent statistically significant LSD groupings from the mean dried biomass data (p < 0.05).

To probe the extent of duckweed cell damage caused by fumonisin exposure, the released electrolytes from the plant to the growth medium were quantitated by conductivity for FB4, FPy4, and FLa4 in comparison to FB1 with DMSO as the negative control. These data are summarized in Figure 3 where a fumonisin concentration of 10.0 µM was used in all cases. This concentration was selected based on the MIC values extracted from the growth inhibition assay (see Figure 2b) in order to ensure an adequate level of phytotoxic activity of FB4 while FPy4 and FLa4 showed minimal growth inhibition at 10.0 µM. After five days of incubation, no significant differences in growth medium conductivity were observed between FB1 (339.5 ± 9.7 µS·cm-1) and FB4 (289.7 ± 13.2 µS·cm-1), whereas the value obtained for FLa4 (57.2 ± 29.4 µS·cm-1) was not significantly different from the control (67.6 ± 28.1 µS·cm-1). A statistically significant larger conductivity value for FPy4 (146.6 ± 14.7 µS·cm-1) was observed as compared to FLa4. At 10.0 µM, the aminated FB1 and FB4 compounds show equivalent cellular damage, whereas the nonaminated fumonisins FPy4 and FLa4 are significantly less phytotoxic (p < 0.05). These


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measurements are in agreement with the growth inhibition results and support the finding that FPy4 is more active than FLa4 at lower fumonisin concentrations. Without an amine functional group, damage to the duckweed fronds was significantly reduced even after five days of exposure to 10.0 µM of fumonisins. In the present experiments, electrolyte leakage from the fronds was similar to that reported for N-acetylated FA1 and FA2 fumonisins in assays with a related duckweed species L. pausicostata.38 These data demonstrate for the first time, that in the presence of TCE side chains, replacement of the amine for a hydroxyl or a ketone results in a dramatic reduction in fumonisins phytotoxic activity.

Figure 3: Conductivity measurements of L. minor growth medium after 5 days of exposure to 10.0 µM of fumonisin. Each compound was tested in quadruplicate. Error bars denote standard error in the mean conductivity measurements. Compounds bearing the same italicized letter are not significantly different by Fisher’s LSD (p < 0.05).

Biosynthetic origins of FPy and FLa fumonisins In order to understand the biosynthetic relationship between FB4, FPy4 and FLa4, their production was monitored over an 8 day fermentation of A. welwitschiae (DAOMC 250207) in


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liquid culture (Figure 4). FB4 was detected two days after inoculation, and reached a maximum of 2.3 µM on day three. Between days two and five, the steepest increase in the concentrations of FPy4 and FLa4 were observed, which coincided with a decrease in the concentration of FB4. A similar decrease in FB4 concentration during a liquid culture fermentation of A. niger was observed by Sørensen et al.39 By day 7, FB4 had largely disappeared corresponding with a small increase in the concentration of FPy4 and FLa4. After 8 days of fermentation, the final concentration of FPy4 was 8-fold greater than FLa4 (Figure 4). The total concentration of FPy4 and FLa4 (1.7 µM) was similar to the peak concentration of FB4 (day three). Furthermore, the decrease in FB4 and corresponding increase in the concentrations of FPy4 and FLa4 between days two and four suggest that an oxidative deamination of FB4 was largely responsible for the observed decrease in FB4.

Figure 4: LC-MS quantitation of FB4, FPy4 and FLa4 during the 8-day fermentation of A. welwitschiae DAOMC 250207 in CYA liquid culture. 100 µL aliquots were removed once per day and analyzed. Data points are the average of 4 replicates with error bars denoting the standard error in the measurements.


