Cloning, characterization, and heterologous expression of a novel glucosyltransferase gene from sophorolipid-producing Candida bombicola

Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3'- and 5'-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty add substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product. Published by Elsevier B.V.