DESIGN, TOTAL SYNTHESIS, AND FUNCTIONAL OVEREXPRESSION OF THE CANDIDA-RUGOSA LIP1 GENE CODING FOR A MAJOR INDUSTRIAL LIPASE

The dimorphic yeast Candida rugosa has an unusual codon usage that ham pers the functional expression of genes derived from this yeast in a c onventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isofo rms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, th e lip1 gene was systematically modified by site-directed mutagenesis t o gain functional expression in Saccharomyces cerevisiae. As alternati ve approach, the gene (1647 bp) was completely synthesized with an opt imized nucleotide sequence in terms of heterologous expression in yeas t and simplified genetic manipulation. The synthetic gene was function ally expressed in both hosts S. cerevisiae and Pichia pastoris, and th e effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the syntheti c gene was functionally overexpressed, allowing for the first time to produce recombinant Lip1 of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombina nt lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.