Isolation of a laccase‐coding gene from the lignin‐degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris

• Published in: Journal of applied microbiology 2018-06, Vol.124 (6), p.1454-1468
• Main Authors: Fonseca, M.I., Molina, M.A., Winnik, D.L., Busi, M.V., Fariña, J.I., Villalba, L.L., Zapata, P.D.
• Format: Article
• Language: English
• Subjects: Amino acids; Antioxidants; Biodegradation; characterization; Cloning; Cloning, Molecular; Decoloring; Decolorization; Degradation; Dyes; Enzyme Stability; Fermentation; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; Fungi; Gene Expression; gene isolation; Gene regulation; Heat shock; Heavy metals; heterologous expression; Kinetics; Laccase; Laccase - chemistry; Laccase - genetics; Laccase - isolation & purification; Laccase - metabolism; Lignin; Lignin - metabolism; Phlebia brevispora; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Polymerase Chain Reaction; Polyporales - chemistry; Polyporales - enzymology; Polyporales - genetics; Protein Sorting Signals; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Regulatory sequences; Secretion; Sodium chloride; Solvents; Textile industry wastewaters; Transcription; Yeast
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.13720

Aims Isolate and characterize a laccase‐encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long‐distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563‐pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α‐factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6‐dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions Lac I‐encoding gene could be successfully sequenced having cis‐acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α‐factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study The cis‐acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.