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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

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Abstract

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.

Key points

• 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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Funding

The authors received funding from the Department of Biotechnology (Government of India) (grant BT/PR13831/BBE/117/68/2015).

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  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India

    Anamika Singh & Atul Narang

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  1. Anamika Singh
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  2. Atul Narang
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AS and AN conceived and designed the research. AS performed the experiments. AS and AN wrote the manuscript. All the authors read and approved the manuscript.

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Correspondence to Atul Narang.

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Singh, A., Narang, A. 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. Appl Microbiol Biotechnol 104, 7801–7814 (2020). https://doi.org/10.1007/s00253-020-10793-8

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