Characterising the metabolic rewiring of extremely slow growing Komagataella phaffii

Retentostat cultivations have enabled investigations into substrate-limited near-zero growth for a number of microbes. Quantitative physiology at these near-zero growth conditions has been widely discussed, yet characterisation of the fluxome is relatively under-reported. We investigated the rewiring of metabolism in the transition of a recombinant protein-producing strain of Komagataella phaffii to glucose-limited near-zero growth rates. We used cultivation data from a 200-fold range of growth rates and comprehensive biomass composition data to integrate growth rate dependent biomass equations, generated using a number of different approaches, into a K. phaffii genome-scale metabolic model. Here, we show that a non-growth-associated maintenance value of 0.65  ${\text{mmol}}_{\mathrm{ATP}}{g}_{\mathrm{CDW}}^{-1}{h}^{-1}$ and a growth-associated maintenance value of 108  ${\text{mmol}}_{\mathrm{ATP}}{g}_{\mathrm{CDW}}^{-1}$ lead to accurate growth rate predictions. In line with its role as energy source, metabolism is rewired to increase the yield of ATP per glucose. This includes a reduction of flux through the pentose phosphate pathway, and a greater utilisation of glycolysis and the TCA cycle. Interestingly, we observed activity of an external, non-proton translocating NADH dehydrogenase in addition to the malate-aspartate shuttle. Regardless of the method used for the generation of biomass equations, a similar, yet different, growth rate dependent rewiring was predicted. As expected, these differences between the different methods were clearer at higher growth rates, where the biomass equation provides a much greater constraint than at slower growth rates. When placed on an increasingly limited glucose diet, the metabolism of K. phaffii adapts, enabling it to continue to drive critical processes sustaining its high viability at near-zero growth rates.