Abstract: Although terpenoids display chemical and structural diversities as well as important biological activities, they are biosynthesized through polymerization of C5 isoprene units according to the “biogenetic isoprene rule.” However, recent observations have identified irregular terpenes, such as C11, C12, C16, and C17, in some microbial species. These irregular terpenes have garnered research interest due to the “Magic Methyl Effect.” These compounds are biosynthesized from the methyl modification of terpene skeletons at a detectable level in Escherichia coli. To explore key factors in the biosynthesis of irregular terpenes, we conducted a screening of C-methyltransferases by UniKP, synthesized methyl-isopentenyl pyrophosphate as a precursor building unit, and polymerized it with C5 isoprene units to generate methyl β-elemene via farnesyl pyrophosphate synthase and germacrene A synthase. Finally, the yield of methyl β-elemene reached 6.98 mg/L, representing a 2.46-fold increase, with a ratio of methylated to native β-elemene increasing by 0.21 through the introduction S-adenosyl-L-homocysteine hydrolase and the optimization of germacrene A synthase. These findings not only expand the chemical diversity of terpenoids but also highlight the evolutionary plasticity of terpene synthases in generating structural novelty.

Key words: C‐methyltransferase, SAM cycle, UniKP, β‐elemene