The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes
Open Access
- 8 October 2011
- journal article
- Published by Springer Science and Business Media LLC in Microbial Cell Factories
- Vol. 10 (1), 78
- https://doi.org/10.1186/1475-2859-10-78
Abstract
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 Aspergillus niger ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed. A bioinformatic analysis of the A. niger 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 in silico amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (mcoA, mcoB, mcoC, mcoD, mcoE, mcoF, mcoG, mcoI, mcoJ and mcoM) were placed under the control of the glaA promoter and overexpressed in A. niger 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)2 tartrate. Aspergillus niger 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)2 tartrate has been found to be the most appropriate for McoB production in A. niger.Keywords
This publication has 53 references indexed in Scilit:
- Comparative genomics of citric-acid-producingAspergillus nigerATCC 1015 versus enzyme-producing CBS 513.88Genome Research, 2011
- The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White BiotechnologyMolecular & Cellular Proteomics, 2010
- Designer laccases: a vogue for high-potential fungal enzymes?Trends in Biotechnology, 2010
- The Yeast Signal Sequence Trap Identifies Secreted Proteins of the Hemibiotrophic Corn PathogenColletotrichum graminicolaMolecular Plant-Microbe Interactions®, 2008
- In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniquesJournal of Biotechnology, 2008
- Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccaseThe Chemical Record, 2007
- Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequencesThe FEBS Journal, 2006
- Feature-based prediction of non-classical and leaderless protein secretion"Protein Engineering, Design and Selection", 2004
- Multiple sequence alignment with the Clustal series of programsNucleic Acids Research, 2003
- Predicting transmembrane protein topology with a hidden markov model: application to complete genomesJournal of Molecular Biology, 2001