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

The constitutive and strong <i>GAP</i> promoter (P_GAP) from the glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>) gene has emerged as a suitable option for protein production in methanol-free <i>Komagataella phaffii</i> (syn. <i>Pichia pastoris</i>) expression systems. Nevertheless, the effect of the transcriptional terminator from the alcohol oxidase 1 gene (T_AOX1) or <i>GAPDH</i> 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 <i>K. phaffii</i> strains, each harboring a single copy of a different combination of regulatory DNA elements (<i>i.e</i>., 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 <i>GAPDH</i> 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 <i>GAPDH</i> 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 <i>GAPDH</i> 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 <i>GAPDH</i> 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 <i>GAPDH</i> 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.