Transcriptomic analysis-driven identification and transcriptional characterization of Komagataella phaffii promoters from highly transcribed endogenous genes across diverse culture conditions

Abstract

The yeast Komagataella phaffii (formerly Pichia pastoris) is a robust host for recombinant protein production, capable of growing at high cell densities, performing post-translational modifications, and efficiently secreting heterologous proteins. However, gene expression still relies mainly on the methanol-inducible AOX1 promoter (PAOX1) and the constitutive GAPDH promoter (PGAP), highlighting the need for alternative promoters with distinct regulatory properties. This study aimed to identify and characterize promoters from the most highly transcribed endogenous genes across diverse culture conditions as strong promoter candidates to expand the genetic toolbox for this yeast. Seven promoters were identified through RNA-seq analysis from genes showing transcript levels comparable to or higher than those of genes regulated by PAOX1 or PGAP under specific culture conditions. These promoters were further transcriptionally characterized by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) under different carbon sources, growth phases, and temperatures through the measurement of transcript levels of their corresponding genes, revealing distinct transcriptional profiles. Two major clusters were identified: one associated with genes upregulated during the stationary phase and the other with genes upregulated during the exponential phase. These transcriptional profiles provided a basis for the rational design of fed-batch strategies tailored to each promoter when driving heterologous gene expression. The 0208 and HSP12 promoters showed particular potential at low specific growth rates, favoring high recombinant protein titers with reduced biomass accumulation. In silico analyses suggested that 0208 encodes a mitochondrial membrane-associated protein with a possible functional relationship to Hsp12. Overall, this work provides well-characterized K. phaffii strong promoter candidates, supporting future biotechnological applications.