Investigating oxalate biosynthesis in the wood-decaying fungus Gloeophyllum trabeum using ¹³C metabolic flux analysis

Abstract

Oxalate synthesis was rigorously investigated in a wood-decaying fungus, Gloeophyllum trabeum, using ¹³C metabolic flux analysis, a method not previously explored in this type of system. Peroxisomal glyoxylate dehydrogenase and cytosolic oxaloacetate acetylhydrolase were found to contribute to the majority of oxalate synthesized under low and high nitrogen conditions, respectively.

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Lignocellulose is the most abundant biomass resource in terrestrial environments,¹ and wood fiber has seen growing use as an industrial feedstock for value-added chemical and biofuel production.² However, the recalcitrant structure of lignocellulosic biomass prevents it from being converted to fermentable sugar efficiently and research in this area is still needed. Brown rot fungi are the only organisms known that can depolymerize crystalline cellulose without removal of lignin.³ Gloeophyllum trabeum, a brown rot fungus with high polysaccharide depolymerization activity has promising industrial application prospects. For example, it has been used in a corn stover pretreatment step for ethanol production,⁴ and also in a saccharification step in ethanol production via simultaneous saccharification and fermentation (SSF).⁵ A non-enzymatic oxidative “chelator-mediated Fenton” (CMF) system is reported to be involved in brown rot fungi cellulose depolymerization processes,⁶ but many aspects of how this system works are unknown. The process involves the secretion of the low molecular weight metabolites, oxalate and hydroquinone/catecholate compounds to first sequester and then reduce iron in a sequence, which results in generation of Fenton chemistry at a distance from the fungal hyphae within the lignocellulose cell wall. A highly reactive hydroxyl radical is generated and has been demonstrated to be responsible for the deconstruction of the wood cell wall in brown rot non-enzymatic decay.^7–9 Previous research on the detection of oxalate and catechols in brown rot fungal liquid culture medium in the absence of wood has demonstrated that these compounds are generated by the fungus rather than being derivatives of wood.^10–12 Due to the important role that oxalate and catecholates play in brown rot fungal degradation mechanisms, study of their biosynthetic pathways will provide novel insight about how brown rot fungi regulate the production of these compounds to break down the recalcitrant structure of lignocellulose biomass.

In this study, we applied ¹³C Metabolic Flux Analysis (¹³C-MFA), a technique that has been widely applied to reveal previously unknown metabolic pathways in various non-model organisms,^13–17 to provide valuable insight into the intracellular metabolism of G. trabeum under different culture conditions.^18–20 We found that the major source for intracellular oxalate synthesis varied under different culture conditions. While peroxisomal glyoxylate dehydrogenase contributed to the majority of oxalate synthesized under low nitrogen conditions, cytosolic oxaloacetate acetylhydrolase was found to be the main pathway for oxalate synthesis when culturing G. trabeum in a high concentration of nitrogen. To our best knowledge, this is the first time that oxalate biosynthesis metabolism in G. trabeum has been rigorously and quantitatively determined by ¹³C trace experiments using the metabolic flux analysis approach. Research in this area will facilitate future work in increasing the degradation efficiency for bioconversion of lignocellulosic biomass by G. trabeum and related organisms.

G. trabeum was cultured statically, in 150 mL flasks at 36 °C in 20 mL liquid medium containing the following basal salts per liter: 2 g NH₄NO₃, 2 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 0.1 g CaCl₂·2H₂O, 0.57 mg H₃BO₄, 0.036 mg CuSO₄·5H₂O,²¹ and 1% glucose. All glassware was acid-washed and Fe and Mn was omitted to promote production of secondary metabolites. After 20 days, the medium in three flasks was homogenized and sterile water was added to bring the total volume to 150 mL. The homogenate was diluted 1 : 40 and a 0.5 mL aliquot was used to inoculate a ¹³C-glucose medium containing low nitrogen (LN), or high nitrogen (HN). These cultures contained 1% ¹³C-glucose (a mixture of 80% [1-¹³C] and 20% [U-¹³C] glucose)¹⁰ and the basal salts listed above with either 25 mM NH₄NO₃ (HN) or 2.5 mM NH₄NO₃ (LN). In separate work with HN and LN cultures using non-labeled glucose, growth was analysed by weighing the dried mycelial mass at 3–5 d intervals, and HPLC of the culture filtrate was also conducted to assess glucose consumption (Table 1). The ¹³C-labeled fungal cultures were harvested during the exponential growth phase, after 14 days. Our cultivation conditions were different to those previously used by Varela and Tien²² in part to avoid uncontrolled levels of iron in the media and so that nitrogen levels could be modified.

