Abstract:The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g−1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The… Show more
“…Black coloured clades were not considered for the analysis. 1—Daniel et al [15]; 2—de Oliveira et al [20]; 3—Linke et al [59]; 4—Cregg et al [14]; 5—Sakai and Tani [79]; 6—Ledeboer et al [55]; 7—Chakraborty et al [13]; 8—Segers et al [81]; 9—Soldevila and Ghabrial [85]Fig. 4Maximum likelihood tree of the dehydrogenase domains in the cellobiose dehydrogenase (CDH) cluster.…”
Background: The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics. Results: To improve the understanding of the GMC superfamily as a whole, we used sequence similarity networks to cluster large numbers of fungal GMC sequences and annotate them according to functionality. Subsequently, different members of the GMC superfamily were analysed in detail with regard to their sequences and phylogeny. This allowed us to define the currently characterised sequence space and show that complete clades of some enzymes have not been studied in any detail to date. Finally, we interpret our results from an evolutionary perspective, where we could show, for example, that pyranose dehydrogenase evolved from aryl-alcohol oxidoreductase after a change in substrate specificity and that the cytochrome domain of cellobiose dehydrogenase was regularly lost during evolution. Conclusions: This study offers new insights into the sequence variation and phylogenetic relationships of fungal GMC/AA3 sequences. Certain clades of these GMC enzymes identified in our phylogenetic analyses are completely uncharacterised to date, and might include enzyme activities of varying specificities and/or activities that are hitherto unstudied.
“…Black coloured clades were not considered for the analysis. 1—Daniel et al [15]; 2—de Oliveira et al [20]; 3—Linke et al [59]; 4—Cregg et al [14]; 5—Sakai and Tani [79]; 6—Ledeboer et al [55]; 7—Chakraborty et al [13]; 8—Segers et al [81]; 9—Soldevila and Ghabrial [85]Fig. 4Maximum likelihood tree of the dehydrogenase domains in the cellobiose dehydrogenase (CDH) cluster.…”
Background: The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics. Results: To improve the understanding of the GMC superfamily as a whole, we used sequence similarity networks to cluster large numbers of fungal GMC sequences and annotate them according to functionality. Subsequently, different members of the GMC superfamily were analysed in detail with regard to their sequences and phylogeny. This allowed us to define the currently characterised sequence space and show that complete clades of some enzymes have not been studied in any detail to date. Finally, we interpret our results from an evolutionary perspective, where we could show, for example, that pyranose dehydrogenase evolved from aryl-alcohol oxidoreductase after a change in substrate specificity and that the cytochrome domain of cellobiose dehydrogenase was regularly lost during evolution. Conclusions: This study offers new insights into the sequence variation and phylogenetic relationships of fungal GMC/AA3 sequences. Certain clades of these GMC enzymes identified in our phylogenetic analyses are completely uncharacterised to date, and might include enzyme activities of varying specificities and/or activities that are hitherto unstudied.
“…Since the first report in 1960 by Farmer et al ( 1960 ), who identified aromatic alcohol oxidase activity in the growth medium of the basidiomycete Polystictus (Trametes) versicolor , AAO activity has been found in cultures of many other basidiomycetes like Pleurotus species (Bourbonnais and Paice 1988 ; Galperin et al 2016 ; Guillen et al 1990 ; Sannia et al 1991 ; Varela et al 2000 ), Bjerkandera adusta (Muheim et al 1990b ; Romero et al 2009 ), Bjerkandera sp. BOS55 (de Jong et al 1992a ), Coprinopsis cinerea (Tamaru et al 2018 ), Phanerochaete chrysosporium (Asada et al 1995 ), Ustilago maydis (Couturier et al 2016 ), and ascomycetous fungi such as Aspergillus terreus (Chakraborty et al 2014 ), Botrytis cinerea (Goetghebeur et al 1992 ), Fusarium proliferatum (Regalado et al 1999 ), Geotrichum candidum (Kim et al 2001 ), and Thermothelomyces thermophilus (Kadowaki et al 2020 ) . Although predominantly found in fungi, enzymes with AAO activities have also been reported in bacteria Sphingobacterium sp.…”
Section: Occurrence and Gmc Superfamilymentioning
confidence: 99%
“…4B ). Differently, C. cinerea r Cc AAO catalyzed oxidation of both vanillyl alcohol and isovanillyl alcohol with similar activities, which were only slightly lower than activity towards benzyl alcohol (Chakraborty et al 2014 ; Tamaru et al 2018 ). AAO of Bjerkandera BOS55 prefers mono- and dichlorinated anisyl alcohols as substrates (de Jong et al 1994 ).…”
Aryl-alcohol oxidases (AAOs) are FAD-containing enzymes that oxidize a broad range of aromatic as well as aliphatic allylic alcohols to aldehydes. Their broad substrate spectrum accompanied by the only need for molecular oxygen as cosubstrate and production of hydrogen peroxide as sole by-product makes these enzymes very promising biocatalysts. AAOs were used in the synthesis of flavors, fragrances, and other high-value-added compounds and building blocks as well as in dye decolorization and pulp biobleaching. Furthermore, AAOs offer a huge potential as efficient suppliers of hydrogen peroxide for peroxidase- and peroxygenase-catalyzed reactions. A prerequisite for application as biocatalysts at larger scale is the production of AAOs in sufficient amounts. Heterologous expression of these predominantly fungal enzymes is, however, quite challenging. This review summarizes different approaches aiming at enhancing heterologous expression of AAOs and gives an update on substrates accepted by these promising enzymes as well as potential fields of their application.
Key points
• Aryl-alcohol oxidases (AAOs) supply ligninolytic peroxidases with H2O2.
• AAOs accept a broad spectrum of aromatic and aliphatic allylic alcohols.
• AAOs are potential biocatalysts for the production of high-value-added bio-based chemicals.
SUMMARYBiomass constitutes an appealing alternative to fossil resources for the production of materials and energy. The abundance and attractiveness of vegetal biomass come along with challenges pertaining to the intricacy of its structure, evolved during billions of years to face and resist abiotic and biotic attacks. To achieve the daunting goal of plant cell wall decomposition, microorganisms have developed many (enzymatic) strategies, from which we seek inspiration to develop biotechnological processes. A major breakthrough in the field has been the discovery of enzymes today known as lytic polysaccharide monooxygenases (LPMOs), which, by catalyzing the oxidative cleavage of recalcitrant polysaccharides, allow canonical hydrolytic enzymes to depolymerize the biomass more efficiently. Very recently, it has been shown that LPMOs are not classical monooxygenases in that they can also use hydrogen peroxide (H2O2) as an oxidant. This discovery calls for a revision of our understanding of how lignocellulolytic enzymes are connected since H2O2is produced and used by several of them. The first part of this review is dedicated to the LPMO paradigm, describing knowns, unknowns, and uncertainties. We then present different lignocellulolytic redox systems, enzymatic or not, that depend on fluxes of reactive oxygen species (ROS). Based on an assessment of these putatively interconnected systems, we suggest that fine-tuning of H2O2levels and proximity between sites of H2O2production and consumption are important for fungal biomass conversion. In the last part of this review, we discuss how our evolving understanding of redox processes involved in biomass depolymerization may translate into industrial applications.
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