Engineering of R-carotene hydroxylase for enhanced astaxanthin production in Saccharomyces cerevisiae

Astaxanthin, a potent antioxidant widely applied in health supplements, cosmetics, and animal feed, faces production challenges in microbial synthesis due to inefficient R-carotene hydroxylase activity. This study identified rate-limiting enzymes and demonstrated that the expressing phytoene desaturase BtCrtI from Blakeslea trispora in Saccharomyces cerevisiae increased R-carotene production 2.8-fold compared to XdCrtI from Xanthophyllomyces dendrorhous. Integrating of XdCrtYB, BtCrtI, CrtE03M, and tHMG1 achieved 208.7 mg/L R-carotene. However, introducing R-carotene ketolase and R-carotene hydroxylase into this strain unexpectedly increased lycopene accumulation, likely due to feedback inhibition by the product and enzyme imbalance. To address this, we utilized a moderate R-carotene producer YJYast-02 for astaxanthin production. Truncating the N-terminal 39 amino acids of R-carotene hydroxylase from Haematococcus pluvialis improved astaxanthin titer by 37.7 %. Rational engineering of the hydrophobicity of the substrate-binding pocket in R-carotene hydroxylase, specifically through mutations such as N183A, C191A and T213A, further increased astaxanthin titer by 15.8 %, 30.5 %, 36.8 %, respectively. The final engineered strain, YJYast-03(t5t39T213A), expressing t5OBKTM30 and t39OcrtZM1T213A, achieved 26.0 mg/L (3.6 mg/g DCW) astaxanthin. This work pioneers the use of molecular docking for engineering R-carotene hydroxylase, offering potential to significantly enhance catalytic activity and optimize astaxanthin biosynthesis in yeast.