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

Claude Jolivalt, Catherine Madzak, Agathe Brault, Eliane Caminade, Christian Malosse, Christian Mougin

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Claude Jolivalt, Catherine Madzak, Agathe Brault, Eliane Caminade, Christian Malosse, et al.. Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. 2nd European Meeting in Oxizymes, Jun 2004, Naples, Italy. 2004. hal-01600125

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<figure><img src="image_1.png" /></figure>
# Expression of laccase IIIb from the white rot fungus *T. versicolor* in the yeast *Yarrowia lipolytica* for environmental applications

CLAUDE JOLIVALT¹, CATHERINE MADZAK²,
AGATHE BRAULT³, ELIANE CAMINADE³, CHRISTIAN MALOSSE³, CHRISTIAN MOGIN³

¹Laboratoire de Synthèse Sélective et Produits Naturels, UMR CNRS 7573, ENSCP, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05,

²Laboratoire de Génétique Moléculaire et Cellulaire, UMR INRA/INA-PG, 78850 Thiverval-Grignon,

³Unité de Phytopharmacie et Médiateurs Chimiques, INRA, route de Saint-Cyr, 78026 Versailles Cedex, France,
email: claude-jolivalt@enscp.jussieu.fr

## 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 [1-3]

- 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:

<figure><img src="image_2.png" /></figure>

- 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

## RESULTS: Expression of LacIIB by *Yarrowia lipolytica*

- The gene encoding LacIIB 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 guaiacol 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.

<figure><img src="image_3.png" /><figcaption>Structure of LacIIB [4]</figcaption></figure>

(atomic coordinates: pdb accession number 1KYA)
* , copper atoms; • and o. glycosylation sites

<figure><img src="image_4.png" /></figure>

Laccase activity detection on agar plates assay.
Numbers refer to different transformant clones of *Y. lipolytica* Po1g strain having received p69TVLB plasmid.

<figure><img src="image_5.png" /></figure>

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 guaiacol.

<figure><img src="image_6.png" /><figcaption>MALDI-TOF spectra</figcaption></figure>

[A] Laccase IIIb from T. versicolor.

[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.

## APPLICATION: Towards the engineering of fungal laccases

### 1- T1 site and neighboring 2,5-xylidine ligand

### 2 - Sequence alignment of laccases from fungi and higher plants

<figure><img src="image_7.png" /></figure>

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 substrates

<figure><img src="image_8.png" /></figure>

- 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)

Fungi: Versicolor = Trametes versicolor; Conireus = Coprinus cinereus; Pccinnaminus = Pycnoporus cinabarinus; Malomycetes = Melanocarpus albomyces

Higher plants: Athaliana = Arabidopsis thaliana; Rivernicifera = Rhus vernicifera; Apeudoplanus = Acer pseudoplatanus

### 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.

### References

[1] C. Jolivalt, S. Brenon, E. Caminade, C. Mougin, M. Ponté (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 isoxafluotide 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