Production of β-carotene from methanol via rDNA-targeted multi-gene integration and selection with increasing antibiotic concentrations in Komagataella phaffii.

Methanol, a C1 compound that can be produced from CO₂ and waste biomass, has attracted increasing attention as a renewable feedstock alternative to petroleum. In recent years, Komagataella phaffii has emerged as a promising microbial host for the methanol-based biosynthesis of value-added compounds through metabolic engineering. Among these compounds, β-carotene-a precursor of vitamin A with antioxidant properties-is widely used in the food, pharmaceutical, and cosmetic industries. In this study, we aimed to develop a high β-carotene-producing strain of K. phaffii using methanol as the sole carbon source. To this end, gene fragments encoding three key genes involved in β-carotene biosynthesis-geranylgeranyl pyrophosphate synthase (crtE), phytoene desaturase (crtI), and lycopene cyclase/phytoene synthase (crtYB)-from Xanthophyllomyces dendrorhous were introduced into K. phaffii using an rDNA-targeted integration strategy. Following transformation, we performed iterative phenotypic screening coupled with stepwise zeocin selection to enrich candidate isolates with higher A450-derived carotenoid specific productivity. The resulting strains, GS115/crt/Z2/01 and GS115/crt/Z4/03, achieved β-carotene titers of 101.8 and 63.6 mg/L, and specific productivities of 14.4 and 17.0 mg/g dry cell weight, respectively. Our results demonstrate the effectiveness of rDNA-targeted multi-gene integration combined with A450-derived phenotypic screening and stepwise zeocin selection as a strategy to identify candidate strains with enhanced target-compound production in K. phaffii. This approach shows promise for expanding the range of valuable chemicals sustainably produced using methanol-based bioprocesses.