De novo biosynthesis of zeaxanthin and 3-hydroxy-β-ionone by engineered Yarrowia lipolytica

Abstract

Zeaxanthin and its derivatives have an increasing market demand owing to their strong antioxidant, anti-cancer, and anti-inflammatory properties. Microbial biosynthesis of zeaxanthin and its derivatives serves as a sustainable and green alternative to extraction from plants and chemical synthesis. In this study, we engineered a β-carotene-producing Yarrowia lipolytica strain by employing multi-level strategies for the efficient accumulation of zeaxanthin and 3-hydroxy-β-ionone from its descendant. The engineered Y. lipolytica strain heterologously expressing the zeaxanthin biosynthetic pathway achieved an initial production titer of 82.36 mg L⁻¹ in shake flask cultivation. Through systematic metabolic engineering strategies including gene copy number amplification, carbon flux redirection, pathway optimization, and cofactor balancing, the zeaxanthin titer was significantly enhanced to 729.15 mg L⁻¹. In a controlled fed-batch bioreactor, the strain produced 2.55 g L⁻¹ zeaxanthin, the highest reported titer to date in yeast. Furthermore, through screening carotenoid cleavage dioxygenases (CCDs), we discovered and identified an efficient pathway for the conversion of zeaxanthin to 3-hydroxy-β-ionone. The final engineered strain produced 416.8 mg L⁻¹ 3-hydroxy-β-ionone in shake flasks and 1.66 g L⁻¹ in fed-batch fermentation. This is the first report of de novo biosynthesis and secretion of 3-hydroxy-β-ionone from a recombinant organism. These results show a significant step towards demonstrating the feasibility of scaling up biologically-derived carotenoids and apocarotenoids of high value.