Engineering low pH-tolerant Komagataella phaffii for industrial-ready itaconic acid production via methanol biorefinery

Methanol biorefinery stands as a cornerstone strategy in the pursuit of sustainable biochemical production from non-food feedstocks. Despite its vast potential, bioconverting methanol into high titer biochemical production remains a formidable challenge. In this study, we have successfully engineered Komagataella phaffii to efficiently produce itaconic acid (IA), one of the top 12 building block chemicals, utilizing methanol as the sole carbon source. Our approach involved strategic metabolic engineering interventions--such as enhancing the supply of cis-aconitate and citrate precursors, minimizing isocitrate diversion, facilitating efficient formaldehyde assimilation, and optimizing IA export mechanisms--culminating in an unprecedented IA titer of 103.8 g/L in a 5-L bioreactor through fed-batch fermentation at pH 5.5. Remarkably, even at a reduced pH of 3.5, the strain still yielded 50.4 g/L of IA, highlighting the industrial viability of methanol biorefinery. This systematic work lays a robust foundation for high-titer production via methanol biorefinery, offering valuable insights for future research and applications.