Cloning and Characterization of a Novel Laccase Gene, fvlac7, Based on the Genomic Sequence of Flammulina velutipes

Abstract

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

Laccase production and metabolic diversity among Flammulina velutipes strains

“…Almost every laccase promoter region brings up to light new insight into the regulation of this enzyme. However, in the available F. velutipes laccase promoter regions, the regulatory sites mentioned above have not been localized yet (Kim et al 2013 ). It should be mentioned that many fungi produce not only extracellular but also intracellular laccase (Nagai et al 2003 ; Xu et al 2012 ), which raises the possibility that those strains unable to secrete extracellular laccase may produce an intracellular one.…”

“…Thanks to presence of various bioactive compounds (polysaccharides, protein-glucan complexes, sterols, lectins, peroxidases, laccases, cellulases, and proteases), F. velutipes can be used in many medical, pharmaceutical, and industrial applications (Hassan et al 2012 ). Moreover, a previous study has reported that F. velutipes belongs to the phylum Basidiomycota and is one of the white rot fungus capable of production of extracellular lignin-modifying laccase (Kim et al 2013 ; Lee and Suh 1985 ; Otsuka Saito et al 2013 ; Zhang et al 2004 ). The aim of this paper was to demonstrate that closely related strains of F. velutipes might differ in laccase production as a response to commonly used inducers.…”

The multigene family of fungal laccases and their expression in the white rot basidiomycete Flammulina velutipes

“…The expression models that exist are unsatisfactory in explaining all the functions of laccases. In addition, a laccase gene fvlac7, was cloned based on the genomic sequence of F. velutipes and heterologously over-expressed in an Escherichia coli system (Kim et al, 2013). Because of its high nutritional and medicinal value and scale of production, F. velutipes has been recognized as a useful model fungal species to study matrix degradation in edible mushrooms (Park et al, 2014).…”

Molecular characterization of laccase genes from the basidiomycete Trametes hirsuta Bm-2 and analysis of the 5′ untranslated region (5′UTR)

“…Laccase isoenzymes show different properties related with substrate specificity, optimum pH and temperature. They oxidize a wide range of diverse phenolic compounds including mono-di-phenols, polyphenols, diamines, and recalcitrant compounds, which affect the environment (Kim et al 2013). Isoenzyme expression is affected by different environmental conditions, i.e., induction by phenols (Terrón et al 2004), lignocellulosic substrates (Park et al 2014), and metals such as copper (Vasina et al 2015).…”