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

BACKGROUND: Specific productivity (qP) 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 qP 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.

Assessment of metabolic variability and diversity present in leaf, peel and pulp tissue of diploid and triploid Musa spp.

Banana (Musa spp.) plants produce many health promoting compounds in leaf, peel and pulp. For a robust metabolic analysis of these tissues, leaf at five developmental stages were compared to assess suitable sampling practices. Results confirmed that the common sampling practise of leaf 3 is applicable for metabolic comparisons. The developed work flow was applied to analyse the metabolite diversity present in 18 different Musa varieties, providing baseline levels of metabolites in leaf, peel and pulp tissue. Correlation analysis was then used to ascertain whether similar trends can be detected in the three plant tissues of the diversity panel. The genome group displayed a dominant role in the composition of the metabolome in all three tissues. This led to the conclusion that a correlation between tissues was only possible within a genome group as the different parental backgrounds caused too great a variation in the metabolomes. It also suggests the metabolome could be used to monitor the interaction/hybridisation of genomes during breeding programmes.