Developing global engineering and synthetic biology tools for process optimisation with Geobacillus spp

There are over 50000 natural terpene structures identified including terpenes such as taxol and artemisinin, from plants, fungi and bacteria. The key enzyme in their production is the terpene synthase/cyclase (TPS). This enzyme's function is the cyclisation step of substrates, such as fernesyl pyrophosphate, to produce the many cyclic terpenes. One of the difficulties of characterising TPSs is that they have very low sequence homologies, sometimes less than 20%, which means that the functions cannot be easily determined from the primary sequence. This make it very difficult to model uncharacterised TPSs from known TPS structures. Although having similar mechanisms for producing the cyclic terpene products. TPSs have different specificities for substrates and products. Many TPSs produce multiple terpenes, for example y-Humulene synthase, which can produce 52 different terpenes. One of the aims of this project is to use structure-guided design of a thermophilic TPS to help to help understand the relationship between the primary and tertiary sequences of a TPS and how this produces specific terpenes. This would enable structure-guided design of TPSs to produce alternative terpenes. This can be done in one of two way, making a mesophilic TPS more thermostable and/or redesigning a thermophilic TPS to make and alternative terpene product.

The second aim of this project is to produce an orthogonal thermophilic MEV pathway. The two alternative pathways used to produce the substrates for terpene production are the mevalonate (MEV) and nonmevalonate (DXP) pathways. While most bacteria use the DXP pathway, several use the MEV pathway. The construction of an orthaganol thermophilic MEV pathway would enable the TPSs produced by the first aim to be inserted to the pathway to produce favourable terpenes. This would create a "plug-and-paly" aspect. Several thermophilic enzymes in this pathway have already been characterised and used to begin the construction of this pathway in the thermophilic organism, Geobacillus, but several enzymes are yet to have thermophilic versions identified.

This project would enable the construction of a thermophilic organism with a functional MEV pathway where a different thermophilic TPS can be inserted to produce a favourable terpene. Having enzymes that are thermostable are beneficial to industrial processes for the production of terpenes due to generally having higher reaction rates than the mesophilic counterparts. Also, many industrial processes run at higher temperatures. Thermophilic enzymes are preferred as it allows for higher substrate solubility, low risk of contamination and lower process costs. Geobacillus can grow on high carbohydrate raw materials, such as agriculture which could be used as feedstocks for terpene production using the thermophilic MEV pathway. Lastly, the ability to understand how structure of a TPS relates to function may enable the redesign of structures to produce alternative favourable terpines.