RESUMO
The URA3 gene of Candida utilis encoding orotidine-5'-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5'-phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae.
Assuntos
Candida/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Orotidina-5'-Fosfato Descarboxilase/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/análise , Teste de Complementação Genética , Dados de Sequência Molecular , Plasmídeos/genética , Transformação GenéticaRESUMO
We report here the development of an auxotrophic transformation system for the food yeast Candida utilis. To facilitate molecular studies in Candida utilis, we isolated auxotrophic strains for uracil biosynthesis by the combination of NTG-mutagenesis and 5-fluorotic acid (FOA) selection. The ura-mutation could be functionally complemented by the homologous URA3 gene. We used both, LiAc and electroporation methods to direct insertions at the ura3 locus through homologous recombination.
Assuntos
Candida/genética , Transformação Genética , Southern Blotting , Candida/crescimento & desenvolvimento , Eletroporação , Proteínas Fúngicas/genética , Vetores Genéticos , Lítio , Mutação , Plasmídeos/genética , Uracila/biossínteseRESUMO
A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry.