Pathway reconstruction of Corynebacterium glutamicum for C30 carotenoid 4,4'-diaponeurosporene production.

4,4′-Diaponeurosporene (4,4′-DIA) is a rare C30 carotenoid with strong antioxidant and immune-enhancing properties. However, microbial production of 4,4′-DIA has been limited by low titers and the simultaneous formation of undesired carotenoid byproducts. In this study, Corynebacterium glutamicum was rationally engineered to enable selective 4,4′-DIA biosynthesis through pathway reconstruction. To redirect metabolic flux toward C30 carotenoids, the native C50 carotenoid biosynthetic gene cluster was deleted and the precursor supply was reconfigured by replacing the core C20 derivative pathway with ispA to produce C15 precursor. A gene module enabling high-level and selective production of 4,4′-DIA was established through the combinatorial expression of crtM and crtN in a constitutive vector system. Pathway analysis predicted crtN to be a bottleneck in 4,4′-DIA biosynthesis, and its expression was strengthened through promoter introduction, leading to improved production level. Deletion of poxB and ldhA combined with ribK overexpression enhanced both 4,4′-DIA biosynthetic gene expression and production, likely due to improvement carbon flux and cofactor supply. The final engineered strain was successfully transferred to a 5-L fed-batch fermentation with a 4,4′-DIA titer of 656.3 mg/L without any inducer. This work demonstrates the selective production of the rare and non-commercial C30 carotenoid 4,4′-DIA in C. glutamicum, establishing it as a robust platform for diversified carotenoid biosynthesis.