Table 1 Growth behaviour of G. trabeum under different nitrogen conditions

Nitrogen conditions	Growth rate (day^−1)	Glucose uptake (g per g DCW per day)
LN	0.14 ± 0.03	0.24 ± 0.002
HN	0.15 ± 0.03	0.15 ± 0.001

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Following harvest, isotopomer analysis of proteinogenic amino acids and intracellular oxalate was conducted using a previously developed protocol.^23–25 In brief, the biomass was hydrolyzed using 6 M HCl (20 h at 100 °C) and the amino acids and intracellular oxalate were silyated using 50 μL tetrahydrofuran and 50 μL N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (Sigma-Aldrich) to form tert-butyl dimethylsilyl (TBDMS) derivatives. Gas Chromatography-Mass Spectrometry (GC-MS) was performed to analyse fungal metabolites using a Shimadzu GC2010 GC with a SH-Rxi-5Sil column and a Shimadzu QP2010 MS. Three types of charged fragments were detected by GC-MS for various amino acids (Table S1†): the [M − 57]⁺ group (containing unfragmented amino acids); and the [M − 159]⁺ or [M − 85]⁺ group (containing amino acids that had lost an α-carboxyl group). For each type of fragments, the labeling patterns, mass distribution vectors (MDVs), were represented by M0, M1, M2, etc., which were fractions of non-labeled, singly-labeled, and doubly-labeled amino acids. The effects of natural isotopes on isotopomer labeling patterns were corrected by previously reported algorithms.²⁶ MDVs were used to calculate the summed fractional labelling (SFL) values which were directly used in the MFA (Biomet Toolbox 2.0,²⁷ based on MATLAB, MathWorks, Inc. MA). The central carbon metabolic model for the MFA was developed based on the KEGG database (http://www.genome.jp/kegg/) and previous reports on metabolic pathways of brown rot fungi,^28,29 which included glycolysis pathways, pentose phosphate pathways, futile pathways, the tricarboxylic acid (TCA) cycle, the glyoxylate cycle in peroxisomes, cytosolic oxaloacetate acetylhydrolase and transport pathways among different cell compartments (Fig. 1A). Of particular interest, two oxalate biosynthesis pathways, i.e., cytosolic oxaloacetate acetylhydrolase (OAH) and peroxisomal glyoxylate dehydrogenase (GLX)³⁰ were examined in our metabolic models (Fig. 1A) to assess oxalate biosynthesis. These two pathways have been reported to contribute to fungal extracellular oxalate accumulation in other species.³¹ Although little research has been done to elucidate the genes associated with oxalate biosynthesis pathways in G. trabeum, the homologs of oxaloacetate acetylhydrolase (fpOAH) and glyoxylate dehydrogenase (fpGLOXDH) in Fomitopsis palustris have been indicated in the G. trabeum genome using Blastp³² with 77% and 67% similarity respectively, which suggested these two pathways could potentially be exploited by G. trabeum for oxalate synthesis. For our metabolic flux analysis, flux estimation was repeated at least 50 times starting with different initial values generated by a genetic algorithm for all fluxes to find a likely global solution. A fit of the simulated and measured summed fractional labelings was determined to be a global solution only after the solution fit was obtained at least twice using this method (Fig. S1†).

