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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.

microbial cell factories

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage