{Reference Type}: Journal Article {Title}: Identification and functional/structural analyses of large terpene synthases. {Author}: Ueda D;Abe T;Fujihashi M;Sato T; {Journal}: Methods Enzymol {Volume}: 699 {Issue}: 0 {Year}: 2024 {Factor}: 1.682 {DOI}: 10.1016/bs.mie.2024.03.017 {Abstract}: Large terpene synthases (large-TSs) are a new family of TSs. The first large-TS discovered was from Bacillus subtilis (BsuTS), which is involved in the biosynthesis of a C35 sesquarterpene. Large-TSs are the only enzymes that enable the biosynthesis of sesquarterpenes and do not share any sequence homology with canonical Class I and II TSs. Thus, the investigation of large-TSs is promising for expanding the chemical space in the terpene field. In this chapter, we describe the experimental methods used for identifying large-TSs, as well as their functional and structural analyses. Additionally, several enzymes related to the biosynthesis of large-TS substrates have been described.