Fig. 1 Metabolic pathway map and key flux distributions for G. trabeum under different C/N ratios. (A) Central carbon metabolic pathways and oxalate synthesis pathways of G. trabeum. Transport fluxes between mitochondria and peroxisome were simplified to a single dash-dot line. (B) The flux ratio between OAH and GLX pathways for LN and HN conditions with the OAH/GLX ratio highlighted. (C) Key metabolic flux distributions for G. trabeum under LN and HN conditions. Abbreviations: GLC: glucose, 6PG: 6-phosphogluconate, ACAL: acetaldehyde, AcCoA: acetyl-CoA, ACE: acetate, AKG: α-ketoglutarate, CIT: citrate, DHAP: dihydroxyacetone phosphate, E4P: erythrose 4-phosphate, EtOH: ethanol, F6P: fructose 6-phosphate, FUM: fumarate, G3P: glyceraldehyde 3-phosphate, G6P: glucose 6-phosphate, GLOX cycle: glyoxylate cycle, GLYC: glycerol, GLOX: glyoxylate, ICIT: isocitrate, MAL: malate, OAAMIT/OAACYT: oxaloacetate (mitochondria/cytosol), OXA: oxalate, PEP: phosphoenolpyruvate, PYRMIT/PYRCYT: pyruvate (mitochondria/cytosol), R5P: ribose 5-phosphate, Ru5P: ribulose 5-phosphate, S7P: sedoheptulose 7-phosphate, SER: serine, SUC: succinate, TCA cycle: tricarboxylic acid cycle, X5P (P5P): xylulose 5-phosphate.

In addition to the MDVs detected by GC-MS, we also used a similar protocol to detect oxalate concentrations for both soluble and insoluble oxalate from the cultivation medium and cell biomass, respectively. To measure the concentrations of soluble oxalate, we dried 5 mL of the medium and followed the same derivatization process for GC-MS analysis. We detected no soluble oxalate and therefore repeated this work with a more sensitive HPLC analysis. Again, no soluble oxalate was detected (at levels down to 0.01 mM), which is consistent with previous results that G. trabeum accumulates less oxalate compared to other brown rot fungi,³³ mainly due to the presence of oxalate decarboxylase, which has previously been shown to decompose oxalate in G. trabeum. To measure the concentrations of insoluble oxalate, we followed similar protocols for biomass hydrolysis followed by derivatization. We found that oxalate was produced at levels of 0.19 ± 0.09 mM and 0.56 ± 0.09 mM under LN and HN conditions, respectively. The observation that greater amounts of oxalate were produced under high nitrogen levels was also consistent with previous research.³⁴

Based on the production of insoluble oxalate, we next investigated the central metabolic flux of G. trabeum when cultured using different nitrogen levels. In general, for most of the central metabolic pathways such as glycolysis and pentose phosphate pathways, the fluxes were similar despite changes in nitrogen culture levels (Fig. 1A). However, when we next evaluated the effects of peroxisomal glyoxylate dehydrogenase (GLX) and cytosolic oxaloacetate acetylhydrolase (OAH) on oxalate synthesis, we found that usage of these two pathways was significantly different when high and low nitrogen conditions were used (Fig. 1B). While the flux ratio of OAH/GLX for the low nitrogen condition was only 0.01; it was as high as 56.2 for the high nitrogen condition (Fig. 1B), indicating that the preferred pathway for oxalate production (OAH or GLX) shifts dramatically when the fungus was grown at different nitrogen levels. Correspondingly, the fluxes of metabolic pathways related to OAH and GLX were also found to be different. For example, the metabolic flux of the futile pathway that supplies cytosolic oxaloacetate from cytosolic pyruvate in the LN condition was dramatically higher than that in the HN condition (Fig. 1C). The varied flux distribution in the peroxisomal glyoxylate dehydrogenase, cytosolic oxaloacetate acetylhydrolase and futile pathways clearly indicated that G. trabeum metabolism was regulated in response to the C/N ratio, which confirmed that the C/N ratio was a key factor in oxalate biosynthesis for this fungus.¹⁰

