Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

Abstract

Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence‐function relationship within the TS family, making product‐outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high‐throughput screening, and machine‐learning that will allow more predictive engineering of these fascinating enzymes in the near future.

Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases

“…This suggests that the role of Ala301 is similar to Phe295 in bLinS, its precise conformation is essential for efficient formation of cineole, and likely plays an important role in the high fidelity observed for bCinS. This is in contrast to what is observed for many other monoterpene synthases, especially those from plant sources, which are generally promiscuous and show a high degree of functional plasticity [13b,21,27,33] …”

“…This is in contrast to what is observed for many other monoterpene synthases, especially those from plant sources, which are generally promiscuous and show a high degree of functional plasticity. [ 13b , 21 , 27 , 33 ]…”

“…Due to the relatively inert active sites employed by many terpene synthases to ‘manage’ the reactive carbocations, the molecular determinants for carbocation control and cyclisation are often unknown. [13b] Previously, we have demonstrated the importance of Asn305 in controlling the final stages of the reaction cascade catalysed by bCinS, [15] but so far little is known about carbocation control to prevent branching in the early stages of the reaction cascade leading to the cyclic α‐terpinyl intermediate.…”

Facile Production of (+)-Aristolochene and (+)-Bicyclogermacrene in Escherichia coli Using Newly Discovered Sesquiterpene Synthases from Penicillium expansum

“…Protein engineering is often used to increase the enzyme activity. However, it is difficult to improve activity of TSs by protein engineering as a result of a remarkable lack of sequence–function relationship knowledge within the TS family and the fact that products of TSs are easily changed by some mutations in the active pockets . Therefore, discovering new TSs with a high activity will greatly promote the large-scale production of bioactive terpenoids.…”

Computer-Informed Engineering: A New Class I Sesquiterpene Synthase JeSTS4 for the Synthesis of an Unusual C10-(S)-Bicyclogermacrene

“…However, the catalytic efficiency of TPSs is generally low, which becomes the bottleneck for using them as biocatalysts for large-scale production of terpenoids in the industry. Efforts towards optimizing the enzyme activities of TPSs to enhance the yields of diverse products such as mono-, sesqui-, or diterpenoids have been reported. − …”