Characterization of new fungal carbohydrate esterase family 1 proteins leads to the discovery of two novel dual feruloyl/acetyl xylan esterases [post]

2020 unpublished
Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important accessory enzymes in the deconstruction of plant biomass. Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database contains both fungal FAEs and AXEs, sharing a high amino acid sequence similarity, even though they target different structural molecules on plant cell wall polysaccharides. Results We recently classified fungal CE1 into five subfamilies . In this study, ten novel fungal CE1 enzymes
more » ... ngal CE1 enzymes from different subfamilies were heterologously produced in Aspergillus niger and characterized to gain insight on relationships among these esterases. The enzymes from CE1_SF1 possess AXE activity, as they hydrolyzed p NP-acetate and released acetic acid from wheat arabinoxylan, but were not active towards FAE substrates. CE1_SF5 showed FAE activity as they hydrolyzed methyl ferulate and other FAE related substrates, and release ferulic acid from wheat arabinoxylan. These FAEs preferred feruloylated arabinoxylan over pectin. Two CE1_SF2, sharing over 70% amino acid sequence identity, possessed the opposite activity. Interestingly, one enzyme from CE1_SF1 and one from CE1_SF5 possess dual feruloyl/acetyl xylan esterase (FXE) activity. These dual activity enzymes showed expansion of substrate specificity. Conclusions The new FXEs from CE1 can efficiently release both ferulic acid and acetic acid from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation. Background Many by-products from agricultural processing industries contain arabinoxylan as a major component. Arabinoxylan comprises of a β-D-(1→4)-linked xylose backbone, which is substituted with α-L-(1→2)and/or α-L-(1→3)-linked arabinose residues. The xylan backbone can be acetylated at the O-2 and/or O-3 positions depending on the types of plants [1] . In commelinid monocots (e.g. wheat, rice and barley), these can be further substituted at the O-5 position by ferulic acid (4-hydroxy-3methoxycinnamic acid) and other hydroxycinnamic acids (e.g. p-coumaric acid) [2] [3] [4] [5] . Ferulic acid is also present in rhamnogalacturonan I (RGI) in pectin, which has a backbone of alternating α-L-(1→2)-4 rhamnose and α-D-(1→4)-galacturonic acid residues. The rhamnose residue can be substituted with α-L-(1→5)-arabinan or β-D-(1→4)-galactan chains, which both can contain terminal ferulic acid residues [6] [7] [8] . Ferulic acid plays a role in defense mechanism against pathogens, due to its antimicrobial property and the di-ferulic acid cross-links between xylan chains increase the physical strength and integrity of plant cell walls [3, 4] . Both acetylation and feruloylation inhibit the action of endo-acting plant cell wall-degrading enzymes and therefore hinder the enzymatic saccharification for biomass valorization, e.g. for bioethanol production and biorefinery processes. Acetyl xylan esterases (AXEs) [EC 3.1.1.72] catalyze the hydrolysis of ester linkages between acetyl groups (in the form of acetylation) and xylan, which leads to the release of acetic acid. Feruloyl esterases (FAEs) [EC 3.1.1.73] catalyze the de-esterification of agro-industrial byproducts which liberates ferulic and other plant phenolic acids [9-11]. AXEs and FAEs facilitate the degradation of complex plant cell wall polysaccharides by removing the ester bonds in plant polymers, providing access to glycoside hydrolases and polysaccharide lyases [12-14]. Apart from being used as accessory enzymes in the saccharification process, AXEs and FAEs are also potential biocatalysts for the synthesis of a broad range of novel bioactive components for the food, cosmetics and pharmaceutical industries [10, 15, 16]. Based on the Carbohydrate-Active enZymes (CAZy) database (http://www.cazy.org [17]), fungal AXEs are primarily classified in Carbohydrate Esterase (CE) families CE1-CE6 and CE16 [18]. While part of the fungal FAEs are grouped in CE1, most FAEs are not classified CAZymes [10, 19]. CE1 FAEs are of interest to industry because of their broad substrate range and high synthetic property for potential antioxidants through transferuloylation [20]. Even though they target structurally different substrates, both FAEs and AXEs from CE1 share a high sequence similarity. We recently classified the fungal members of CE1 into five subfamilies (CE1_SF1-SF5) based on phylogenetic analysis [21]. CE1_SF1 and CE1_SF2 are closely related and split from the same node. However, CE1_SF1 contains characterized AXEs, while CE1_SF2 contains characterized FAEs. CE1_SF4 and CE1_SF5 are also split from a common node, and CE1_SF5 contains characterized FAEs, whereas CE1_SF4 has only uncharacterized members. CE1_SF3 is the smallest branch, which contains only 5 sequences from basidiomycetes with no predicted secreted signal peptide. It is distantly related to the other fungal CE1 members (Esterase, PHB depolymerase, IPR010126), but related to the Esterase D/S-formylglutathione hydrolase sequences (IPR014186) according to InterPro classification [22]. In comparison with bacterial CE1 enzymes, fungal CE1 enzymes mainly belong to the Esterase_phb family based on the ESTHER database, a classification for proteins with an α/β-hydrolase fold [23], which is distantly related to bacterial enzymes belonging to Antigen85c family (<20% sequence identity) [24]. Thus far, two crystal structures of fungal CE1 enzymes have been reported, i.e. Anaeromyces mucronatus AmCE1 (PDB: 5CXU [25]) and Aspergillus luchuensis AlAXEA (formerly Aspergillus awamori) (PDB: 5X6S [24]). In this study, we characterized ten fungal CE1 proteins covering CE1_SF1, SF2 and SF5 (Fig. 1 , Table 1 ). These enzymes were heterologously produced in an Aspergillus niger strain derived from N400 (CBS 120.49, ATCC 9029, FGSC A1143) and biochemically characterized using both simple model and complex plant biomass substrates to validate their activity and substrate preference. Our results provide new insights into the differences in activity of fungal CE1 enzymes, and shed light on the relationship between these CE1 FAEs and other fungal FAEs. Results And Discussion Model substrates do not provide conclusive functional assignment for CE1 enzymes In this study, we selected ten previously uncharacterized fungal CE1 enzymes covering three CE1 subfamilies for characterization. Four of these enzymes belonged to CE1_SF1, two to CE1_SF2 and four to CE1_SF5 (Table 1) . We also included two characterized fungal CE1 enzymes from Aspergillus niger (AxeA) [26, 27] and Chaetomium thermophilum (AxeA) [28], and an enzyme that did not belong to any CE1 subfamily, but was a tannase-related FAE candidate (FAE_SF2 in [10]) to validate the substrate preference. Heterologous production of CE1 enzymes was performed using A. niger strain CSFG_6005 as a host [29]. The protein concentration was estimated using a densitometric method, showing that A. niger AxeA was produced at the highest level (Table 1) . Most CE1_SF1 enzymes efficiently hydrolyzed pNP-acetate, but not pNP-ferulate and four methyl hydroxycinnamates (methyl
doi:10.21203/rs.3.rs-17222/v1 fatcat:k67exjjg7jgj3lgvydx2suq4uy