Yarrowia lipolytica, a yeast expression system adapted to the genetic engineering of complex proteins: directed mutagenesis of a fungal laccase

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