Awakening of the RuMP cycle for synthetic methylotrophy in the thermophile Parageobacillus thermoglucosidasius

Given sustainability and scalability concerns of using sugar feedstocks for microbial bioproduction of bulk chemicals, widening the feedstock range for microbial cell factories is of high interest. Methanol is a one-carbon alcohol that stands out as an alternative feedstock for the bioproduction of chemicals, as it is electron-rich, water-miscible and can be produced from several renewable resources. Bioconversion of methanol into products under thermophilic conditions (>50°C) could be highly advantageous for industrial biotechnology. However, thermophilic natural methylotrophic microorganisms are typically not suitable for this purpose. Hence, we set out to implement synthetic methanol assimilation in the emerging thermophilic model organism Parageobacillus thermoglucosidasius. We engineered P. thermoglucosidasius to be strictly dependent for its growth on methanol assimilation via the core of the highly efficient ribulose monophosphate (RuMP) cycle, while co-assimilating ribose. Surprisingly, this did not require heterologous expression of RuMP enzymes. Instead, by laboratory evolution we awakened latent, native enzyme activities to form the core of the RuMP cycle. We obtained fast methylotrophic growth in which ∼15% of biomass was strictly obtained from methanol. This work paves the way for developing a thermophilic bioproduction platform based on renewable methanol.