Journal
JOURNAL OF BIOTECHNOLOGY
Volume 352, Issue -, Pages 59-67Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2022.05.010
Keywords
GAP promoter; Gene expression levels; Physiological response; Pichia pastoris; Volumetric phytase productivity
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Funding
- Programa de Apoyo a la Investigacion Cientifica y Tecnologica (PAICYT) of the Universidad Autonoma de Nuevo Leon, Mexico [CN595-18]
- Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico [CB-2016-286093]
- CONACYT
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In this study, we constructed a constitutive P. pastoris expression system to extracellularly produce the phytase FTEII. Optimization of the culture conditions led to increased phytase productivity, and the results showed that the regulation of specific growth rate and temperature played a key role in enhancing phytase production, rather than increasing heterologous gene transcription.
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 (Q(p)) 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 Q(p) to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Q(p). The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Q(p), which was correlated with an upregulation of the KAR2 and PSA1-1/MPG1 genes rather than increased heterologous gene transcription.
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