Enhancing 3-hydroxypropionic acid production in Komagataella phaffii via methanol and formate co-feeding

Komagataella phaffii is a promising cell factory that can use CO 2 derived methanol, a sustainable carbon (C 1 ) source, for chemical production. Introducing the ß-alanine biosynthetic pathway alongside a NADP + -dependent formate dehydrogenase (FDH) in K. phaffii enables the production of the platform chemical 3-hydroxypropionic acid (3-HP). Co-feeding of formate and methanol (MeOH) was systematically explored to enhance cellular reducing power and improve 3-HP biosynthesis. Implementing a pulsed formate strategy alongside MeOH resulted in up to a 20.7% increase in 3-HP per gram of MeOH (Yg 3-HP g MeOH -1 ) compared to MeOH alone in shake flask cultivations. This co-feeding strategy likely enhanced NADPH availability through formate oxidation via the introduced NADP⁺-dependent FDH, thereby improving redox balance, as supported by simulation studies based on the K. phaffii genome-scale metabolic model. Similar improvements were demonstrated in repeated batch cultivations at 1-L bioreactor scale, where a formate pulse every 4-hour led to a 25.8% increase in Yg 3-HP g MeOH -1 and a 41% increase in volumetric productivity over the control without formate (only MeOH). In addition to the feeding strategy, pH regulation also played a crucial mechanistic role: strict pH control at 5 inhibited growth, due to the predominance of undissociated formic acid, whereas allowing the pH to rise to 6-7 favoured dissociation and supported higher productivity. These findings elucidate the pH dependent nature of formate assimilation and highlight the potential of coupling MeOH and formate co-utilization with dynamic feeding and pH strategies to enhance bioproduction in K. phaffii .