Evaluation of Ogataea polymorpha DUR31 TPP riboswitch as a tool to downregulate gene expression in the yeast...

Komagataella phaffii (syn. Pichia pastoris) is a methylotrophic yeast widely established as host for recombinant protein production and increasingly used as cell factory for C1-based metabolites. Nevertheless, synthetic biology tools for regulating protein expression in this host remain limited, relying largely on promoters of genes from carbon metabolism. Riboswitches, i.e. mRNA elements that regulate translation or splicing via ligand-dependent folding, offer a complementary layer of regulation, yet remain underused in K. phaffii synthetic biology. In the methylotrophic yeast Ogataea polymorpha, an intron within the DUR31 gene acts as a thiamine pyrophosphate (TPP) riboswitch, downregulating gene expression through alternative splicing, and retaining a premature stop codon in response to exogenous thiamine. We evaluated this TPP riboswitch in K. phaffii as a tool to modulate expression from the glycolytic GAP promoter. Using EGFP and destabilised UBIYΔkGFP* as reporters in combination with flow cytometry, we show that the orthogonal riboswitch is functional in K. phaffii. Like many other described riboswitches, the system exhibits substantial basal (leaky) expression and cannot be considered a tight molecular on/off switch. RT-PCR confirmed the presence of both spliced and unspliced transcripts in the cells regardless of external thiamine supplementation. Unexpectedly, inserting an additional exon upstream of the riboswitch intron abolished detectable downstream protein production, indicating that splicing efficiency is strongly influenced by the local sequence context of the splice sites.