CLONING AND DISRUPTION OF THE BETA-ISOPROPYLMALATE DEHYDROGENASE GENE(LEU2) OF PICHIA-STIPITIS WITH URA3 AND RECOVERY OF THE DOUBLE AUXOTROPH

Transformation of Pichia stipitis is required to advance genetic studi es and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (Ps URA3) to disrupt P. stipitis LEU2 in a P. s tipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX2 6) was selected for resistance to 5'-fluoroorotic acid to obtain P. st ipitis FPL-UC7 (ura3-3). A URA3:lacZ ''pop-out'' cassette was construc ted containing PsURA3 flanked by direct repeats from segments of the l acZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyc es cerevisiae LEU2, and a disruption cassette was constructed by repla cing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC 7 (ura3-3) was transformed with the disruption cassette, and a site-sp ecific integrant was identified by selecting for the Leu(-)Ura(+) phen otype. The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mut ants. Excision of the flanked Ps URA3 gene resulted in the Leu(-)Ura(- ) phenotype. The double auxotrophs are stable and can be transformed a t a high frequency by PsLEU2 or PsURA3 carried on autonomous-replicati on-sequence-based plasmids.