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.


INTRODUCTION
-Fungal laccases (EC 1.10.3.2) are involved in lignin transformation,morphogenesis, pathogenesis and fungal virulence, as well as in the oxidation of numerous xenobiotics [1][2][3] -A wide range of their substrates are of environmental interest, and are recognized pollutants of waters and soils -Laccases are potential useful tools to improve the chemical quality of polluted media through bioremediation [1,3] Our objective is the design of laccases with: -a high efficiency in catalysis (high redox potential) -a wider variety of substrates -a pH of activity optimized for natural media Our approach is based on the knowledge of the cavity enclosing a weak reducing substrate of the enzyme, the arylamine 2,5-xylidine, and on the replacement of neighboring interacting residues

3-Strategy of directed mutagenesis
Three mutants have been obtained comprising: -a residue with another negative chain: D206E -a residue with a polar uncharged chain: D206N -a residue with an uncharged chain: D206A -Their catalytic properties are currently studied.

-Sequence alignment of laccases from fungi and higher plants
-Biological roles of laccases from fungi and higher plants appear to be opposite: fungal laccases are involved in ligninolysis whereas enzymes from plants catalyze the initial polymerization of monolignols during lignification -Fungal laccases have a negatively charged residue at the 206 position (Asp, D or Glu, E), whilst those of higher plants have an asparagine (Asn, N) Laccase activity detection on agar plates assay.
Numbers refer to different transformant clones of Y. lipolytica Po1g strain having received p69TVLB plasmid.
Electrophoresis analysis of recombinant laccase IIIb.
[A] Samples were boiled before loading on the gel.Lane 1 -Laccase IIIb from T. versicolor.Lane 2 -Concentrated crude cell free extract from a transformant (clone 4) of Yarrowia lipolytica expressing laccase IIIb.
[B] Samples were not boiled before loading on the gel.Lanes 3 and 6 -Laccase IIIb from T. versicolor.Lanes 4 and 7 -Concentrated crude cell free extract from a transformant (clone 4) of Yarrowia lipolytica expressing laccase IIIb.Lane 5 -Molecular weight markers.Lanes 1 to 5 were stained with Coomassie bleu (R250).Laccase activity was detected in lanes 6 and 7 by coloration with guaïacol.
[B] Analysis of the band corresponding to the activity detected in electrophoresis pattern of crude supernatant from a p69TVLB-derived transformant (clone 4) of Yarrowia lipolytica expressing laccase IIIb.
T. versicolor Y. lipolytica [1] C. Jolivalt, S. Brenon, E. Caminade, C. Mougin, M. Pontié (2000) Immobilization of laccase from Trametes versicolor on a modified PVDF microfiltration membrane: characterization of the grafted support and application in removing a phenylurea pesticide in wastewater, J. Membrane Sci.180:103-113.[2] C. Mougin, F.-D. Boyer, E. Caminade and R. Rama (2000).Cleavage of the diketonitrile derivative of the herbicide isoxaflutole by extracellular fungal oxidases,J.Agric.Food Chem.48:4529-4534.[3] C. Mougin, C. Jolivalt, P. Briozzo, C. Madzak (2003) Fungal laccases: from structure-activity studies to environmental applications.Environ.Chem.Lett.1:145-148.[4] T. Bertrand, C. Jolivalt, P. Briozzo, E. Caminade, N. Joly, C. Madzak and C. Mougin (2002) Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics, Biochemistry 41:7325-7333 -The gene encoding LacIIIb has been cloned in Escherichia coli using the pGEM-T plasmid -It has been sequenced and comprises 1563 bases (sequence accession number AF414109), including a peptide signal -It was then expressed in the yeast Yarrowia lipolytica using the pINA1269 vector (Patent INRA/INA-PG), thus allowing the production of an active form of the recombinant enzyme (transformation of the substrate ABTS to a green oxidized form on solid culture media and oxidation of guaïcol after electrophoretic separation).After tryptic digestion of the transformed Y. lipolytica extract, the mass spectrum contains the most significative ions detected and assigned to T. versicolor laccase peptides.RESULTS: Expression of LacIIIb by Yarrowia lipolytica APPLICATION: Towards the engineering of fungal laccases Two charged or polar residue interact with the amino group of the xylidine: -His (H458) coordinates the copper that functions as the primary electron acceptor -Asp (D206) possesses acido-basic properties that could be responsible for the pH-dependent activity towards : pdb accession number 1KYA) • , copper atoms; • and o, glycosylation sites 2,5-xylidine Figure 1