Isolation and sequence analysis of the orotidine-5'-phosphate decarboxylase gene (URA3) ofCandida utilis. Comparison with the OMP decarboxylase gene family

The nucleotide sequence of the URA3 gene encoding orotidine-5 -phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking. Deduced amino acid sequence showed strong homologies (59-85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains. Very close upstream from the URA3 gene, the 3 -end of a gene encoding a Gea2-like protein was identified. A non-revertible C. lusitaniae ura3 mutant was selected on the basis of 5-fluoroorotic acid resistance. The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism. The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100-200 transformants per µg of DNA. Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus. The GeneBank Accession No. for C. lusitaniae URA3 gene is AF450297.

Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker

François

Chapeland-Leclerc

Villard

et al. 2003

Cloning, characterization and functionality of the orotidine‐5′‐phosphate decarboxylase gene (URA3) of the glycolipid‐producing yeast Candida bombicola

Bogaert

Maeseneire

Schamphelaire

et al. 2007

Candida bombicola is a yeast species known to synthesize glycolipids. Although these glycolipids find several industrial, cosmetic and pharmaceutical applications, very little is known about the genetics of C. bombicola. A basic tool for genetic study and modification is the availability of an efficient transformation and selection system. In order to develop such a system, the URA3 gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes for an enzyme of 262 amino acids and shows high homology with the known orotidine-5 -phosphate decarboxylases of several other yeast species. The functionality of the gene was proved by complementation of a URA3-negative Saccharomyces cerevisiae strain. The GenBank Accession No. of the sequence described in this article is DQ916828.

Development of a transformation and selection system for the glycolipid‐producing yeast Candida bombicola

Bogaert

Maeseneire

Develter³

et al. 2008

Sophorolipids are surface-active compounds synthesized by the non-pathogenic yeast Candida bombicola. Over recent decades much effort has been spent to optimize culture conditions in order to improve the yield and production process. As far as we know, however, hardly any attention has been given to the genetics of the producing yeast strain itself and there are no published results available on the genetic engineering of C. bombicola. Nevertheless, this can be a useful tool for the study of the sophorolipid synthesis pathway and open up perspectives for improved production. A first step is the development of a suitable transformation and selection method. This article describes the creation and selection of an uracil auxotrophic C. bombicola mutant, which can be transformed back to prototrophy with the species' own orotidine 5 -phosphate decarboxylase or URA3 gene. Successful transformation was confirmed by a PCR-based method discriminating between the wild-type and mutated URA3 gene.