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

从植物、真菌和细菌中鉴定出超过50000种天然萜烯结构，包括紫杉醇和青蒿素等萜烯。它们的关键酶是萜烯合成酶/环化酶（TPS）。这种酶的功能是底物的环化步骤，如焦磷酸铁烯基，以产生许多环萜烯。表征tps的困难之一是它们具有非常低的序列同源性，有时小于20%，这意味着不能容易地从初级序列确定功能。这使得从已知的TPS结构中模拟未表征的TPS变得非常困难。虽然有相似的机制产生环萜烯的产物。tps对基材和产品具有不同的特异性。许多tps产生多种萜烯，例如y-Humulene合成酶，它可以产生52种不同的萜烯。该项目的目的之一是使用结构导向设计的嗜热性TPS，以帮助理解TPS的一级和三级序列之间的关系，以及它如何产生特定的萜烯。这将使tps的结构导向设计能够产生替代萜烯。这可以通过两种方式之一来完成，使中温性TPS更耐热和/或重新设计嗜热性TPS来制造替代萜烯产品。该项目的第二个目标是产生一个正交的嗜热MEV途径。用于生产萜烯生产底物的两种替代途径是甲羟戊酸（MEV）和非甲羟戊酸（DXP）途径。虽然大多数细菌使用DXP途径，但有几种使用MEV途径。正戊醇嗜热MEV途径的构建将使第一个目标产生的tps插入到该途径中以产生有利的萜烯。这将创造一个“即插即用”的方面。该途径中的几种嗜热酶已经被表征，并用于在嗜热生物Geobacillus中开始构建该途径，但仍有几种酶尚未确定嗜热版本。该项目将能够构建具有功能MEV途径的嗜热生物，其中可以插入不同的嗜热TPS以产生有利的萜烯。具有耐热性的酶有利于生产萜烯的工业过程，因为它们通常比中温性的对应物具有更高的反应速率。此外，许多工业过程在更高的温度下运行。首选嗜热酶，因为它允许更高的底物溶解度，低污染风险和更低的工艺成本。地杆菌可以生长在高碳水化合物的原料上，如农业，这些原料可以用作使用嗜热MEV途径生产萜烯的原料。最后，了解TPS结构如何与功能相关的能力可能使结构的重新设计能够产生替代的有利衍生物。

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.