Multiplex engineering of Candida tropicalis for efficient production of astaxanthin.

Astaxanthin, a red ketocarotenoid, exhibits strong antioxidant, anti-inflammatory, pigmentation, and immunomodulatory activities. In this study, a high-β-carotene-producing Candida tropicalis strain was engineered for efficient astaxanthin biosynthesis using multiplex engineering strategies. First, promoter engineering was applied to balance the expression of β-carotene ketolase and hydroxylase, and an optimized promoter combination was identified, leading to an astaxanthin titer of 126.7  mg/L. Subsequently, subcellular compartmentalization was implemented by targeting the key enzymes to the cytosol, peroxisomes, and lipid droplets, followed by co-expression across multiple compartments, which raised the astaxanthin titer to 192.0  mg/L. Further enhancement was achieved through endoplasmic reticulum engineering to expand membrane-associated enzyme capacity and lipid engineering to improve product storage, increasing astaxanthin production to 249.8  mg/L. Finally, fermentation optimization significantly improved the shake-flask performance, yielding 320.1  mg/L astaxanthin. In a 5-L bioreactor, the astaxanthin titer reached 3096.2  mg/L, comparable to the highest reported production to date. This work represents the first efficient biosynthesis of astaxanthin in C. tropicalis and provides a novel chassis and technical framework for the industrial production of astaxanthin.