Ebner_2025_Microb.Cell.Fact_24_228

BACKGROUND: Expanding the promoter toolbox of Komagataella phaffii (K. phaffii) in terms of strength and regulatory flexibility can significantly enhance bioprocess efficiency for recombinant protein and metabolite production. The most frequently used promoters are still derived from the methanol utilization (MUT) pathway or genes of the central metabolism. However, the hazards and costs associated with methanol have prompted the search for alternative promoters, including engineered variants. A key limitation remains, many available promoters are still growth-coupled, tying production to biomass accumulation and shortening process duration. Promoters with growth-decoupled expression are therefore highly desirable. In this context, the recently described P(DH) promoter is of interest due to its methanol independence, strong expression, and growth-decoupled regulation. RESULTS: In order to identify potential activator sites of the P(DH), a systematic semi-rational block-scanning approach was used, employing single-base and sequence block walking mutagenesis. The strength of 152 systematically generated variants was characterized using the intracellular reporter eGFP. Variants showed altered strengths and regulatory patterns with fluorescence levels spanning approximately 10-150% of the parental promoter. Subsequently, the best-performing variants were combined to multi-combination variants, which showed activities up to 250% of the parental P(DH). Selected variants were also evaluated with the industrially relevant and secreted enzyme CalB, a lipase from Candida antarctica. Lipase product titers were approx. 2-fold higher than with the parental native promoter sequence and also outperformed the typical state-of-the-art benchmark and constitutive and growth-coupled GAP promoter (P(GAP)). CONCLUSIONS: Creating and characterizing variants of the P(DH) sequence supported the elucidation of the sequence-function relationships of this promoter. In addition, the surprisingly beneficial effects of a synthetic 10 bp sequence stretch opened up opportunities for further engineering of this system and extended the toolbox of efficient vector parts for methanol-free and growth-decoupled protein production with K. phaffii. Those additional promoter sequences will also support the construction of stable engineered strains with a balanced expression of multiple genes, as needed for e.g. multienzyme pathways and synthetic biology applications.