# Expanding the substrate scope of a bacterial monoterpene synthase for the production of sesquiterpenoid and diterpenoid products

## TL;DR

Researchers modified a bacterial enzyme to produce larger terpenoids by changing its active site, enabling it to work with longer substrates.

## Contribution

The first successful engineering of a monoterpene synthase to accept diterpene substrates through rational mutations.

## Key findings

- Combinatorial mutations expanded the enzyme's activity to sesquiterpenoids and diterpenoids.
- The triple mutant W58A-F74A-F179A showed highest activity with diterpene substrates.
- The modified enzyme retained its core 1,6 cyclisation cascade across all substrates.

## Abstract

1,8‐cineole synthase from Streptomyces clavuligerus (bCinS) is the only known bacterial terpene synthase that shows exclusive activity towards the monoterpene substrate geranyl diphosphate (GPP; C10). Unlike most plant terpene synthases, bCinS is a high‐fidelity enzyme producing 1,8‐cineole as the predominant product (> 95%). A large number of bulky aromatic residues in the active site steer the carbocationic intermediates down a single path and restrict the conversion of larger prenyl‐diphosphate substrates. Previously, we have shown that a single Phe‐to‐Ala mutation (F74A or F179A) allows bCinS to convert farnesyl diphosphate (FPP; C15) into sesquiterpenoid products, including sesquicineole and germacrene A. Here, we made combinatorial mutations of aromatic active site residues to further expand the substrate scope of bCinS. The F74A‐F179A double variant was not only more active than the wild type but showed increased activity towards FPP over GPP, with sesquicineole and cineole as the main products from these substrates, respectively. Computational active site volume analysis identified an additional residue, W58A, that unlocked activity towards the diterpene substrate geranylgeranyl diphosphate (GGPP; C20), with the W58A‐F74A‐F179A triple variant showing the highest activity on this substrate. Remarkably, these key variants all appear to use the same 1,6 cyclisation cascade to form their main products from GPP, FPP, and GGPP. These results show that even high‐fidelity terpene synthases such as bCinS can be engineered to accept different prenyl‐pyrophosphate substrates without affecting the fundamental reaction cascade.

We have converted the only known true bacterial monoterpene synthase, cineole synthase from Streptomyces clavuligerus (bCinS, C10 substrate), to a highly competent sesquiterpene synthase (C15) with a minimum number of rational mutations. By comparison with diterpene synthases (C20), we were then able to bestow diterpene synthase activity on bCinS. This is the first time the doubling of the substrate length has been reported for a monoterpene synthase.