Cloning and disruption of the beta-isopropylmalate dehydrogenase gene (LEU2) of Pichia stipitis with URA3 and recovery of the double auxotroph

Applied Microbiology and Biotechnology
P LuThomas W Jeffries

Abstract

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5'-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ "pop-out" cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu- Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu- Ura- phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replic...Continue Reading

Citations

Jan 7, 2005·Applied Microbiology and Biotechnology·Yong-Su JinThomas W Jeffries
Apr 29, 2009·Applied Microbiology and Biotechnology·Cheng ZhongYing-Jin Yuan
Oct 31, 2006·Applied and Environmental Microbiology·Marja IlménMerja Penttilä
Mar 2, 2002·Applied and Environmental Microbiology·Yong-Su JinThomas W Jeffries
Feb 24, 2012·Microbial Cell Factories·Balaji BalagurunathanRajagopalan Srinivasan
Apr 28, 2004·FEMS Microbiology Reviews·U Klinner, B Schäfer
Apr 12, 2015·Bioresource Technology·Liang WeiJianping Wen
Aug 8, 2009·FEMS Yeast Research·Thomas W Jeffries, Jennifer R Headman Van Vleet
Apr 11, 2017·ACS Synthetic Biology·Mingfeng CaoZengyi Shao
Jul 17, 2004·Metabolic Engineering·Yong-Su Jin, Thomas W Jeffries
Jun 20, 2018·Biotechnology Journal·Mingfeng CaoZengyi Shao
Apr 19, 2003·FEMS Yeast Research·Thomas WartmannGotthard Kunze
May 29, 1999·Applied and Environmental Microbiology·J Y Cho, T W Jeffries
Aug 25, 2017·ACS Synthetic Biology·Mingfeng CaoZengyi Shao

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