Yarrowia lipolytica, a yeast expression system adapted to the genetic engineering of complex proteins: directed mutagenesis of a fungal laccase
Résumé
Numerous heterologous proteins have been successfully produced
in Y. lipolytica, a dimorphic yeast with high secreting capacities
Abstracts
513
(Madzak J. Biotechnol 2004; 109: 63), including complex metalloproteins
such as laccases (Jolivalt AMB 2005; 66: 450; Madzak
FEMSYR 2005; in press). Laccases are multicopper oxidases used
in industrial oxidative processes, with potential applications in
depollution (Mougin. Environ Chem Lett 2003; 1: 145). The design
of recombinant laccases fully adapted to industrial applications
will be possible using genetic engineering.Y. lipolytica expression
system enables high transformation efficiency, as well as control
of both copy number and integration locus of transformants.
The successful production of active Trametes versicolor laccase
(Jolivalt, 2005) has been a preliminary step towards engineering
this enzyme for environmental applications. Crystal structure of
T. versicolor laccase (Bertrand, Biochem 2002; 41: 7325) enlighted
the interaction of amino acid 206 (Aspartate) with the substrate.
This Aspartate is conserved among laccases from basidiomycetes.
We tested the effects of its replacement by Glutamate (conserved
among ascomycetes), Asparagine (conserved among plants),
or Alanine. Mutated recombinant laccases were expressed in
Y. lipolytica, using an expression/secretion vector which allows
the precise targeting of monocopy integration events at a docking
platform into the recipient strain genome (Madzak JMMB 2000;
2: 207). This system reproducibly provides transformants carrying
a unique expression cassette, integrated at a precisely known site.
We were thus able to analyze the consequences of each mutation
on laccase activity on various substrates. This work exemplifies
the potential of Y. lipolytica expression system for the genetic
engineering of complex proteins.
Domaines
Sciences du Vivant [q-bio]
Origine : Fichiers produits par l'(les) auteur(s)
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