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Considerable effort has been made to elucidate the biosynthesis of FB1 from genetic and biochemical perspectives.40 In F. verticillioides, it is well understood that, after the 18-carbon polyketide synthase (PKS), the FUM8 gene in the fumonisin biosynthetic gene cluster encodes an α-oxoamine enzyme. This enzyme introduces the amine functionality to FB1 by catalyzing the condensation of L-alanine to the dimethyl stearoyl molecule attached to PKS by an acyl carrier protein (ACP; see Figure 5, pathway 1a).41 The FC fumonisins, where the alkyl backbone contains only 19 carbons, are produced when Fum8p uses glycine as a substrate rather than Lalanine for FB biosynthesis.42 Although unlikely, the hypothesis that an unknown enzyme or Fum8p itself could condense pyruvic acid (Figure 5, pathway 2) in order to form FPy4 or L-lactic acid to form FLa4 was explored. Pathways 1a and 2 were investigated using stable isotope labelling experiments with either L-alanine (13C3,15N) or sodium pyruvate (13C3). In light of the rapid production of FB4 by A. welwitschiae DAOMC 250207 (Figure 4), these labelled compounds were added to separate cultures 24 hours after inoculation and their incorporation into either FB4, FPy4 or FLa4 were monitored. Addition of L-alanine (13C3,15N) resulted in changes to the isotopic profiles of all the fumonisins measured, suggesting that FPy4 and FLa4 are derived from FB4 post-biosynthesis. However, to a lesser extent, the addition of sodium pyruvate (13C3) also shifted the isotope profiles for these three compounds (see Figure S14 in the Supporting Information). The conversion of sodium pyruvate (13C3) to L-alanine (13C3) was observed concurrently with the biosynthesis of fumonisins meaning that the change in the isotope profiles could again be a result of pathway 1a/1b. In contrast, when L-alanine (13C3,15N) was added to fermentation cultures, the sodium pyruvate (13C3) molecule was not observed. These data suggest that the proposed pathway 1a/1b is used for the biosynthesis of FPy and FLa fumonisins. However, the conversion between pyruvate and L-alanine does not allow us to


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unambiguously rule out pathway 2, although the nature of the Fum8p enzyme makes it unlikely.41 O O CO2

OH O

HO

O

OH

Fum8p pathway (1a) CH3

HO

O

O HO

L-ala

HO

O

O

CH3

NH2

FB4

O

NH2 ACP

dimethyl stearoyl-S-PKS

pathway (1b)

transaminase

S O

O

O O

HO O pyruvic acid

HO

O

Fum8p pathway (2) CO2

OH O

CH3

HO O HO

O

OH

O

O

CH3

FPy4

O

Figure 5: Proposed pathways for the biosynthesis of FPy4 in A. welwitschiae either via a nonspecific Fum8p enzyme (pathway 1a or 2) or an oxidative deamination of FPy4 post-biosynthesis (pathway 1b).

It has been shown that A. niger/A. welwitschiae do not produce FB1 and FB3 due to the absence of the FUM2 gene, which is responsible for fumonisin C10 hydroxylation in F. verticillioides.43 Therefore, pathway 1b (see Figure 5) was directly explored by feeding FB1 into lysed cells of DAOMC 250207 at a concentration of 10 µM. Since a decrease in the FB4 concentration was observed by day four (see Figure 4), the cells used for the FB1 feeding study were harvested after 4 days of fermentation in liquid culture. Following overnight incubation, a LC-MS peak corresponding to the theoretical m/z of [FPy1-H]- (±3 ppm) was observed earlier in the chromatogram than the previously reported isobaric metabolite FPy6 (see Figure 6). The product ion spectrum of FB1 showed the neutral loss of the TCE side chains, all hydroxyl groups, and the amine yielding a characteristic fragment ion at m/z 299.2718. In contrast, the


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characteristic ion of FPy1 is m/z 297.2564 due to the loss of the ketone as H2O following deprotonation of the fumonisin backbone, resulting in an additional unsaturation.26 The FPy1 peak was not observed in the control sample, which did not contain FB1, nor was it present in the FB1 standard itself. We concluded from this experiment that FB1 was transformed to FPy1 via pathway 1b, thereby providing evidence for a post-biosynthetic modification of FB fumonisins to FPy fumonisins. FLa1 was not detected in this experiment and, when comparing the FLa4 concentration relative to that of FPy4 over time (Figure 4), it is proposed that the FLa series of fumonisins are not produced directly from FBs but are likely the result of a reduction of the ketone in FPy fumonisins. The mechanisms involved in the oxidative deamination of fumonisins by A. welwitschiae are not clear and warrant further investigation.

