Efficient production of itaconic acid from the single carbon substrate methanol with engineeredKomagataella phaffii

Background Amidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single carbon feedstocks, derived from CO 2 , represent promising substrates for biotechnological applications. Especially methanol is gaining prominence for bio-production of commodity chemicals. Results In this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus ( cadA , mttA and mfsA ) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g·L -1 after five days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies we further boosted the itaconic acid production reaching titers of 55 g·L -1 after less than five days of methanol feed, whilst increasing the product yield on methanol from 0.06 g·g - 1 to 0.24 g·g -1 . Conclusion Our results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.