Heterologous Expression and In Vivo Functional Analysis of OsTPS2 in Yeast, Tobacco, and Rice

Casbene-type macrocyclic diterpenes possess a range of biological activities, with casbene and neocembrene serving as key precursor compounds. While there is an abundance of research on casbene synthase, in-depth studies in vivo on neocembrene synthase are relatively scarce. This study presents the first functional validation in vivo of OsTPS2 as a primary neocembrene synthase in rice, as demonstrated through both transient tobacco expression and yeast-induced expression systems. Comparative analysis with the OsTPS28 gene revealed that OsTPS2 produces a higher content of neocembrene. Furthermore, the overexpression of OsTPS2 in rice resulted in an increased neocembrene content within the leaves. Additionally, both OsTPS2 and OsTPS28 were found to induce the synthesis of geranylgeraniol in yeast. Geranylgeraniol exhibits antifungal properties against Rhizoctonia solani and Phomopsis sp., with EC₅₀ values of 56.67 ± 3.78 µg/mL and 89.75 ± 12.75 µg/mL, respectively. Geranylgeraniol also displayed antifungal activity against Alternaria solani (Ell. et Mart.) Jones et Grout, albeit to a lesser extent. The findings of this study provide a scientific foundation for the metabolic engineering of neocembrene and offer novel perspectives on enhancing the nutritional value of rice.