title: Development of a transformation system for the flavinogenic yeast Candida famata.
abstract: Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.
authors.affiliation: Institute of Cell Biology, Drahomanov Street 14/16, Lviv 79005, Ukraine.
authors.forename: Andriy A
authors.initials: AA
authors.lastname: Voronovsky
authors.forename: Charles A
authors.initials: CA
authors.lastname: Abbas
authors.forename: Lyubov R
authors.initials: LR
authors.lastname: Fayura
authors.forename: Barbara V
authors.initials: BV
authors.lastname: Kshanovska
authors.forename: Kostyantyn V
authors.initials: KV
authors.lastname: Dmytruk
authors.forename: Kateryna A
authors.initials: KA
authors.lastname: Sybirna
authors.forename: Andriy A
authors.initials: AA
authors.lastname: Sibirny
mesh_terms: D001483:Base Sequence; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics; D006136:GTP Cyclohydrolase / Q000235:genetics; D005800:Genes, Fungal; D005819:Genetic Markers; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D005819:Genetic Markers; C076364:ARS protein, Candida maltosa; D006136:GTP Cyclohydrolase; D007930:Leucine