Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) P_AOX1 -driven recombinant producer strains with different target gene dosage

Background: The P _AOX1 -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. Results: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two P _AOX1 -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 P _AOX1 -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 q _p ) on Crl1 production with no significant detrimental effects on physiological capabilities. Conclusions: P _AOX1 -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 P _AOX1 -driven gene expression - heterologous genes included -, thus directly influencing the production kinetics of recombinant protein.