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Protein production in  Pichia pastoris  is often based on the methanol‐inducible P  AOX1   promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P  GTH1   promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P  GTH1  . Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P  GTH1   variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P  GTH1   variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P  AOX1   strain in terms of titer and process performance.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris