| Summary: | <i>Komagataella phaffii</i> yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast <i>K. phaffii</i> was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when <i>K. phaffii</i> was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by <i>K. phaffii</i> even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of <i>AOX1</i> gene promoter with proline can be partially reversed by the deletion of the <i>KpPUT4.2</i> gene.
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