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 producedin Y. lipolytica, a dimorphic yeast with high secreting capacitiesAbstracts513(Madzak J. Biotechnol 2004; 109: 63), including complex metalloproteinssuch as laccases (Jolivalt AMB 2005; 66: 450; MadzakFEMSYR 2005; in press). Laccases are multicopper oxidases usedin industrial oxidative processes, with potential applications indepollution (Mougin. Environ Chem Lett 2003; 1: 145). The designof recombinant laccases fully adapted to industrial applicationswill be possible using genetic engineering.Y. lipolytica expressionsystem enables high transformation efficiency, as well as controlof both copy number and integration locus of transformants.The successful production of active Trametes versicolor laccase(Jolivalt, 2005) has been a preliminary step towards engineeringthis enzyme for environmental applications. Crystal structure ofT. versicolor laccase (Bertrand, Biochem 2002; 41: 7325) enlightedthe 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 (conservedamong ascomycetes), Asparagine (conserved among plants),or Alanine. Mutated recombinant laccases were expressed inY. lipolytica, using an expression/secretion vector which allowsthe precise targeting of monocopy integration events at a dockingplatform into the recipient strain genome (Madzak JMMB 2000;2: 207). This system reproducibly provides transformants carryinga unique expression cassette, integrated at a precisely known site.We were thus able to analyze the consequences of each mutationon laccase activity on various substrates. This work exemplifiesthe potential of Y. lipolytica expression system for the geneticengineering of complex proteins