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

FONSECA, MARÍA ISABEL; MELISA MOLINA; DANIANA WINNIK; MARÍA VICTORIA BUSI; FARIÑA, JULIA INÉS; VILLALBA, LAURA; ZAPATA, PEDRO DARÍO

JOURNAL OF APPLIED MICROBIOLOGY

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2018 vol. 2018 p. 1 - 15

1364-5072

AimsIsolate 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 analyze the biotechnological application potential.Methods and Resultslac 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 in a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in Pichia 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 solvent and nine synthetic dyes could be degraded to different degrees.ConclusionsLac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lacI 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 a NaCl and various solvents and degraded some recalcitrant synthetic dyes.Significance and Impact of StudyThe 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 forlaccase 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.