Discovery and Functional Analysis of a Salicylic Acid Hydroxylase from Aspergillus niger.

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.

Colonies of the fungus Aspergillus niger are highly differentiated to adapt to local carbon source variation.

Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi-omics approach, adaptation of A. niger colonies to spatially separated and compositionally different plant biomass substrates. This demonstrated a high level of intra-colony differentiation, which closely matched the locally available substrate. The part of the colony exposed to pectin-rich sugar beet pulp and to xylan-rich wheat bran showed high pectinolytic and high xylanolytic transcript and protein levels respectively. This study therefore exemplifies the high ability of fungal colonies to differentiate and adapt to local conditions, ensuring efficient use of the available nutrients, rather than maintaining a uniform physiology throughout the colony.

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.

Temporal transcriptome analysis of the white-rot fungus Obba rivulosa shows expression of a constitutive set of plant cell wall degradation targeted genes during growth on solid spruce wood.

The basidiomycete white-rot fungus Obba rivulosa, a close relative of Gelatoporia (Ceriporiopsis) subvermispora, is an efficient degrader of softwood. The dikaryotic O. rivulosa strain T241i (FBCC949) has been shown to selectively remove lignin from spruce wood prior to depolymerization of plant cell wall polysaccharides, thus possessing potential in biotechnological applications such as pretreatment of wood in pulp and paper industry. In this work, we studied the time-course of the conversion of spruce by the genome-sequenced monokaryotic O. rivulosa strain 3A-2, which is derived from the dikaryon T241i, to get insight into transcriptome level changes during prolonged solid state cultivation. During 8-week cultivation, O. rivulosa expressed a constitutive set of genes encoding putative plant cell wall degrading enzymes. High level of expression of the genes targeted towards all plant cell wall polymers was detected at 2-week time point, after which majority of the genes showed reduced expression. This implicated non-selective degradation of lignin by the O. rivulosa monokaryon and suggests high variation between mono- and dikaryotic strains of the white-rot fungi with respect to their abilities to convert plant cell wall polymers.

Comparative analysis of basidiomycete transcriptomes reveals a core set of expressed genes encoding plant biomass degrading enzymes.

Basidiomycete fungi can degrade a wide range of plant biomass, including living and dead trees, forest litter, crops, and plant matter in soils. Understanding the process of plant biomass decay by basidiomycetes could facilitate their application in various industrial sectors such as food & feed, detergents and biofuels, and also provide new insights into their essential biological role in the global carbon cycle. The fast expansion of basidiomycete genomic and functional genomics data (e.g. transcriptomics, proteomics) has facilitated exploration of key genes and regulatory mechanisms of plant biomass degradation. In this study, we comparatively analyzed 22 transcriptome datasets from basidiomycetes related to plant biomass degradation, and identified 328 commonly induced genes and 318 repressed genes, and defined a core set of carbohydrate active enzymes (CAZymes), which was shared by most of the basidiomycete species. High conservation of these CAZymes in genomes and similar regulation pattern in transcriptomics data from lignocellulosic substrates indicate their key role in plant biomass degradation and need for their further biochemical investigation.

The physiology of Agaricus bisporus in semi-commercial compost cultivation appears to be highly conserved among unrelated isolates.

The white button mushroom Agaricus bisporus is one of the most widely produced edible fungus with a great economical value. Its commercial cultivation process is often performed on wheat straw and animal manure based compost that mainly contains lignocellulosic material as a source of carbon and nutrients for the mushroom production. As a large portion of compost carbohydrates are left unused in the current mushroom cultivation process, the aim of this work was to study wild-type A. bisporus strains for their potential to convert the components that are poorly utilized by the commercial strain A15. We therefore focused our analysis on the stages where the fungus is producing fruiting bodies. Growth profiling was used to identify A. bisporus strains with different abilities to use plant biomass derived polysaccharides, as well as to transport and metabolize the corresponding monomeric sugars. Six wild-type isolates with diverse growth profiles were compared for mushroom production to A15 strain in semi-commercial cultivation conditions. Transcriptome and proteome analyses of the three most interesting wild-type strains and A15 indicated that the unrelated A. bisporus strains degrade and convert plant biomass polymers in a highly similar manner. This was also supported by the chemical content of the compost during the mushroom production process. Our study therefore reveals a highly conserved physiology for unrelated strains of this species during growth in compost.

The molecular response of the white-rot fungus Dichomitus squalens to wood and non-woody biomass as examined by transcriptome and exoproteome analyses.

The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post-)genomic analyses have shown that species that occupy a large variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well-adapted to the prevailing substrate. White-rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white-rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non-woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non-woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat.

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.

Aromatic metabolism of filamentous fungi in relation to the presence of aromatic compounds in plant biomass.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and ß-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose.

The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-acting enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable transcript levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cellulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expression levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus.

An improved and reproducible protocol for the extraction of high quality fungal RNA from plant biomass substrates.

Isolation of high quantity and quality RNA is a crucial step in the detection of meaningful gene expression data. Obtaining intact fungal RNA from complex lignocellulosic substrates is often difficult, producing low integrity RNA which perform poorly in downstream applications. In this study we developed an RNA extraction method using CsCl centrifugation procedure, modified from previous reports and adapted for isolation of RNA from plant biomass. This method provided high level of integrity and good quantity of RNA which were suitable for reliable analyses of gene expression and produced consistent and reproducible results.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

Agaricus bisporus and related Agaricus species on lignocellulose: production of manganese peroxidase and multicopper oxidases.

