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

Komagataella phaffii is a promising cell factory that can use CO₂ derived methanol, a sustainable carbon (C₁) 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.