Integration of strain and process optimization to increase autotrophic growth of engineered Komagataella phaffii

Synthetic autotrophs are a promising platform for sustainable bioproduction using CO₂ as substrate. The methylotrophic yeast Komagataella phaffii has been engineered to use CO₂ as the sole carbon source by integration of the Calvin-Benson-Bassham (CBB) cycle, based on its native methanol assimilating xylulose monophosphate (XuMP) cycle. Initial growth rates were low, but could be doubled by adaptive laboratory evolution (ALE). Beneficial mutations led to a decrease of CBB cycle reactions, indicating further limitations. During this study, temperature was identified as one of the key process parameters to improve autotrophic growth. For this reason, a new round of adaptive laboratory evolution was performed at the identified optimal cultivation temperature of 25°C, resulting in clones growing up to 50 % faster compared to the control strain. Whole genome re-resequencing followed by reverse engineering helped to identify first key mutations of the evolved strains. In addition, targeted engineering was performed by increasing the copy number of the key gene of the CBB cycle, RuBisCO, which is the bottleneck for carbon fixation. Combining this strategy together with optimal temperature conditions for cultivation, boosted maximum specific growth rates of the autotrophic K. phaffii strain. In comparison to ALE, the targeted engineering still is lagging behind a bit. Starting from the initial condition, growth was improved more than 2.5-fold in this study reaching a maximum of 0.025 h^-1.