Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum

Abstract Astaxanthin, a versatile C40 carotenoid prized for its applications in food, cosmetics, and health, is a bright red pigment with powerful antioxidant properties. To enhance astaxanthin production in Corynebacterium glutamicum , we employed rational pathway engineering strategies, focused on improving precursor availability and optimizing terminal oxy-functionalized C40 carotenoid biosynthesis. Our efforts resulted in an increased astaxanthin precursor supply with 1.5-fold higher β-carotene production with strain BETA6 (18 mg g −1 CDW). Further advancements in astaxanthin production were made by fine-tuning the expression of the β-carotene hydroxylase gene crtZ and β-carotene ketolase gene crtW , yielding a nearly fivefold increase in astaxanthin (strain ASTA**), with astaxanthin constituting 72% of total carotenoids. ASTA** was successfully transferred to a 2 L fed-batch fermentation with an enhanced titer of 103 mg L −1 astaxanthin with a volumetric productivity of 1.5 mg L −1 h −1 . Based on this strain a pathway expansion was achieved towards glycosylated C40 carotenoids under heterologous expression of the glycosyltransferase gene crtX . To the best of our knowledge, this is the first time astaxanthin-β- d -diglucoside was produced with C. glutamicum achieving high titers of microbial C40 glucosides of 39 mg L −1 . This study showcases the potential of pathway engineering to unlock novel C40 carotenoid variants for diverse industrial applications.