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，包括萜烯如紫杉醇和青蒿素。其生产中的关键酶是萜烯合酶/环化酶（TPS）。这种酶的功能是底物的环化步骤，如焦磷酸铁酯，以产生许多环状萜烯。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一级序列确定功能。这使得很难从已知的TPS结构中对未表征的TPS进行建模。尽管具有类似的产生环状萜烯产物的机制。TPS对底物和产物具有不同的特异性。许多TPS产生多种萜烯，例如γ-蛇麻烯合酶，其可以产生52种不同的萜烯。该项目的目的之一是使用嗜热TPS的结构指导设计，以帮助理解TPS的一级和三级序列之间的关系以及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中的一种来完成，使嗜温TPS更热稳定和/或重新设计嗜热TPS以制备和替代萜烯产物。用于产生萜烯生产的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，但有几种细菌使用MEV途径。正丁醇嗜热MEV途径的构建将使得由第一目的产生的TPS能够插入到该途径中以产生有利的萜烯。这将创建一个“即插即用”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中开始该途径的构建，但几种酶尚未鉴定出嗜热版本。该项目将使具有功能性MEV途径的嗜热生物的构建成为可能，其中不同的嗜热TPS可以插入以产生有利的萜烯。具有热稳定的酶对用于生产萜烯的工业过程是有益的，因为其通常具有比嗜温对应物更高的反应速率。此外，许多工业过程在更高的温度下运行。嗜热酶是优选的，因为它允许更高的底物溶解度、低的污染风险和更低的工艺成本。土芽孢杆菌可以在高碳水化合物原料上生长，例如农业，其可以用作使用嗜热MEV途径生产萜烯的原料。最后，了解TPS结构与功能的关系的能力可以使结构的重新设计能够产生替代的有利萜类。

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.