Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (m(S)) and maximum biomass yield on glucose (Y-X/S(max)) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (mu) of 0.025 h(-1) to near-zero specific growth rates (mu < 0.001 h(-1)). At these near-zero specific growth rates, viability remained at least 97%. The value of m(S) at near-zero growth rates was 3.1 +/- 0.1 mg glucose per g biomass and h, which was 3-fold lower than the m(S) estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low mu, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while mu progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes. IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.