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• W2777118615 startingPage "1037" @default.
• W2777118615 abstract "Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate PAOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1, and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing PAOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms." @default.
• W2777118615 created "2018-01-05" @default.
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• W2777118615 date "2018-01-29" @default.
• W2777118615 modified "2023-10-14" @default.
• W2777118615 title "Methanol independent induction in <i>Pichia pastoris</i> by simple derepressed overexpression of single transcription factors" @default.
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• W2777118615 doi "https://doi.org/10.1002/bit.26529" @default.
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• W2777118615 hasPublicationYear "2018" @default.
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