| Autor: |
Lu, P., Davis, B. P., Hendrick, J., Jeffries, T. W. |
| Zdroj: |
Applied Microbiology & Biotechnology; Feb1998, Vol. 49 Issue 2, p141-146, 6p |
| Abstrakt: |
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-replication-sequence-based plasmids. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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