Figure 6: LC-MS chromatograms of DAOMC 250207 incubated without FB1 (a) and with 10 µM FB1 (b). A new peak is observed in (b) corresponding to FPy1. In both (a) and (b), FPy6 is observed, which corresponds to the deamination product of FB6, a naturally occurring fumonisin from Aspergillus. The product ion spectrum of [FB1+H]+ (c) and [FPy1+NH4]+ (d) show the characteristic neutral losses of the TCE side chains and of hydroxyl groups as H2O.


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In conclusion, the newly characterized fumonisins, FPy4 and FLa4, are the first naturally biosynthesized fumonisins reported without an amine group. The isolation and characterization of these new compounds was critical for understanding fumonisin toxicity as it has long been hypothesised that the amine was responsible for their biological effects. Using a duckweed bioassay we showed significant reduction in growth inhibition and cellular damage by these nonaminated fumonisins when compared to the fumonisin B mycotoxins. This study allowed, for the first time, an unambiguous assessment of the toxicological effects caused by the presence or absence of the amine functional group with intact TCE substituents. Experiments monitoring the concentrations of FPy4 and FLa4 during fermentation in liquid culture provided insight into the significance of these metabolites and, more importantly, suggested a post-biosynthetic oxidative deamination of FB4. This hypothesis was further supported using both 13C and 15N stable isotope labelling experiments as well as by the transformation of FB1 to FPy1 during feeding experiments. This work has shown that some Aspergillus species can produce an enzyme that is naturally capable of detoxifying fumonisins, including those produced by Fusarium, which could have many practical applications in the food production system. Methods General Procedures All NMR spectra were acquired on a Varian INOVA 600 MHz Spectrometer equipped with a Varian 13C enhanced HCN cold probe. 1D and 2D NMR experiments were performed at 25°C in CD3OD (Cambridge Isotope Labs) and isotropic chemical shifts (δ) are referenced to TMS at 0.00 ppm using the residual solvent peak for methanol (3.31 ppm for 1H and 49.00 ppm for

13

C) with water suppression. NMR spectra were analyzed using Mnova NMR software

(Mestrelab Research, ver. 10.0). HRMS data were acquired on a Thermo Q-Exactive Orbitrap


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mass spectrometer coupled to an Agilent 1290 HPLC. Silica gel chromatography was performed using a Büchi automatic flash chromatography system with 25 g Sepacore C18 columns. Semipreparative HPLC fractionation was performed on an Agilent 1200 HPLC using a Phenomenex Gemini 5 micron C18 column (150 × 10 mm). Fractions containing desired fumonisins were monitored by LC-MS. All solvents used (EtOAc, MeCN, and MeOH) were HPLC grade. Unless otherwise stated, all chemical reagents and microbiological media were purchased from SigmaAldrich (Oakville, ON, Canada). Specific rotation measurements were performed on a Rudolph Research Analytical Autopol IV automatic polarimeter. Conductivity (in µS.cm-1) was measured on a Mettler Toledo SevenExcellence bench meter equipped with an InLab 751-4mm conductivity microprobe at 25°C. A one-way analysis of variance (ANOVA; GLM procedure, Fisher’s LSD) using Statistical Analysis System software (SAS; ver. 2.03, SAS Institute, Cary, NC) was used to test for significant differences in duckweed dried biomass and conductivity measurements. Fungal Strains and Fermentation The isolation of the fumonisin-producing Aspergillus welwitschiae strain (DAOMC 250207) is described elsewhere.27 Prior to fermentation, DAOMC 250207 was transferred via three-point inoculation to a CYA agar plate and grown for 7 days. Red table grapes (total 5 kg) were purchased from a local grocery store (London, ON). The grapes were surface sterilized first by rinsing in tap water three times followed by a rinse with distilled water. One final rinse was performed using sterilized distilled water and grapes were placed under UV irradiation for a total of 20 minutes turning them over once after 10 minutes. Grapes were divided evenly into 2 L presterilized Erlenmeyer flasks capped with a foam plug (300 g per flask). For each flask, a 2 × 2 cm agar plug of DAOMC 250207 was homogenized in 20 mL of sterile distilled water and used


