Abstract
BackgroundThe PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear.ResultsThe influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities.ConclusionsPAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression—heterologous genes included—, thus directly influencing the production kinetics of recombinant protein.
Highlights
The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii)
Effect of increasing CRL1 gene dosage on culture physiological state Increasing the dosage of heterologous genes is known to affect homeostasis in P. pastoris cultivations through restrictions in protein processing [35, 36]
No methanol accumulation was observed under any conditions, but no D values above 0.095 h−1 were used in order to avoid washout
Summary
The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). In the last two decades, Komagataella phaffii, which was formerly known as Pichia pastoris, has emerged as a promising host for recombinant protein production (RPP) [1,2,3,4,5,6]. Promoter sequence analysis has allowed several binding sites for transcription factors (TFs) to be identified Most such TF were previously known and have been related to stress response, glucose repression and oxygen consumption [18]. Such systems do not need methanol to trigger MUT genes because their TF genes have been derepressed by genetic engineering
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