Pichia pastoris protease-deficient and auxotrophic strains generated by a novel, user-friendly vector toolbox for gene deletion

Abstract

Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.

Keywords: P. pastoris; auxotrophic strains; gene disruption; knockout plasmids; proteases-deficient strains.

Figure 1

Knockout plasmids harbouring different Pichia pastoris selection markers constructed and applied during this study. (a) pPpKC1. (b) pPpKC2–4. Indicated are the unique restriction sites NdeI and PstI for marker exchange

Figure 2

Schematic representation of the experimental procedure for gene deletion and its confirmation. (a) By performing two PCR reactions, the 3′‐ and 5′‐homology regions of the respective target gene were amplified separately. The two PCR products were joined by oe‐PCR, creating a unique restriction site (SmaI) for subsequent linearization. (b) The SfiI‐restricted 3′‐ and 5′‐homology regions were cloned into the knockout vector. The final knockout vector was linearized using SmaI prior to transformation into Pichia pastoris. (c) Homologous recombination replaced the target open reading frame (ORF) with the linear knockout cassette. The correct integration was verified by amplifying region I containing the 5′‐homology (primer pair P5/PAox1SeqR) and region II containing the 3′‐homology (primer pair PucSeqF/P6). Clones with positive results for both PCRs were selected for marker recycling. The removal of the integrated marker cassette was verified by amplification of regions III and IV. FRT, flippase recombination target site; P1–P8, primers

Figure 3

Verification of successful gene knockout by PCR analysis. The results for wild type and knockout strains are shown using knockout specific primer pairs P5/P6 (PCR III of Figure 2). Marker, GeneRuler DNA Ladder Mix (Thermo Scientific); WT, wild type CBS 7435

Figure 4

Growth behaviour of Pichia pastoris auxotrophic strains. Cells were cultivated in 96‐well deep‐well plates containing 250‐μl BMD media supplemented with the respective amino acids at 28°C, 320 rpm and 80% humidity for 24 hr. Equal number of cells (OD₆₀₀ = 0.5) were pinned onto BMD/BYPD plates (supplemented with or without respective amino acids for selective growth), and plates were incubated for 3–4 days at 28°C

Figure 5

Growth rate analysis of Pichia pastoris wild type, pro3Δ, and pha2Δ strains. The strains were cultivated in 300‐ml baffled shake flasks at 28°C and 120 rpm in media as indicated, and OD₆₀₀ was measured regularly. Experiments were performed in triplicates

Figure 6

Phenotype of CBS 7435 pha2Δ (Pp7029). Growth on (a) buffered minimal media and (b) buffered minimal media supplemented with phenylalanine (150 μg/ml) after ~10 days of incubation at 28°C

Figure 7

CBS 7435 his4Δ kex2Δ (Pp6910) shows growth defect on YPD plates. Knockout strains were grown to OD₆₀₀ of 1 in YPD, diluted 10‐fold, and spotted on YPD plates. Depicted are results for knockout strains yps7Δ, kex1Δ, kex2Δ, prtPΔ, ctseΔ, kpx8Δ, and kpx4Δ. All other protease knockout strains behaved similar as the wild type