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

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 orotatephosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycinresistance 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.

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

“…P. pastoris strain YJN165 (ura5) (Nett and Gerngross, 2003) was used for construction of yeast strains. PCR reactions were performed according to suppliers' recommendations, using ExTaq (TaKaRa), Taq Poly (Promega) or Pfu Turbo  (Stratagene).…”

“…All disruption vectors were derived from plasmid pJN653 (Figure 1), which was constructed in a similar way to the previously published pJN267 (Nett and Gerngross, 2003). The only differences Table 3.…”

“…It is known that the heterologous ScURA3 marker does not fully complement the ura3 strains of P. pastoris (Lin Cereghino et al, 2001;Nett and Gerngross, 2003), therefore we replaced the auxotrophic marker cassette with the PpURA5-blaster cassette (Nett and Gerngross, 2003) in the following way: plasmid pJN665 was cut with SwaI and Bgl II, and plasmids pJN666, pJN667, pJN668, pJN669, pJN670 and pJN671 were cut with SwaI and XhoI to release the ScURA3 marker cassette. The isolated plasmid backbones were then made blunt, using T4 DNA ploymerase, and a blunt DNA fragment containing the PpURA5-blaster cassette that had been obtained by digestion of pJN396 (Nett and Gerngross, 2003) with EcoRI and SphI and treatment with T4 DNA polymerase was ligated in. This resulted in the PpURA5-blaster marked knockout plasmids pJN675 (ARG1 ), pJN676 (ARG2 ), pJN677 (ARG3 ), pJN678 (HIS1 ), pJN679 (HIS2 ), pJN680 (HIS5 ) and pJN681 (HIS6 ).…”

“…However, for completion of the project, several more markers are needed. We have also recently reported the cloning and use as an auxotrophic marker of the PpURA5 gene (Nett and Gerngross, 2003), which, like the PpURA3 gene, can be used repeatedly after counterselection on medium containing 5-fluoroorotic acid (5FOA). However, in contrast to Saccharomyces cerevisiae ura auxotrophic strains, the P. pastoris ura3 and ura5 auxotrophs have significantly reduced growth rates.…”

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

“…Several vectors are available which have auxotrophic markers for elements of the arginine, adenine, histidine, uracil and methionine biosynthetic pathways in P. pastoris (Lin Cereghino et al, 2001;Nett and Gerngross, 2003;Nett et al, 2005;Thor et al, 2005). However, these vectors require sequential transformation for the selection of more than one expression cassette and concomitant expression of several heterologous genes.…”