A Positive Selection of Methylotrophic Yeast Hansenula Polymorpha Containing URA 3 Auxotrophic Mutation

Methylotrophic yeast Hansenula polymorpha is an interesting eukaryotic system used in biotechnology. Yeast cells have been mutagenized with NTG (N-methyl-N'-nitro-N-nitrosoguanidine) to obtain strains defective in URA genes. The mutated cells were enriched with uracil auxotrophs by 5-FOA (S-Fluoro-orotic acid). From all H. polymorpha uracil auxotrophs 20.5% ura37minus; and ura5− mutant strains H. polymorpha were obtained. URA− mutants were transformed with shuttle vector YepBU36 containing the URA3 gene of S. cerevisiae as a selective marker in yeast, ARS of pL36 (a plasmid design for propagation in H. polymorpha leu2 arg4 strains) for rapid identification of ura3 mutants. 11 ura strains were transformed with YepBU36, 5 of them showed appearance of the uracil prototrofity, identifying them as ura3− mutants.