Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity

• Published in: Journal of biotechnology 2022-06, Vol.352, p.59-67
• Main Authors: Herrera-Estala, Ana Lucía, Fuentes-Garibay, José Antonio, Guerrero-Olazarán, Martha, Viader-Salvadó, José María
• Format: Article
• Language: English
• Subjects: GAP promoter; Gene expression levels; Physiological response; Pichia pastoris; Volumetric phytase productivity
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2022.05.010

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1–1/MPG1 genes rather than increased heterologous gene transcription. •A low specific growth rate and temperature increases extracellular volumetric productivity.•The increased extracellular volumetric productivity is correlated with an upregulation of KAR2 and PSA1-1 genes.•The increased secretion process was caused by an increase in the Kar2p/BiP chaperone and cell viability.