# 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

## TL;DR

This study explores how different transcriptional terminators affect gene expression and protein production in yeast, showing that terminator choice and growth conditions significantly influence outcomes.

## Contribution

The study reveals how TAOX1 and TGAP terminators differentially regulate gene expression and protein secretion in Komagataella phaffii under varying growth conditions.

## Key findings

- TAOX1 enhances mRNA stability and protein secretion at high growth rates, while TGAP improves post-transcriptional activity at low growth rates.
- The highest extracellular Yp/x was achieved with the PGAP-TAOX1 strain at a low growth rate using glucose.
- TAOX1 induces competitive regulation between heterologous and endogenous genes, boosting GAPDH transcription.

## Abstract

The constitutive and strong GAP promoter (PGAP) 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 (TAOX1) or GAPDH gene (TGAP) within the heterologous gene structure on the transcriptional activity in a PGAP-based strain and the impact on the extracellular product/biomass yield (Yp/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., PGAP-TAOX1 or PGAP-TGAP 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 Yp/x values. The results indicate that the regulation of transcription for both heterologous and endogenous GAPDH genes, the extracellular Yp/x values, and translation and/or heterologous protein secretion were influenced by the PGAP-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 Yp/x values showed an increasing trend as the culture progressed, with the highest values observed in glucose cultures, and in the PGAP-TAOX1-based strain. The presence of TAOX1 or TGAP 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 (PGAP). TAOX1 induced competitive regulation of transcriptional activity between the two genes, resulting in enhanced transcriptional activity of the GAPDH gene. Moreover, TAOX1 led to increased mRNA stability and triggered distinct metabolic downregulation mechanisms due to carbon source depletion compared to TGAP. TAOX1 enhanced translation and/or heterologous protein secretion activity at a high specific growth rate (0.253 h−1), while TGAP 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 Yp/x was obtained with the PGAP-TAOX1-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.