High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris

Abstract

Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media ( a w >0.9). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae , was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene ( AOX1 ). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 g l −1 (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.