The Mut+ Strain of Komagataella Phaffii (pichia Pastoris) Expresses PAOX1 5 and 10 Times Faster Than Muts and Mut− Strains: Evidence That Formaldehyde Or/and Formate Are True Inducers of PAOX1
Applied microbiology and biotechnology(2020)
摘要
The methylotrophic yeast Komagataella phaffii is among the most popular hosts for recombinant protein synthesis. Most recombinant proteins have been expressed in the wild-type Mut+ host strain from the methanol-inducible alcohol oxidase (AOX) promoter PAOX1. Since methanol metabolism has undesirable consequences, two additional host strains, Muts (Δaox1) and Mut− (Δaox1Δaox2), were introduced which consume less methanol and reportedly also express recombinant protein better than Mut+. Both results follow from a simple model based on two widespread assumptions, namely methanol is transported by diffusion and the sole inducer of PAOX1. To test this model, we studied 14C-methanol uptake in the Mut− strain and β-galactosidase expression in all three strains. We confirmed that methanol is transported by diffusion, but in contrast to the literature, Mut+ expressed β-galactosidase 5- and 10-fold faster than Muts and Mut−. These results imply that methanol is not the sole inducer of PAOX1—metabolites downstream of methanol also induce PAOX1. We find that formate or/and formaldehyde are probably true inducers since both induce PAOX1 expression in Mut− which cannot synthesize intracellular methanol from formate or formaldehyde. Formate offers a promising substitute for methanol since it does not appear to suffer from the deficiencies that afflict methanol. • This is the first study to systematically compare all three Mut phenotypes as host strains. • Mut+ strain expresses 5- and 10-fold faster than Muts and Mut− strains. • Methanol is transported by diffusion in Komagataella phaffii. • Formate and formaldehyde are true and strong inducers of PAOX1 expression
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关键词
Komagataella phaffii (Pichia pastoris),Mut phenotype,AOX induction,Methanol,Methanol uptake,Formate,Inducer
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