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Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). A. niger cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form cis,cis-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of A. niger grown on salicylic acid to identify genes (shyA and crcA) involved in salicylic acid metabolism. Heterologous production in Escherichia coli followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that cis,cis-muconic acid can be produced from salicylic acid. In addition, the in vivo roles of shyA, dhbA, and crcA were studied by creating A. niger deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid. IMPORTANCE Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as cis,cis-muconic acid. cis,cis-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, cis,cis-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce cis,cis-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.

Additional file 1: Fig S1. Visualization of VdhA, VhyA and MhdA by SDS-PAGE. Fig. S2. Conversion of aromatic compounds by the reference (a), ∆vdhA (b), ∆vhyA (c), and ∆mhdA (d) after 24 h of incubation. Concentrations of the detected compounds can be found in Table 1. Error bars represent the standard deviation between three biological replicates. Fig. S3. Maximum likelihood (ML; 500 bootstraps) phylogenetic tree of A. niger VdhA compared to selected fungal genomes. The scale bar shows a distance equivalent to 0.2 amino acid substitutions per site. Values over 50% bootstrap support are shown with ML values in black, Neighbor Joining values in purple and Minimum Evolution values in blue. Characterized enzymes are in bold. Blue font represents ascomycete fungi, red font basidiomycete fungi, green font bacteria, and orange font Saccharomycetes. Fungal species names are followed by protein IDs from JGI ( http://genome.jgi-psf.org/programs/fungi/index.jsf ). Fig. S4. Maximum likelihood (ML; 500 bootstraps) phylogenetic tree of A. niger MhdA compared to selected fungal genomes. The scale bar shows a distance equivalent to 0.2 amino acid substitutions per site. Values over 50% bootstrap support are shown with ML values in black, Neighbor Joining values in purple and Minimum Evolution values in blue. In bold are characterized enzymes. Blue font represents ascomycete fungi, red font basidiomycete fungi, green font bacteria, black font plants and pink font Homo sapiens. Fungal species names are followed by protein IDs from JGI ( http://genome.jgi-psf.org/programs/fungi/index.jsf ). Fig. S5. 1H-NMR spectrum of 4-oxo-monomethyl adipate (D2O). The structure corresponds to 4-oxo-monomethyl adipate with D2 at C5 position.

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and biomass valorization. AXEs catalyze the hydrolysis of acetyl group from xylan, while FAEs release ferulic and other hydroxycinnamic acids from xylan and pectin. Previously, we reported a phylogenetic analysis for the fungal members of CE1, establishing five subfamilies (CE1_SF1–SF5). Currently, the characterized AXEs are in the subfamily CE1_SF1, whereas CE1_SF2 contains mainly characterized FAEs. These two subfamilies are more related to each other than to the other subfamilies and are predicted to have evolved from a common ancestor, but target substrates with a different molecular structure. In this study, four ascomycete enzymes from CE1_SF1 and SF2 were heterologously produced in Pichia pastoris and characterized with respect to their biochemical properties and substrate preference toward different model and plant biomass substrates. The selected enzymes from CE1_SF1 only exhibited AXE activity, whereas the one from CE1_SF2 possessed dual FAE/AXE activity. This dual activity enzyme also showed broad substrate specificity toward model substrates for FAE activity and efficiently released both acetic acid and ferulic acid (∼50%) from wheat arabinoxylan and wheat bran which was pre-treated with a commercial xylanase. These fungal AXEs and FAEs also showed promising biochemical properties, e.g., high stability over a wide pH range and retaining more than 80% of their residual activity at pH 6.0–9.0. These newly characterized fungal AXEs and FAEs from CE1 have high potential for biotechnological applications. In particular as an additional ingredient for enzyme cocktails to remove the ester-linked decorations which enables access for the backbone degrading enzymes. Among these novel enzymes, the dual FAE/AXE activity enzyme also supports the evolutionary relationship of CE1_SF1 and SF2.

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A.terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes.

The aromatic compound benzoic acid and its derivatives are frequently used preservatives in food and beverages due to their strong antimicrobial properties. Benzoic acid naturally occurs in plants and serves as a building block for primary and secondary metabolites. Several Aspergilli are able to degrade benzoic acid to p-hydroxybenzoic acid by p-hydroxylation followed by m-hydroxylation to protocatechuic acid. The aromatic ring of protocatechuic acid is then cleaved and further converted through the oxoadipate pathway where it is used as a carbon source. The benzoic acid pathway is well studied in the filamentous fungus Aspergillus niger; however, only one enzyme, benzoate-4-monooxygenase, has been characterized. In this study, three novel enzymes of the benzoic acid pathway were identified and characterized. Transcriptome analysis of A. niger on p-hydroxybenzoic acid, protocatechuic acid, and the related compounds p-coumaric acid and caffeic acid revealed two genes of unknown function, which were highly expressed on all four aromatic compounds. Analysis of A. niger deletion mutants of these genes revealed their essential role in the utilization of benzoic acid, p-hydroxybenzoic acid, and protocatechuic acid. Biochemical analysis confirmed that the corresponding enzymes function as the p-hydroxybenzoate-m-hydroxylase and protocatechuate 3,4 ring-cleavage dioxygenase in the benzoic acid pathway. In addition, a hydroxyquinol 1,2 ring-cleavage dioxygenase, involved in an alternative protocatechuic acid metabolic pathway, was identified.

Distillers’ grain (DG) is one of the lignocellulosic agricultural waste streams having a high potential for biofuel production, however it is causing health and environmental problems in Southwest region of China because of improper treatment before being used for landfill. Here, we aimed to identify fungal strains that efficiently hydrolyze DG and explored the possibility to further improve the hydrolysis of DG and other agricultural wastes by co-culturing these fungi. The tested fungal strains included 11 strains that were isolated from DG (Maotai Town, Guizhou, China) and 19 strains that were selected from the CBS collection of Westerdijk Fungal Biodiversity Institute. Aspergillus niger CBS 110.42 and CBS 554.65 and Phanerochaete chrysosporium CBS 246.84 showed overall best cellulose and xylan degrading activity, whereas high laccase and peroxidase activities were detected for Oidiodendron echinulatum CBS 113.65 and Bjerkandera adusta CBS 143380, respectively. Furthermore, A. niger CBS 554.65 co-cultured with Trichoderma reesei CBS 383.78 and A. niger CBS 110.42 with P. chrysosporium CBS 246.84 showed the best improvement (up to 2- and 3-fold higher of β-glucosidase and β-xylosidase activities). These fungi and the combinations have strong potential to be further developed for local bioenergy production.