Characterization and functional reconstruction of a highly productive germacrene A synthase from <i>Liriodendron chinense</i>

Plants produce a large array of natural products which play important roles in flavours, fragrances and medicines. However, some high-value plant intermediate metabolites cannot be directly extracted from plants. The tulip tree (Liriodendron chinense) in the Magnoliaceae family is rich in sesquiterpenes. Upon characterizing the functions of 11 Liriodendron chinense terpene synthases, we discovered that LcTPS3 could produce high yields of (+)-germacrene A, which was shown to be a central scaffold in sesquiterpene biosynthesis. This compound can be completely transformed into β-elemene at high temperature, a broad-spectrum antitumor drug widely used in clinical treatment. By expressing LcTPS3 in a precursor-providing Saccharomyces cerevisiae chassis and with the aid of metabolic engineering, the fermentation yield of (+)-germacrene A has been achieved at 14.71 g/L. Site-directed mutagenesis experiments and molecular dynamics simulations revealed that the A280V suppresses the cyclization of substrate by influencing the conformation of the enzyme-substrate. The Y282L facilitates secondary cyclization to produce α-guaiene by shortening the distance between the catalytic residue Y531 and the substrate. These insights underscore the high plasticity of LcTPS3 and suggest that its targeted engineering could unlock the synthesis of a wider array of valuable sesquiterpenes.