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

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Abstract
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. Copyright © 2003 John Wiley & Sons, Ltd.