Determinants of Product Outcome in Two Sesquiterpene Synthases from the Thermotolerant Bacterium <i>Rubrobacter radiotolerans</i>

Rubrobacter radiotolerans nerolidol synthase (NerS) and trans-α-bergamotene synthase (BerS) are among the first terpene synthases (TPSs) discovered from thermotolerant bacteria, and, despite sharing the same substrate, make terpenoid products with different carbon scaffolds. Here, the potential thermostability of NerS and BerS was investigated, and NerS was found to retain activity up to 55 °C. A library of 22 NerS and BerS variants was designed to probe the differing reaction mechanisms of NerS and BerS, including residues putatively involved in substrate sequestration, cation-π stabilisation of reactive intermediates, and shaping of the active site contour. Two BerS variants showed improved in vivo titres vs the WT enzyme, and also yielded different ratios of the related sesquiterpenoids (E)-β-farnesene and trans-α-bergamotene. BerS-L86F was proposed to encourage substrate isomerisation by cation-π stabilisation of the first cationic intermediate, resulting in a greater proportion of trans-α-bergamotene. By contrast, BerS-S82L significantly preferred (E)-β-farnesene formation, attributed to steric blocking of the isomerisation step, consistent with what has been observed in several plant TPSs. Our work highlights the importance of isomerisation as a key determinant of product outcome in TPSs, and shows how a combined computational and experimental approach can characterise TPSs and variants with improved and altered functionality.