Abstract Formate is an exciting potential microbial feedstock as it can be derived from CO 2 and electricity. Despite this, limited progress has been made in engineering formatotrophy in yeasts, and no yeasts grow using formate naturally. Here we use metabolic modelling to find two potential formatotrophy pathways in Yarrowia lipolytica . We then use C13 tracer analysis and computationally guided growth experiments to show that wild-type Y. lipolytica possesses strong formate dissimilation and a cyclical C1 pathway with similar architecture to the synthetic serine–threonine cycle, which it uses to co-assimilate formate and glycerol. Messenger RNA sequencing shows that formate exposure results in increased oxidative stress and changes in the tricarboxylic acid cycle, redox and C1 metabolism. Following this, we use model-guided adaptive laboratory evolution to produce a formatotrophic strain of Y. lipolytica using the eukaryotic serine–threonine cycle. We then use further messenger RNA sequencing to show that formatotrophy is supported by changes in adenosine triphosphate and reactive oxygen species metabolism. Subsequently, we engineer nicotinamide adenine dinucleotide phosphate (NADPH) and reactive oxygen species metabolism to create a strain with substantially improved growth. This strain reaches about 10% of the theoretical maximum biomass yield, highlighting its potential for additional engineering approaches. Finally, we show that beta-carotene production from formate is possible in our engineered strain, opening the door to formatotrophic eukaryote bioprocesses.
Newell et al. (Fri,) studied this question.