Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications.
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.
Jolivalt
Claude
C
Laboratoire de Synthèse Sélective Organique et Produits Naturels, ENSCP, UMR CNRS 7573, 11, rue Pierre et Marie Curie, 75231, Paris Cedex 05, France. claude-jolivalt@enscp.jussieu.fr
Madzak
Catherine
C
Brault
Agathe
A
Caminade
Eliane
E
Malosse
Christian
C
Mougin
Christian
C
D001483:Base Sequence; D001673:Biodegradation, Environmental; D001709:Biotechnology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004785:Environmental Pollutants / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D007700:Kinetics; D042845:Laccase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic; D025062:Yarrowia / Q000235:genetics
D018076:DNA, Complementary; D004271:DNA, Fungal; D004785:Environmental Pollutants; D011994:Recombinant Proteins; D042845:Laccase