The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes

<p>Abstract</p> <p>Background</p> <p>Many filamentous fungal genomes contain complex groups of multicopper oxidase (MCO) coding genes that makes them a good source for new laccases with potential biotechnological interest. A bioinformatics analysis of the <it>Aspergillus niger </it>ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed.</p> <p>Results</p> <p>A bioinformatic analysis of the <it>A. niger </it>ATCC 1015 genome revealed the presence of 13 MCO genes belonging to three different subfamilies on the basis of their phylogenetic relationships: ascomycete laccases, fungal pigment MCOs and fungal ferroxidases. According to <it>in silico </it>amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (<it>mcoA</it>, <it>mcoB</it>, <it>mcoC</it>, <it>mcoD</it>, <it>mcoE</it>, <it>mcoF</it>, <it>mcoG</it>, <it>mcoI</it>, <it>mcoJ </it>and <it>mcoM</it>) were placed under the control of the <it>glaA </it>promoter and overexpressed in <it>A. niger </it>N593. Enzyme activity plate assays with several common laccase substrates showed that all genes are actually expressed and code for active MCOs. Interestingly, expressed enzymes show different substrate specificities. In addition, optimization of fungal pigment MCOs extracellular production was investigated. The performance of the widely used glucoamylase signal sequence (ssGlaA) in McoA secretion was studied. Results obtained suggest that ssGlaA do not yield higher levels of secreted McoA when compared to its native secretion signal. Also, McoB synthesis was investigated using different nitrogen sources in minimal medium liquid cultures. Higher yields of extracellular McoB were achieved with (NH₄)₂tartrate.</p> <p>Conclusions</p> <p><it>Aspergillus niger </it>is a good source of new laccases. The different substrate specificity observed in plate assays makes them interesting to be purified and biochemically compared. The homologous signal sequence of McoA has been shown to be a good choice for its extracellular overexpression. From the nitrogen sources tested (NH₄)₂tartrate has been found to be the most appropriate for McoB production in <it>A. niger</it>.</p

Fungal glucuronoyl esterases : Genome mining based enzyme discovery and biochemical characterization

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.4-O-Methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl D-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.Peer reviewe

Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.Peer reviewe