Engineering Komagataella phaffii to produce lycopene sustainably from glucose or methanol.

Lycopene, a potent carotenoid with high antioxidant capacity and extensive applications, holds significant potential for sustainable production via microbial engineering, particularly with the rising interest in methanol as an ideal non-grain feedstock for a carbon-negative economy. In this study, Komagataella phaffii was systematically engineered to enhance lycopene production using glucose and renewable methanol as alternative carbon sources. Firstly, we demonstrated that the cytoplasmic FPP could penetrate into the peroxisome, and thus achieved the dual-localized lycopene synthesis. Subsequently, the cytoplasmic FPP pool was expanded by dynamically regulating squalene synthase and enhancing the mevalonate pathway, and FPP was redirected to lycopene synthesis via assembling critical enzymes. Furthermore, the synthesis of lycopene from methanol was improved by reprogramming the methanol metabolic pathway. In the above process, we found that the engineered strains would degrade significantly in the process of passing culture. Comparative transcriptomic analysis revealed that nitrogen metabolism genes contributed significantly to strain degeneration, and a gene (PAS_chr2-2_0003) that positively influenced lycopene synthesis was identified. Finally, two strains were successfully engineered: strain zw327, which produced 8.4 g/L lycopene from glucose, and strain zw352, which achieved 10.2 g/L from methanol and glycerol. The latter represents the highest reported titer from methanol to date, underscoring the potential of K. phaffii as a robust one-carbon platform for industrial terpenoid biosynthesis.