Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology

Abstract

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts.

Figure 1. A simplified scheme of the glycerol assimilation pathway in yeast.

Glycerol kinase 1 (Gut1p) knocked out in this study is involved in the first step of glycerol metabolism. Gpp1p and Gpp2p: glycerol-3-phosphatases 1 and 2, Gpd1p and Gpd2p: glycerol 3-phosphate dehydrogenases 1 and 2, Dak1 and Dak2p: dihydroxyacetone kinases 1 and 2, Gcy1p: putative NADP⁽⁺⁾ coupled glycerol dehydrogenase, DHA: dihydroxyacetone, DHAP: dihydroxyacetone phosphate, G3P: Glycerol-3-phosphate, “?”: the enzyme responsible for the conversion of DHAP to DHA is not verified. Under aerobic conditions, mitochondrial Gut2p (glycerol-3-phosphate dehydrogenase 2) can convert G3P to DHAP. Figure modified from .

Figure 2. Integration cassette composition and function.

a) KU70 disruption cassette based on the S. cerevisiae FLP recombinase system. On both sides the flipper cassette with AOX1 promoter (P_AOX1), FLP recombinase (FLP), CYC1 terminator (CYC1_TT) and Zeocin™ resistance cassette are surrounded by recombinase target sequences (FRT) and locus specific integration sequences (5′int and 3′int). Cassette components are not drawn to scale. b) After methanol induced (P_AOX1) FLP production and subsequent FRT recognition leading to cassette excision only one FRT (34 bp) is left in the locus in between the 3′ and 5′ integration sequences. c) The lengths of the homologous sequences at 5′ and 3′ ends of the disruption cassettes used to compare the homologous recombination frequencies in wt and ku70 deletion strains varied from 100 bp to 1350 bp in the HIS4 locus. Zeocin™ resistance cassette was placed in between the homologous sequences. Cassette components are not drawn to scale.