Comparative structural engineering of Euglena phytoene synthase reveals functional residues in carotenoid biosynthesis

Carotenoids are high-value natural pigments providing antioxidant benefits in healthcare and various industries. Phytoene synthase (PSY/CrtB), which catalyzes the first committed step in carotenoid biosynthesis, is a key determinant of metabolic flux and a major target for enzyme engineering. Although functional amino acid substitutions have been extensively characterized in plant PSYs, it remains unclear whether these structure–function relationships are conserved across evolutionarily distant microalgae. Here, we tested the transferability of plant-derived functional mutations to phytoene synthase from Euglena gracilis (CrtB), an industrially relevant microalga with secondary plastids. Structural comparison using the crystal structure of Staphylococcus aureus CrtM as a reference revealed a conserved structural framework between tomato PSY2 and CrtB. Guided by this analysis, we introduced 14 site-directed variants, including substitutions and deletions, at ten positions in CrtB corresponding to residues implicated in plant PSY functional variation. We evaluated the variants in an Escherichia coli system for carotenoid (zeaxanthin diglucoside) production. This platform enabled quantitative assessment of total carotenoid levels. We identified EgcrtB variants with enhanced (Y197A, E121H) or reduced (L349F) carotenoid productivity, suggesting that plant-informed residue selection could facilitate the identification of functionally relevant residues in E. gracilis CrtB. These findings demonstrate the potential transferability of PSY structure–function principles across distant lineages and highlight the utility of E. coli –based platforms for scalable enzyme engineering of microalgal carotenoid biosynthesis.