Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications

Abstract

Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.

Induction by Hypoxia of Heterologous-Protein Production with the Kl PDC1 Promoter in Yeasts

“…In fact, applying the two-stage strategy, cells reach a final biomass level similar to that of fully aerated cultures, and as a consequence, volumetric production results consistently increased for all the proteins assayed. We also could observe the accumulation in the growing medium of satisfactory amounts of laccase, a protein that is usually poorly expressed in yeasts compared to the expression levels obtained with fungi (9,12). Moreover, levels of laccase much higher than those reported in the present paper have been obtained with plasmid pLC12 with K. lactis strains different from the one described here (M. M. Bianchi, personal communication), suggesting the possibility of further process improvement.…”

Heterologous laccase production and its role in industrial applications

“…59 Hyper-glycosylated laccases are often produced in such conventional yeasts. First results of heterologous expression of fungal laccases in the non-conventional yeasts Kluyveromyces lactis, 60 Yarrowia lipolytica, 61 and Pichia methanolica 62 have become available in 2005, with yields comparable to those obtained using other yeasts and a limited extent of glycosylation. A complete list of heterologously expressed laccases in yeasts is reported in Table 3.…”

Accurate Stabilities of Laccase Mutants Predicted with a Modified FoldX Protocol

“…Two recent mutant stability data sets of the high-redoxpotential fungal laccases (HRPL), TvLIIIb, a widely studied fungal laccase from Trametes versicolor (white-rot fungi), 34 and PM1L, 35 have rendered such an investigation possible. The TvLIIIb data set consists mostly of single-point mutants, 36 whereas the PM1L data set contains nine multisite mutants.…”