Submitted to: Gene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2014
Publication Date: 2/22/2014
Citation: Solaiman, D., Liu, Y., Moreau, R.A., Zerkowski, J.A. 2014. Cloning, characterization, and heterologous expression of a novel glucosyltransferase gene from sophorolipid-producing Candida bombicola. Gene. 540:46-53. DOI: 10.1016/j.gene.2014.02.029.
Interpretive Summary: Sophorolipid (SL) is a natural product produced by a yeast strain called Candida bombicola. Sophorolipid has many important potential uses. It can serve variously as an environmentally friendly surfactant (or detergent), antimicrobial agent, ingredient for formulation of foods, and stabilizer of nanoparticles. Its components (or molecular parts) are also valued in the biomass conversion and the oleochemical arenas. The types of sophorolipid produced by Candida bombicola are limited in variety. One way to increase the variety of sophorolipid is to change the property of the enzyme (i.e., a protein that speeds up a reaction) responsible for making SL. In turn, genetic engineering is a powerful tool to change the property of an enzyme. As a first step in our research effort to increase the variety of SL, we have isolated and studied a gene responsible for making the kind of enzyme used by organism to produce molecules like the SL. This paper describes the technical details of the isolation and study of this gene, which we call “glucosyltransferase.” The outcome of this work provides an important groundwork to proceed with the making of new organisms to produce new SL.
Technical Abstract: 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 yeast glucosyltransferases. An amplified sequence (amplicon) of 700 base-pair was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional 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 Kluyveromyces lactis for expression in an intracellular or secreted form.