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to inoculate the grapes. The fermentation was performed over a period of 21 days in darkness at 30°C. For FB1 and FB4 standards, mycotoxins were produced by inoculating Fusarium verticillioides (MRC 826, DAOMC 250206) on field corn (local producer, London, ON). 100 g of field corn was soaked in 200 mL deionized H2O overnight. The excess water was removed and the 1 L Erlenmeyer flask was capped with a foam plug and autoclaved. For each flask, a 2 × 2 cm agar plug of MRC 826 (DAOMC 250206) was homogenized in 20 mL of sterile distilled water and inoculated over the corn. Fermentation of the fungus occurred in darkness for a period of 10 days at 25°C. Extraction and Purification of FPy4 and FLa4 At the end of the fermentations, 700 mL of MeOH was added to each flask and subsequently sonicated for one hour. Longer sonication/shaking or a second extraction did not yield any additional fumonisins. The grapes were then filtered and the methanolic filtrates were pooled together. The MeOH was removed with rotary evaporation under vacuum and the aqueous residue was diluted to a total of 2 L with deionized water. The resulting solution was pH adjusted to ~3 with 1 M HCl and extracted three times with EtOAc. The organic layers containing the new fumonisins were pooled (total 6 L), dried using Na2SO4, and the EtOAc was removed under rotary evaporation. The black residue (~20 g) was subjected to reversed phase (C18) flash column chromatography (2 g per injection) using a 20 minute gradient MeOH (+0.1% v/v trifluoroacetic acid (TFA))/H2O program (50 to 100% MeOH over 12 minutes) at a flow rate of 25 mL/min. A total of 30 fractions were collected and screened using LC-MS. Fractions containing FPy4 and


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FLa4 were pooled and the MeOH was removed by rotary evaporation. The remaining water (100 mL) was pH adjusted to ~10 using 6 M KOH and extracted 3×100 mL of EtOAc. The organic layer was discarded and the aqueous layer containing the fumonisins was re-acidified to a pH of ~3 using concentrated HCl and extracted 3×100 mL of EtOAc. The aqueous layer was discarded, the organic layer containing the fumonisins was dried with Na2SO4, and the EtOAc was removed under rotary evaporation. The residue was then subjected to semi-preparative HPLC purification using a 20 minute H2O +0.1% v/v TFA/MeCN +0.1% v/v TFA gradient elution method (0 to 100% MeCN over 13 minutes) to afford pure fractions containing FPy4 and FLa4. FB2, FB6 and their equivalent non-aminated analogues were previously detected by LC-MS26 but were not produced by this strain in sufficient quantities for NMR characterization. FB1 and FB4: To each 100 g of infected corn, 500 mL of MeOH was added and the mixture was sonicated for 1 hour at 25°C and filtered. The combined filtrates were dried to completeness under reduced pressure and the residual oil was subjected to the flash column chromatography conditions described above. Fractions containing FB1 and/or FB4 were identified by LC-MS and were combined and evaporated under reduced pressure. The residue was then further fractionated by semi-preparative HPLC using the same elution method as for FPy4 and FLa4 in order to yield standards of FB1 and FB4. Samples for NMR characterization and for biological assays were freeze-dried prior to analysis. Fumonisin B4 : yellow oil; [α]20D –31.7 (c 0.02, MeOH); 1H and

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C NMR data, Table S1;

HRMS m/z 690.40619 [M+H]+ (calcd for C34H60O13N, 690.40592). Fumonisin Py4 : yellow oil; [α]20D –19.0 (c 0.05, MeOH); 1H and

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HRMS m/z 689.37469 [M+H]+ (calcd for C34H57O14, 689.37428).


