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50000多种天然萜类结构，包括紫杉醇和青蒿素等萜类化合物。它们产生的关键酶是萜烯合成酶/环化酶(TPS)。这种酶的作用是底物的循环步骤，如焦磷酸亚铁，以产生许多环萜烯。鉴定TPS的困难之一是它们的序列同源性很低，有时不到20%，这意味着不能容易地从一级序列确定功能。这使得从已知的TPS结构中模拟未表征的TPS变得非常困难。尽管具有类似的用于生产环萜烯产品的机制。TPS对底物和产品有不同的特性。许多TPSS能产生多种萜类，如γ-胡萝卜烯合成酶，它能产生52种不同的萜类。该项目的目的之一是使用结构指导设计的高温TPS，以帮助了解TPS的一级和三级序列之间的关系，以及这如何产生特定的萜类。这将使TPSS的结构导向设计能够生产替代萜烯。这可以通过两种方法中的一种来完成，使中温TPS更耐热和/或重新设计嗜热TPS来制造和替代萜类产品。本项目的第二个目标是生产正交的嗜热MEV途径。用于生产萜烯底物的两种替代途径是甲氧丙戊酸(MEV)和非甲氧丙戊酸(DXP)途径。虽然大多数细菌使用DXP途径，但有几种细菌使用MEV途径。构建一条正构烷醇嗜热的MEV途径将使第一个目标产生的TPSS能够插入该途径以产生有利的萜类化合物。这将创建一个“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有机体--地杆菌中开始构建这一途径，但几种酶还没有鉴定出嗜热版本。该项目将使构建具有功能MEV途径的嗜热有机体成为可能，其中可以插入不同的嗜热TPS来产生有利的萜类。具有耐热性的酶有利于工业生产萜类化合物，因为它的反应速率通常比中温酶高。此外，许多工业过程在更高的温度下运行。嗜热酶是首选，因为它允许更高的底物溶解度，低污染风险和较低的工艺成本。土杆菌可以生长在高碳水化合物的原料上，例如农业，这些原料可以作为利用高温MEV途径生产萜类的原料。最后，理解TPS的结构如何与功能相关的能力可以使结构重新设计，以生产替代的有利松节油。

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.