Engineered β-Carotene Hydroxylase with Excellent Thermostability Promotes Zeaxanthin Production in Yeast

Zeaxanthin, as an important natural pigment and nutrient, is applied in food and cosmetics industries. Zeaxanthin is converted from β-carotenoid by the β-carotene hydroxylase (CrtZ). Limited by the thermostability of CrtZ, zeaxanthin and its derivatives tend to be fermented in Saccharomyces cerevisiae at low temperatures. In this study, CrtZ mutants with improved thermostability and catalytic efficiency were designed via the position-specific scoring matrix (PSSM), and the beneficial mutants were verified in vitro. Moreover, molecular dynamics simulations revealed the thermostability mechanisms of the mutants. Subsequently, CrtZ mutant M83L was introduced into the S. cerevisiae chassis, and the zeaxanthin titer increased by 121.2% compared with that of the CrtZ, reaching 156.8 mg/L in shake flask fermentation at 30 °C. Furthermore, the oxidoreductase RFNR/FD3 system was introduced to match the overexpressed M83L, and the zeaxanthin titer further increased by 138.9% (reaching 374.6 mg/L). Ultimately, 814.6 mg/L zeaxanthin was produced in S. cerevisiae in 5.0 L fed-batch fermentation at 30 °C, which is the highest reported titer in S. cerevisiae. This study not only provides a useful strategy to increase the thermostability of key enzymes but also describes an efficient platform for the biosynthesis of zeaxanthin and its high-value derivatives.