New developments in Yarrowia lipolytica yeast expression system and its application to genetic engineering of heterologous proteins

More than 100 heterologous proteins from various phylogenetic origins have been successfully produced in Yarrowia lipolytica yeast expression system, which offers reliable performances in glycosylation, folding and secretion of complex proteins. We present here a short review of the major tools developed in this yeast, with focus on vectors for targeted monocopy integration (YLEX expression kit, Yeastern Biotech Co, Taiwan), a system particularly adapted to genetic engineering of heterologous proteins. In order to examplify the various applications of Yarrowia expression system, a number of current collaborative works on wholecell biocatalysis, genetic engineering of secreted enzymes, and surface-display of heterologous proteins (arming yeasts) will be briefly resumed. Some applications, like high throughput screening, require improving Yarrowia transformation methods for higher efficiency, without sacrificing the high percentage of correctly targeted integrations. For that purpose, we have developed a sensor system for homologous integration, based on E. coli lacZ gene expression, which allowed us to evaluate some chemicals increasing transformation efficiency for maintenance of a high percentage of correct integration. Dithiothreitol was found to be not only more efficient than dimethyl-sulfoxide in increasing transformation efficiency, but also compatible with homologous integration, which was not the case of the latter that promoted out-of-site integration. An optimized transformation protocol using dithiothreitol has been developed, which ensures very high transformation efficienc