Sh ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis

Pichia stipitis is widely studied for its capacity to ferment d-xylose to ethanol. Strain improvement has been facilitated by recent completion of the P. stipitis genome. P. stipitis uses CUG to code for serine rather than leucine, as is the case for the universal genetic code thereby limiting the availability of heterologous drug resistance markers for transformation. Development of a modified selectable marker for resistance to bleomycin (Sh ble) and efficient excision of the marker after integration (loxP/Cre) should facilitate functional genomics and metabolic engineering in this yeast. The Sh ble marker did not code for an active protein in P. stipitis until four CUG codons were mutagenized to TTG, which is properly translated as leucine in yeasts that use the alternative yeast nuclear genetic code. The 18 CTG codons in Cre were mutagenized in a similar manner and the system was used to delete XYL2. The resulting xyl2Δ mutant did not use xylose as a carbon source.