Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5′-phosphate decarboxylase gene ( URA3 ) and uracil auxotrophic mutants

Abstract

A transformation system for Hansenula fabianii J640, a commonly used wastewater treatment yeast, was constructed. As a host cell, a uracil auxotrophic mutant designated as H. fabianii J640 u-1, which was confirmed to have a mutation at the locus of the gene for orotidine-5'-phosphate (OMP) decarboxylase (URA3), was obtained by positive selection using 5-fluoroorotic acid. A plasmid named pHFura3, which includes a 795-bp open-reading frame of the OMP decarboxylase H. fabianii, was obtained by complementation of the Escherichia coli pyrF mutant, pHFura3 could transform H. fabianii J640 u-1 by a non-homologous and frequently multicopy integration into the host genomic DNA.

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

“…The URA3 gene encodes for the enzyme orotidine 5′‐phosphate decarboxylase (OMP DCase), which catalyses the last step in the biosynthesis of pyrimidine (Rose et al , 1984). Transformation systems using the URA3 gene have been developed in many Candida species, such as C. albicans (Kelly et al , 1987), C. tropicalis (Haas et al , 1990), C. boidinii (Sakai et al , 1991), C. maltosa (Ohkuma et al , 1993), C. glabrata (Zhou et al , 1994), C. utilis (Rodriguez et al , 1998), and related teleomorphs such as Pichia ( Kodamaea ) ohmeri (Piredda and Gaillardin, 1994), P. fabianii (Kato et al , 1997) and Yarrowia lipolytica (Juretzek et al , 2001). Depending on its expression or non‐expression, the URA3 gene allows two different selection systems (Boeke et al , 1984), which have made it attractive for the development of the URA3 ‐blaster disruption cassette, first in Saccharomyces cerevisiae (Alani et al , 1987) and C. albicans (Fonzi and Irwin, 1993), then in many yeast species.…”

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

“…All the S. cerevisiae strains used (Table 1 ) were kindly provided by Dr. N. Ogawa at Stanford University. Wastewater treatment yeast strains H. fabianii J640 (Kato et al 1997 ), H. anomala J224-1 (Saito et al 1990 ), Cryptococcus sp. S-2 (Iefuji et al 1994a ), and Geotrichum sp.…”

“…We therefore attempted to select wastewater treatment yeast strains whose acid phosphatase activities are regulated by phosphorus concentration as they are in S. cerevisiae . Two yeast strains, Hansenula fabianii J640 (Kato et al 1997 ) and Hansenula anomala J224-1 (Saito et al 1990 ) were selected as candidates for creating mutants that efficiently remove phosphorus. Both strains were originally isolated in our laboratory as wastewater treatment yeasts.…”

Breeding of a new wastewater treatment yeast by genetic engineering

“…We previously constructed an expression system based on this strain (Kato et al 1997). A uracil auxotrophic mutant of H. fabianii J640, named H. fabianii J640 u-1, lacking orotidin-5'-phosphate decarboxylase, was obtained.…”

“…We constructed a plasmid, pHFura3, that contains the gene encoding orotidine-5'-phosphate decarboxylase of H. fabianii J640. In the previous study (Kato et al 1997), by employing H. fabianii J640 u-1 as a host strain and pHFura3 as a vector plasmid, we constructed a transformation system of H. fabianii J640.…”