Abstract
The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2–3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors thank Glen D. Wheeler for his stylistic suggestions in the preparation of the manuscript. Y.R.-del-P. and A.L.H.-E. thank CONACYT for their fellowship.
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This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico (Grant No CB-2016-286093) and the Universidad Autónoma de Nuevo León, Mexico (Grant Nos. CN1599-21 and CN604-22, PAICYT).
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All authors contributed to the study conception and design. Material preparation and data collection were performed by Y.R.-del-P. and A.L.H.-E. All authors performed the data analysis and interpretation. The first draft of the manuscript was written by Y.R.-del-P., J.M.V.-S. and M.G.-O. All further improvements and the final version of the manuscript were prepared by J.M.V.-S. and M.G.-O. All authors read and approved the final manuscript.
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Robainas-del-Pino, Y., Viader-Salvadó, J.M., Herrera-Estala, A.L. et al. Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator. World J Microbiol Biotechnol 39, 246 (2023). https://doi.org/10.1007/s11274-023-03682-5
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DOI: https://doi.org/10.1007/s11274-023-03682-5