Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris.

A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544.