Rewiring methanol metabolism in Komagataella phaffii through implementation and evolution of a heterologous RuMP cycle

Biotechnology holds great potential for sustainable industrial production, with methanol emerging as a promising alternative carbon source due to its availability, cost-effectiveness, and high degree of reduction. Natural methylotrophic microorganisms, such as Komagataella phaffii , are well-suited for methanol-based processes. However, the native xylulose monophosphate (XuMP) cycle in K. phaffii is less energy-efficient than the bacterial ribulose monophosphate (RuMP) cycle, which requires fewer ATP molecules for methanol assimilation. In this study, we introduced the bacterial RuMP cycle as the sole methanol assimilation pathway in K. phaffii . The resulting strain, RuMPi, grew on methanol as its sole carbon and energy source, achieving a specific growth rate of μ = 0.007 ± 0.001 h -1 . Optimization and adaptive laboratory evolution (ALE) improved the strain’s performance, resulting in the final strain, RuMPi_mc_fba-ta_evo, with a growth rate of μ = 0.030 ± 0.001 h -1 . The final strain exhibited biomass yields and methanol uptake rates comparable to the wild type at lower growth rates. This work demonstrates the feasibility of engineering K. phaffii with a heterologous RuMP cycle and provides insights for optimizing methanol utilization in methylotrophic yeasts for industrial applications. • Engineering of Komagataella phaffii to use a heterologous RuMP cycle for methanol assimilation. • RuMP cycle implemented as the sole methanol utilization pathway with an initial growth rate μ = 0.007 ± 0.001 h −1 . • Optimization and ALE improved growth to μ = 0.030 ± 0.001 h −1 (RuMPi_mc_fba-ta_evo). • Final strain showed biomass yields and methanol uptake comparable to WT at low growth rates. • Demonstrates feasibility of RuMP-based methanol utilization and informs industrial strain design.