While it is clear that the conversion of oxaloacetate to oxalate by peroxisomal glyoxylate dehydrogenase occurred in the peroxisome, oxalate synthesis via cytosolic oxaloacetate acetylhydrolase could potentially be from several sources since oxaloacetate, the precursor of oxalate in synthesis via cytosolic oxaloacetate acetylhydrolase, can be generated by either of three pathways in different regions of the cell: (1) the futile pathway, where pyruvate is converted to oxaloacetate in the cytosol to supply the TCA cycle in mitochondria; (2) the TCA cycle, where oxaloacetate is generated from malate in mitochondria; and (3) the glyoxylate cycle, where oxaloacetate is generated in the peroxisome. However, based on ¹³C-MFA, we found the flux for oxaloacetate synthesis in the glyoxylate cycle was trivial under both LN and HN conditions (Fig. 1B), indicating that glyoxylate was mainly used to produce oxalate directly rather than being converted to oxaloacetate in G. trabeum. Therefore, it is likely that the oxalate produced by action of cytosolic oxaloacetate acetylhydrolase is generated directly from oxaloacetate synthesized in the futile cycle or the TCA cycle, rather than from oxaloacetate that is synthesized in the glyoxylate cycle. In addition, in prior research with a related fungus, F. palustris^11,35 under high nitrogen conditions (although with a different nitrogen source than in our research), cytosolic oxaloacetate acetylhydrolase was the dominant precursor enzyme active in oxalate synthesis. Both F. palustris and G. trabeum have previously been reported to utilize the non-enzymatic chelator-mediated Fenton (CMF) system in lignocellulose deconstruction, and therefore it is possible that these two fungi would adopt a similar regulatory system for oxalate synthesis as it is required by CMF chemistry. It has been reported that high levels of ammonium ions may inhibit the activity of glyoxylate dehydrogenase, the key enzyme for oxalate production in the glyoxylate cycle,^36,37 which could explain the limited role of the peroxisomal glyoxylate dehydrogenase on oxalate synthesis when nitrogen (ammonium) concentrations were high.

To further investigate the regulation of cytosolic oxaloacetate acetylhydrolase and peroxisomal glyoxylate dehydrogenase, we performed qPCR to determine the expression level of the genes that encode these two enzymes. We followed an established protocol³⁸ to extract the total RNAs of G. trabeum grown under both LN and HN conditions using a FastRNA spin kit (MP Biomaterials, USA), and measured the mRNA levels. It was found that there was no significant difference in gene expression levels between the LN and HN conditions for both cytosolic oxaloacetate acetylhydrolase gene and peroxisomal glyoxylate dehydrogenase gene (Fig. S2†). Comparing the distinct flux results between LN and HN conditions, this indicates that the regulation of the pathways for oxalate synthesis was not executed at the transcriptional level. Considering the complex regulatory machinery in eukaryotic metabolism (e.g., G. trabeum), peroxisomal glyoxylate dehydrogenase and cytosolic oxaloacetate acetylhydrolase could be regulated in translational, post-translational or even at the metabolic level (e.g., ammonium inhibition). A systematic investigation of the regulatory mechanism is beyond the scope of this manuscript but will be investigated by our group in the future.

In sum, this study examined the effects of peroxisomal glyoxylate dehydrogenase and cytosolic oxaloacetate acetylhydrolase on oxalate biosynthesis in G. trabeum using ¹³C trace experiments and metabolic flux analysis; approaches not previously explored in fungi for oxalate production. As revealed by the metabolic flux results, the pathway selected for oxalate biosynthesis was highly dependent on the C/N ratio, with cytosolic oxaloacetate acetylhydrolase being the dominant pathway for oxalate synthesis under low C/N (i.e., high nitrogen) conditions, and peroxisomal glyoxylate dehydrogenase contributing to oxalate synthesis under high C/N (i.e., low nitrogen) conditions. Combined with transcriptional analysis, it was determined that such metabolic reprogramming was not transcriptionally controlled. Overall, ¹³C-MFA proved to be a useful approach in evaluating the pathway usage in G. trabeum for oxalate production and could help improve our understanding of oxalate metabolism in wood-decaying fungi and to facilitate further improvement of lignocellulose bioconversion technology in other fungal species.

Acknowledgements

This study was supported by start-up fund (#175323) from Virginia Tech. This work was also supported in part by the USDA-HATCH Project S-1041 VA-136288, and by The Research Council of Norway 243663/E50 BioMim.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra19203j

‡ LZ and WG contributed to this work equally.

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