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 [59].
Table 1.
Strains of P. pastoris used and constructed during this work.
Table 2.
Excision cassette structures.
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 (PAOX1), FLP recombinase (FLP), CYC1 terminator (CYC1TT) 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 (PAOX1) 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.
Table 3.
Homologous recombination frequency (HRFb) in wt CBS7435 and ku70 deletion strains.
Table 4.
Selection marker integration.
Table 5.
New shuttle vectors constructed during this study.