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

Juan A Tamayo Ramos1, Sharief Barends23, Raymond MD Verhaert2 and Leo H de Graaff1*

Author Affiliations

1 Fungal Systems Biology, Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands

2 ProteoNic BV, Niels Bohrweg 11-13, 2333CA Leiden, The Netherlands

3 Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA

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Microbial Cell Factories 2011, 10:78  doi:10.1186/1475-2859-10-78

Published: 8 October 2011

Abstract

Background

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.

Results

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.

Conclusions

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:
Multicopper oxidase; laccase; Aspergillus niger; secretion