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  • Methanol-Utilization-Deficient Strains

    Most P. pastoris host strains grow on methanol at the wild-type rate (Mut+, methanol utilization plus phenotype). However, two other types of host strains are available that vary with regard to their ability to utilize methanol because of deletions in one or both AOX genes. Strains with AOX mutations are sometimes better producers of foreign proteins than wild-type strains (Cregg et al. 1987; Tschopp et al. 1987; Chiruvolu et al. 1997). Additionally, these strains require only small amounts of methanol for induction and, therefore, do not have the potential methanol feeding problems that large-scale fermentations of Mut+ strains can experience. KM71 (his4 arg4 aox1Δ::SARG4) is a strain in which AOX1 has been partially deleted and replaced with the S. cerevisiae ARG4 gene (Cregg and Madden 1987). Since the strain must rely on the weaker AOX2 for methanol metabolism, it grows slowly on this carbon source (Muts, methanol utilization slow phenotype). Another strain, MC100–3 (his4 arg4 aox1Δ::SARG4 aox2Δ::Phis4), is deleted for both AOX genes and is totally unable to grow on methanol (Mut–, methanol utilization minus phenotype) (Cregg et al. 1989; Chiruvolu et al. 1997). All of these strains, including the Mut– strain, retain the ability to grow at wild-type rates on other carbon sources and induce expression at high levels from the AOX1 promoter.

    We do not recommend that new P. pastoris users utilize these AOX-mutated strains as a first choice for their expression projects but try them only after they have performed initial and reasonably successful expression experiments with a wild-type host to see if these mutant host are better producers or in more convenient for their specific usage.

    • Chiruvolu V, Cregg JM, Meagher MM (1997) Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations. Enzyme Microb Technol 21: 277–283
    • Cregg JM, Madden KR (1987) Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Biological Research on Industrial Yeasts (Stewart GG, Russell I, Klein RD and Hiebsch RR, Eds),Vol. 2, CRC Press, Boca Raton, FL, pp 1–18
    • Cregg JM,Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholz RG, Madden KR, Kellaris PA, Davis GR, Smiley BL, Cruze J,Torregrossa R,Velicelebi G, Thill GP (1987) High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/Technology 5: 479–485
    • Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9: 1316–1323
    • Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the LacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15: 3859–3876

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