title: The yeast Yarrowia lipolytica as a generic tool for molecular evolution of enzymes.
abstract: It has been 20 years since strains of the yeast Yarrowia lipolytica were developed for the expression of recombinant proteins as alternative host to the commonly used yeasts, Pichia pastoris and Saccharomyces cerevisiae. Recently, a new strain, JMY1212, was engineered for protein evolution. With this new strain, a very high reproducibility in protein expression level was demonstrated, thus enabling its use for both rational and directed evolution strategies. Indeed, the coefficient of variation was shown to be of 10.7% for the whole process when all the steps are optimized, i.e. transformation of this strain with the gene of interest, cell growth, and protein production under oleic acid induction, and until activity screening assay. The object of this article is to summarize the fruit of these works and show the interest of Y. lipolytica strain JMY1212 for molecular evolution of enzymes, for both rational and directed evolution strategy. Lipase Lip2 from Y. lipolytica is taken here as an example to describe both strategies of molecular evolution. In these two methods, a first step of PCR creates either one targeted (rational design) or various random mutations (directed evolution), and is followed by the incorporation of the expression cassette in the genome of Y. lipolytica. An easy and direct comparison of variant properties is then allowed thanks to the extracellular and reproducible production of variants.
authors.affiliation: Equipe de Catalyse et Ingénierie Moléculaire Enzymatique, Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, INSA, UPS, INP, LISBP, Toulouse, France. sophie.duquesne@insa-toulouse.fr
authors.forename: Sophie
authors.initials: S
authors.lastname: Duquesne
authors.forename: Florence
authors.initials: F
authors.lastname: Bordes
authors.forename: Franck
authors.initials: F
authors.lastname: Fudalej
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.forename: Alain
authors.initials: A
authors.lastname: Marty
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016681:Genome, Fungal; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019301:Oleic Acid / Q000378:metabolism; D010957:Plasmids; D015203:Reproducibility of Results; D014170:Transformation, Genetic; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D019301:Oleic Acid; D008049:Lipase