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Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris
Author(s) -
Nett Juergen H.,
Gerngross Tillman U.
Publication year - 2003
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1049
Subject(s) - pichia pastoris , biology , plasmid , genetics , gene , multiple cloning site , cloning (programming) , microbiology and biotechnology , computational biology , recombinant dna , expression vector , computer science , programming language
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 lac Z– PpURA3 – lac Z and lac Z– PpURA5–lac Z 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.