Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates

Background Specific productivity (q P ) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (μ). Understanding the factors limiting productivity at extremely low μ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris ) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions. Results By the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h −1 . Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average q P at the end of the retentostat was estimated at 0.019 mg g −1 h −1 . Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival. Conclusions In conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.