Metabolic engineering of Corynebacterium glutamicum producing four-color C30 carotenoids

Microbial production of carotenoids has predominantly focused on C40 compounds, whereas C30 carotenoids remain underexplored despite their diverse coloration and functional potential. In this study, we engineered Corynebacterium glutamicum as a platform for the selective production of structurally diverse C30 carotenoids. To improve C30 carotenoid biosynthesis, promoter optimization was applied to key pathway genes, additional chromosomal copies of selected sigma factors were introduced, and competing metabolic pathways were sequentially eliminated. Through these engineering strategies, four C30 carotenoids, 4,4′-diaponeurosporene (yellow), 4,4′-diapolycopene (orange), 4,4′-diapolycopendial (violet), and 4,4′-diapolycopendioic acid (pink), were successfully produced with high target-product selectivity. In 5-L fed-batch cultivation, the engineered strains achieved up to 399.4 mg/L (10.11 mg/g DCW) of 4,4′-diaponeurosporene, 309.0 mg/L (8.34 mg/g DCW) of 4,4′-diapolycopene, 218.1 mg/L (6.25 mg/g DCW) of 4,4′-diapolycopendial, and 118.3 mg/L (3.48 mg/g DCW) of 4,4′-diapolycopendioic acid. Nearly complete selectivity was observed for 4,4′-diaponeurosporene, whereas approximately 90%, 83%, and 70% of the total carotenoid corresponded to 4,4′-diapolycopene, 4,4′-diapolycopendial, and 4,4′-diapolycopendioic acid, respectively. These results establish a C. glutamicum platform capable of producing multiple structurally distinct C30 carotenoids with different color profiles and demonstrate its potential as a microbial host for the biosynthesis of rare C30 carotenoids.