Abstract
Lycopene is a high-value carotenoid with broad applications in the food, pharmaceutical, and cosmetic industries. The oleaginous yeast Rhodotorula toruloides naturally synthesizes mixed carotenoids but fails to accumulate lycopene, primarily due to the bifunctional activity of lycopene cyclase/phytoene synthase Car2, which efficiently converts lycopene into downstream cyclic carotenoids. Here, we engineered R. toruloides for efficient lycopene production using a combinatorial strategy that includes pathway blocking, heterologous enzyme screening, precursor enhancement, and fermentation optimization. CRISPR/Cas9-mediated CAR2 knockout abolished endogenous phytoene synthesizing and lycopene cyclizing activities. Heterologous complementation with exogenous phytoene synthase (CrtB) restored phytoene biosynthesis, and the resultant loss of cyclase activity blocked lycopene cyclization, facilitating robust intracellular lycopene accumulation. Screening three heterologous phytoene synthases identified EgCrtB from Euglena gracilis as the superior enzyme. Co-overexpression of endogenous rate-limiting enzymes involved in the mevalonate pathway further boosted precursor supply. Combined with nitrogen-limited cultivation, which synergistically enhanced lycopene accumulation, the final engineered strain achieved a lycopene content of 0.74 mg/g DCW and a titer of 7.24 mg/L. This work verifies that metabolic pathway modification combined with fermentation regulation can effectively drive lycopene synthesis and accumulation in R. toruloides, offering an early-stage proof-of-concept for further advancing lycopene bioproduction in oleaginous yeast.