Securing a furan-based biorefinery: disclosing the genetic basis of the degradation of hydroxymethylfurfural and its derivatives in the model fungus Aspergillus nidulans

Abstract

Hydroxymethylfurfural (HMF) is a promising lignocellulosic-derived source for the generation of diverse chemical building blocks constituting an alternative to fossil fuels. However, it remains unanswered if ubiquitous fungi can ensure their efficient decay, similar to that observed in highly specialised fungi. To disclose the genetic basis of HMF degradation in aspergilli, we performed a comprehensive analysis of Aspergillus nidulans ability to tolerate and to degrade HMF and its derivatives (including an HMF-dimer). We identified the degradation pathway using a suite of metabolomics methods and showed that HMF was modified throughout sequential reactions, ultimately yielding derivatives subsequently channelled to the TCA cycle. Based on the previously revealed hmfFGH gene cluster of Cupriavidus basilensis, we combined gene expression of homologous genes in Aspergillus nidulans and functional analyses in single-deletion mutants. Results were complemented with orthology analyses across the genomes of twenty-five fungal species. Our results support high functional redundancy for the initial steps of the HMF degradation pathway in the majority of the analysed fungal genomes and the assignment of a single-copy furan-2,5-dicarboxylic acid decarboxylase gene in A. nidulans. Collectively our data made apparent the superior capacity of aspergilli to mineralise HMF, furthering the environmental sustainability of a furan-based chemistry.

Fig. 1

Relevant building blocks obtained from HMF.

Fig. 2

Biodegradability of HMF and its derivatives along the incubation time in A. nidulans submerged cultures. The growth media (MMG) were supplemented with the IC₅₀ concentration of HMF (A), BHMF (B), FDCA (C) or OBMF (D) and their extracellular concentrations measured daily during the first 7 days and after 14 days (mM). The chromatographic detection of specific transformation products along the incubation time is also depicted comprising both extracellular (continuous lines, mM) and intracellular (dotted lines, arbitrary units) sub‐products. FDCA biodegradability was reassessed also in MM supplemented with acetate instead of glucose (C, dashed line).

Fig. 3

Schematic representation of the HMF degradation pathway based on the results obtained in this work and adapted from Ran and colleagues (2014) (A). Proposed pathway of transformation of OBMF based on the results obtained in this work (compounds in brackets were not confirmed by MS/MS analysis) (B).

Fig. 4

Relative expression of genes putatively involved in HMF, BHMF and FDCA transformation (AN4212, AN7164, AN12147 and AN12148), determined by qRT‐PCR. Aspergillus nidulans was exposed to HMF, BHMF or FDCA for 30 (white), 60 (grey) or 120 min (black). The y axes represent the fold‐change of gene expression relative to time zero (before exposure). β‐tubulin gene was used as internal control. The asterisks mark significant difference in expression of treatment when compared to the control for each exposure time (*P < 0.05; **P < 0.01, ***P < 0.001).

Fig. 5

Products formed after 7 days of incubation with IC₅₀ concentration of HMF in glucose or acetate (marked as underlined) media by Aspergillus nidulans wild type (WT pyrG ⁺) and the mutants ΔAN4212 and ΔAN7164 (A); and with IC₅₀ concentration of FDCA in acetate medium by WT pyrG ⁺ and the mutant ΔAN7164 (B). Only the molecules of which the identification could be validated using HPLC (RT and spectra) and ¹H‐NMR, and within the HPLC method quantification limits, are shown. The schematic representation of the HMF degradation pathway for A. nidulans is also displayed (C).