Biotechnological, microbiological, and genetic studies of Agaricus species other than A. bisporus, the white button mushroom, have been limited so far. To expand the knowledge in the genus Agaricus, six novel wild-type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. All the selected Agaricus species produced extracellular manganese peroxidase (MnP) and laccase activities in semi-solid rye bran cultures. Moderate MnP activities were measured for A. bisporus, A. bernardii and A. campestris. The highest laccase activities were obtained for A. bisporus and A. campestris. On soy medium, the highest mycelial tyrosinase activity was determined for A. bernardii. For A. bisporus, addition of copper caused no increase in laccase or tyrosinase activities on soy or malt extract media. Hyphal growth rate of the isolates was studied on lignocellulose amended agar plates. Fastest growth was obtained for A. bisporus on wheat bran and birch leaf litter agar. Except for A. bernardii, hyphal growth rates correlated well with MnP and laccase production levels between Agaricus species. Molecular taxonomy of the novel Agaricus spp. positioned them to distinct phylogenetic clusters with species-level identity. In conclusion, our data point to the importance of both MnP and multicopper enzymes in Agaricus spp. while growing on lignocelluloses.

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 µkat/µg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

The transcriptomic response of two basidiomycete fungi to plant biomass is modulated by temperature to a different extent.

Many fungi show a strong preference for specific habitats and growth conditions. Investigating the molecular mechanisms of fungal adaptation to varying environmental conditions is of great interest to biodiversity research and is important for many industrial applications. In this study, we compared the transcriptome profiles of two previously genome-sequenced white-rot wood-decay fungi, Trametes pubescens and Phlebia centrifuga, during their growth on two common plant biomass substrates (wheat straw and spruce) at two temperatures (15 °C and 25 °C). The results showed that both fungi partially tailored their molecular responses to different types of carbon sources, differentially expressing genes encoding polysaccharide degrading enzymes, transporters, proteases and monooxygenases. Notably, more lignin modification related AA2 genes and cellulose degradation related AA9 genes were differentially expressed in the tested conditions of T. pubescens than P. centrifuga. In addition, we detected more remarkable transcriptome changes to different growth temperature in P. centrifuga than in T. pubescens, which reflected their different ability to adapt to the temperature fluctuations. In P. centrifuga, differentially expressed genes (DEGs) related to temperature response mainly encode protein kinases, trehalose metabolism, carbon metabolic enzymes and glycoside hydrolases, while the main temperature-related DEGs identified in T. pubescens are only the carbon metabolic enzymes and glycoside hydrolases. Our study revealed both conserved and species-specific transcriptome changes during fungal adaptation to a changing environment, improving our understanding of the molecular mechanisms underlying fungal plant biomass conversion at varying temperatures.

L-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate.

Catabolism of amino acids is a central process in cellular nitrogen turnover, but only a few of the mechanisms involved have been described from basidiomycete fungi. This study identified one such mechanism, the l-amino acid oxidase (Lao1) enzyme of Hebeloma cylindrosporum, by 2D gel separation and MS. We determined genomic DNA sequences of lao1 and part of its upstream gene, a putative pyruvate decarboxylase (pdc2), and cloned the cDNA of lao1. The two genes were also identified and annotated from the genome of Laccaria bicolor. The lao1 and pdc2 gene structures were conserved between the two fungi. The intergenic region of L. bicolor possessed putative duplications not detected in H. cylindrosporum. Lao1 sequences possessed dinucleotide-binding motifs typical for flavoproteins. Lao1 was less than 23 % identical to Lao sequences described previously. Recombinant Lao1 of H. cylindrosporum was expressed in Escherichia coli, purified and refolded with SDS to gain catalytic activity. The enzyme possessed broad substrate specificity: 37 l-amino acids or derivatives served as effective substrates. The highest activities were recorded with l-glutamate, but positively charged and aromatic amino acids were also accepted. Michaelis constants for six amino acids varied from 0.5 to 6.7 mM. We have thus characterized a novel type of Lao-enzyme and its gene from the basidiomycete fungus H. cylindrosporum.

Comparative Analysis of Enzyme Production Patterns of Lignocellulose Degradation of Two White Rot Fungi: Obba rivulosa and Gelatoporia subvermispora.

The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi, Obba rivulosa and Gelatoporia subvermispora, during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications.

Fractionation of Technical Lignin from Enzymatically Treated Steam-Exploded Poplar Using Ethanol and Formic Acid.

Lignocellulosic biorefineries produce lignin-rich side streams with high valorization potential concealed behind their recalcitrant structure. Valorization of these residues to chemicals, materials, and fuels increases the profitability of biorefineries. Fractionation is required to reduce the lignins' structural heterogeneity for further processing. We fractionated the technical biorefinery lignin received after steam explosion and saccharification processes. More homogeneous lignin fractions were produced with high ß-O-4' and aromatic content without residual carbohydrates. Non-toxic biodegradable organic solvents like ethanol and formic acid were used for fractionation and can be adapted to the existing biorefinery processes. Macromolecular properties of the isolated fractions were carefully characterized by structural, chemical, and thermal methods. The ethanol organosolv treatment produced highly soluble lignin with a reasonable yield, providing a uniform material for lignin applications. The organosolv fractionation with formic acid and combined ethanol-formic acid produced modified lignins that, based on thermal analysis, are promising as thermoresponsive materials.

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity.

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.