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Fumonisin La4 : yellow oil; [α]20D –5.8 (c 0.04, MeOH); 1H and

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13


HRMS m/z 691.38965 [M+H]+ (calcd for C34H59O14, 691.38993). Duckweed (Lemna minor) assays The Lemna minor strain used for the duckweed bioassays was obtained from the Canadian Phycological Culture Centre (Waterloo ON, CPCC 490) and grown in foam plugcapped 250 mL Erlenmeyer flasks containing 100 mL of Hoagland’s E+ medium44 in a growth chamber under constant illumination of 70 µmol·m-2·s-1 at 25°C. For the duckweed growth inhibition assay, one colony containing three fronds of duckweed was placed in each well of a 24-well plate containing 1.5 mL of Hoaglands E+ medium44 without sucrose or tartaric acid as described by Vesonder et al.32 The fumonisin stock solutions were prepared in DMSO such that their final test concentrations were 5.0, 2.5, 1.3, 0.6, and 0.3 µM for FB1 and FB4 and 40.0, 20.0, 10.0, 5.0, and 2.5 µM for FPy4 and FLa4 in the test wells. In all cases, the fumonisins were introduced into the media with 10 µL of DMSO. The experiment was performed in triplicate over a period of 7 days and duckweed biomass was oven-dried overnight at 80°C prior to weighing. For the conductivity assay, stock solutions of FB1, FB4, FPy4, and FLa4 were prepared in DMSO such that their final concentration in each well was 10.0 µM. The assay was performed in a 24-well plate containing 2 mL of Hoagland’s E+ medium without sucrose or tartaric acid. In each test well, three fronds of actively growing Lemna minor were placed along with 10 µL of the fumonisin stock solutions. The experiment was performed in quadruplicate and, after 5 days of incubation in the aforementioned growing conditions, the medium from each individual well was transferred to a vial and freeze-dried overnight. Each residue was re-suspended in 2 mL of


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deionized water for conductivity measurements. This procedure was done to account for small fluctuations in the volume of each well after 5 days. FPy and FLa biosynthesis experiments: In 20 mL of sterilized distilled water, six 8 mm plugs of a 7-day old culture of DAOMC 250207 were homogenized. 1 mL of the mixture was inoculated into 10 mL of CYA liquid broth in 50 mL test tubes capped with foam plugs. For experiments that monitored FB4, FPy4, and FLa4 concentrations over time, 100 µL aliquots were removed every 24 hours over the course of the 8-day fermentation and diluted with an additional 100 µL of MeOH prior to LC-MS analysis. For stable isotope labelling experiments, 3 mg of L-alanine (13C3,

15

N; CDN Isotopes, Pointe-

Claire, Canada) or sodium pyruvate (13C3; Cambridge Isotope Labs, Tewksbury, MA) dissolved in 100 µL of sterile distilled water were added to separate test tubes after 24 hours of fermentation. In all cases, 100 µL aliquots were removed every 24 hours over the course of the 8 day fermentation process and analyzed by LC-MS at 140 000 resolution in positive ionization mode (scan range m/z 660-760). Isotopically enriched pyruvate and L-alanine were also monitored with LC-MS by polarity switching between positive and negative ionization modes throughout the first 2 minutes of the chromatographic method (scan range m/z 70-110). For FB1 incubation experiments, mycelium from CYA liquid cultures of DAOMC 250207 was harvested after four days of incubation. The tissue was frozen under liquid nitrogen and ground with a mortar and pestle. 464 µL of phosphate-buffered saline solution (PBS, pH = 7.4) was added to two 2 mL micro centrifuge tubes each containing 10 mg of the ground material. In one tube, 36 µL of a 100 µg·mL-1 PBS solution of FB1 was added to obtain a final concentration of 10 µM. A second control incubation without FB1 was also prepared. The


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samples were placed overnight on an Eppendorf thermomixer at 30°C shaking at 600 rpm. The supernatant was removed and filtered through a 13 mm, 0.45 µm PTFE membrane syringe filter prior to LC-MS analysis.


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Supporting Information 1D 1H and 13C NMR spectra for FPy4, and FLa4; 2D 1H–1H DQFCOSY, 1H–13C HSQC, 1H–13C H2BC, 1H–13C HMBC spectra for FPy4, and FLa4; key NMR correlations for FPy4 and FLa4; isotope patterns for labelling experiments.

Acknowledgments J. D. Miller (Carleton University) is acknowledged for helpful comments and suggestions regarding this manuscript; G. Shaw (Western University) for technical support with NMR spectroscopy; K. Seifert (AAFC-ORDC) for expert advice. We also thank the EmTOX Research network on emerging mycotoxins. This project was funded by an AAFC GF2 grant to MWS.


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Mechanistic Insight into the Biosynthesis and Detoxification of

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