Semi-rational design and engineering of chicory-derived germacrene A synthase for enhanced (−)-β-elemene production in Saccharomyces cerevisiae

In this study, we applied a semi-rational engineering strategy to improve the catalytic performance and operational stability of germacrene A synthase from Cichorium intybus (CiGASlo), a key biocatalyst for stereochemically defined (–)-β-elemene biosynthesis. An AlphaFold2-predicted model was first validated by confidence-score analysis, stereochemical-quality assessment, and structural comparison with a related plant germacrene A synthase. Molecular docking was then used to define substrate-proximal residues around farnesyl pyrophosphate (FPP), while alanine scanning and HotSpot Wizard 3.0 were used as stability- and mutational-tolerance filters rather than as direct predictors of catalytic activity. Experimental screening of a focused mutation library identified several variants with improved activity, including D337H, L415W, D477H, E485Y, R488A, W305A, and the double variant W305A/L415W. Experimental screening identified W305A/L415W as the variant with the highest specific activity under the fixed assay conditions (0.95 ± 0.03 U·mg − 1 ; 69.6% above wild type). Subsequent characterization showed that L415W, rather than the double variant, had the more favorable K m, k cat/ K m, and numerical thermal profile. Expression of W305A/L415W in the engineered yeast strain produced 5.44 g·L − 1 (−)-β-elemene under continuous feeding, demonstrating feasibility of this strain configuration without establishing superiority over an L415W-containing strain. This work provides a combined enzyme-engineering and fermentation-validation strategy for improving plant terpene synthases in microbial cell factories.