Background: Precision fermentation offers a sustainable alternative production route for proteins but still suffers from moderate productivities and low yields. Especially compared to biomass yields, recombinant protein yields on substrate are very low. Uncoupling recombinant protein production from growth would allow higher product yields, but requires that productivity is maintained. So far, two-phase production processes mostly rely on inducers to activate recombinant protein production after an initial growth phase, e.g., a change in carbon source. On large scale, specific growth rates can be controlled by nutrient availability, and we aim to use this as trigger to uncouple recombinant protein production from growth.

Results: We investigated the correlation between low specific growth rates (0.02 h^{- 1} < µ < 0.1 h^{- 1}) and specific recombinant protein production rates, both for intracellularly accumulating and secreted proteins. By comparing two differently regulated promoters, the strong, constitutive P_TEF1 and stress-induced P_HSP12, we show that recombinant protein production rates and yields in Saccharomyces cerevisiae can be partially uncoupled from growth. The optimal strategy thereby differs for intracellular and secreted production. The P_HSP12 resulted in increased product yields of intracellular protein at very low growth rates, including a 10-fold increase in intracellular protein titer, while titers remained virtually constant for the benchmark P_TEF1. The P_TEF1 on the other hand led to increased protein secretion rates and efficiencies at lower specific growth rates cumulating in higher extracellular protein titers.

Conclusion: Our results demonstrate that promoter selection plays a critical role in production performance under slow growing conditions. Moreover, it highlights that optimising intracellular and extracellular recombinant protein production requires distinct, strategy-specific approaches.