The Biosynthesis of the Monoterpene Tricyclene in <i>E. coli</i> through the Appropriate Truncation of Plant Transit Peptides
Meijia Zhao | Shaoheng Bao | Jiajia Liu | Fuli Wang | Ge Yao | Penggang Han | Xiukun Wan | Chang Chen | Hui Jiang | Xinghua Zhang | Wenchao Zhu
Tricyclene, a tricyclic monoterpene naturally occurring in plant essential oils, holds potential for the development of medicinal and fuel applications. In this study, we successfully synthesized tricyclene in <i>E. coli</i> by introducing the heterologous mevalonate (MVA) pathway along with <i>Abies grandis</i> geranyl diphosphate synthase (GPPS) and <i>Nicotiana sylvestris</i> tricyclene synthase (TS) XP_009791411. Initially, the shake-flask fermentation at 30 <sup>◦</sup>C yielded a tricyclene titer of 0.060 mg/L. By increasing the copy number of the TS-coding gene, we achieved a titer of 0.103 mg/L. To further enhance tricyclene production, optimal truncation in the N-terminal region of TS XP_009791411 resulted in an impressive highest titer of 47.671 mg/L, approximately a 794.5-fold improvement compared to its wild-type counterpart. To the best of our knowledge, this is the highest titer of the heterologous synthesis of tricyclene in <i>E. coli</i>. The SDS-PAGE analysis revealed that lowering induction temperature and truncating the random coil N-terminal region effectively improved TS solubility, which was closely associated with tricyclene production levels. Furthermore, by truncating other TSs, the titers of tricyclene were improved to different degrees.
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