Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation

Abstract

An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii. This system uses a uracil auxotrophic mutant of C. boidinii as the host strain in combination with its URA3 gene as the selectable marker. First, the C. boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli. Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance. Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained. All of these strains could be transformed to Ura+ phenotype with a C. boidinii URA3-harboring plasmid linearized within the Candida DNA. The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations. These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C. boidinii.

Physiological role of S-formylglutathione hydrolase in C1 metabolism of the methylotrophic yeast Candida boidinii

“…C. boidinii S2 (Tani et al, 1985) was the origin of the chromosomal DNA and was used as the wild-type strain. C. boidinii TK62 (ura3) (Sakai et al, 1991) was used as the host for transformation. Yeast cultures were grown on the synthetic MI medium described previously (Sakai et al, 1991), with the carbon and nitrogen sources as follows: 1?5 % (v/v) methanol, 2 % (w/v) glucose, 0?76 % (w/v) NH 4 Cl, 0?5 % (w/v) methylamine hydrochloride and 0?5 % (w/v) choline chloride.…”

The Pro-peptide ofStreptomyces mobaraensisTransglutaminase Functions incisand intransto Mediate Efficient Secretion of Active Enzyme from Methylotrophic Yeasts

“…BMMY medium, which consisted of 1.34% (w/v) yeast nitrogen base without amino acids, 0.5% (v/v) methanol and 4 Â 10 À5 % (w/v) biotin in 0.1 M potassium phosphate buffer, pH 6, was routinely used for TGase expression in P. pastoris. BMYPM medium, in which 0.76% NH 4 Cl, 1% (w/v) yeast extract, 1% (w/v) Bacto-peptone, and 1% methanol was added to synthetic MI medium (BM medium) as described previously, 19) was routinely used for TGase expression in C. boidinii. Yeast strains were aerobically grown at 28 C in a shaking flask.…”

“…C. boidinii strain S2 18) was used as the wild-type strain. C. boidinii strains TK62 (ura3) 19) and BUL (ura3, leu2) 20) were also used as hosts for transformation.…”

Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii

“…C. boidinii TK62 (ura3 ) was used as the host for transformation (Sakai et al, Formaldehyde detoxification by alcohol dehydrogenases in C. boidinii 343 1991). Yeast cultures were grown on synthetic MI medium described previously (Sakai et al, 1991), with carbon sources as follows: 1.5% (v/v) methanol, 2% (w/v) glucose, and 1% (v/v) ethanol. Cultivation was performed under aerobic conditions at 28…”