Effect of AOX1 and GAP transcriptional terminators on transcript levels of both the heterologous and the GAPDH genes and the extracellular Yp/x in GAP promoter-based Komagataella phaffii strains

The constitutive and strong GAP promoter (P-GAP) from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene has emerged as a suitable option for protein production in methanol-free Komagataella phaffii (syn. Pichia pastoris) expression systems. Nevertheless, the effect of the transcriptional terminator from the alcohol oxidase 1 gene (T-AOX1) or GAPDH gene (T-GAP) within the heterologous gene structure on the transcriptional activity in a P-GAP-based strain and the impact on the extracellular product/biomass yield (Y-p/x) has not yet been fully characterized. In this study, we engineered two K. phaffii strains, each harboring a single copy of a different combination of regulatory DNA elements (i.e., P-GAP-T-AOX1 or P-GAP-T-GAP pairs) within the heterologous gene structure. Moreover, we assessed the impact of the regulatory element combinations, along with the carbon source (glucose or glycerol) and the stage of cell growth, on the transcript levels of the reporter gene and the endogenous GAPDH gene in the yeast cells, as well as the extracellular Y-p/x values. The results indicate that the regulation of transcription for both heterologous and endogenous GAPDH genes, the extracellular Y-p/x values, and translation and/or heterologous protein secretion were influenced by the P-GAP-transcriptional terminator combination, with the carbon source and the stage of cell growth acting as modulatory factors. The highest transcript levels for the heterologous and endogenous GAPDH genes were observed in glucose cultures at a high specific growth rate (0.253 h(-1)). Extracellular Y-p/x values showed an increasing trend as the culture progressed, with the highest values observed in glucose cultures, and in the P-GAP-T-AOX1-based strain. The presence of T-AOX1 or T-GAP within the heterologous gene structure activated distinct gene regulatory elements in each strain, leading to differential modulation of gene regulation for the heterologous and the GAPDH genes, even though both genes were under the control of the same promoter (P-GAP). T-AOX1 induced competitive regulation of transcriptional activity between the two genes, resulting in enhanced transcriptional activity of the GAPDH gene. Moreover, T-AOX1 led to increased mRNA stability and triggered distinct metabolic downregulation mechanisms due to carbon source depletion compared to T-GAP. T-AOX1 enhanced translation and/or heterologous protein secretion activity at a high specific growth rate (0.253 h(-1)), while T-GAP was more effective in enhancing post-transcriptional activity at a low specific growth rate (0.030 h(-1)), regardless of the carbon source. The highest extracellular Y-p/x was obtained with the P-GAP-T-AOX1-based strain when the culture was carried out at a low specific growth rate (0.030 h(-1)) using glucose as the carbon source. The optimization of regulatory elements and growth conditions presents opportunities for enhancing the production of biomolecules of interest.