NREL Partnership: Engineering enzymes for improved ethylene production in C.necator

乙烯是一种小碳氢化合物气体，广泛用于化学工业。其全球年产量目前超过1.5亿吨，超过任何其他有机化合物。乙烯目前是由乙烷的蒸汽裂解生产的，而乙烷会产生大量的二氧化碳，导致全球变暖。乙烯是最常见的塑料——聚乙烯的单体，全球年产量约为8000万吨。因此，开发一种可持续的或碳中性的乙烯生产替代品势在必行。目前有三种乙烯合成途径，本项目将重点研究在丁香假单胞菌和指状青霉菌中发现的另一种乙烯合成途径，该途径利用a-酮戊二酸酯（AKG）和精氨酸在乙烯形成酶（EFE）的催化下进行反应。该项目旨在利用增强的定向进化方法，结合系统生物学，组学数据挖掘和计算生物学；进化EFE酶，提高酶的溶解度和活性，从而使生产绿色乙烯所需的生产力得到实质性提高。定向进化需要高通量筛选，该项目将通过开发乙烯生物传感器来解决这一问题。宿主菌株的转录组将在乙烯存在和不存在的情况下进行评估。响应乙烯的转录激活将通过RNA测序获得，并用作乙烯浓度的比例表达响应。然后，选定的候选物将用于调节一个必需基因的表达，从而建立一个响应乙烯浓度的生长筛选，能够筛选多达108-12个变体的文库。此外，乙烯敏感候选启动子转录的GFP表达将作为报告因子进行评估，以实现基于facs的多达108种酶变体的筛选。我们还将利用大肠杆菌中的EnvZ/OmpR双组分系统获得已知的乙烯生物传感器。最后，该项目将致力于阐明目前缺乏的EFE的明确蛋白质结构。通过结合诺丁汉大学SBRC和NREL各自的优势，我们有机会在从生物质中获取化学品和燃料的新细菌途径方面取得重大进展。

Ethylene is a small hydrocarbon gas, widely used in the chemical industry. Its annual worldwide production currently exceeds 150 million tonnes, surpassing any other organic compound. Ethylene is currently produced from steam cracking of ethane which produces vast quantities of CO2, contributing to global warming. Ethylene is the monomer for the most common plastic, polyethylene, and annual global production is approximately 80 million tons. Therefore, unlocking a sustainable or carbon neutral alternative to ethylene production is imperative. There are currently three pathways for ethylene synthesis this project will focus on the alternative ethylene synthesis pathway, found in Pseudomonas syringae and Penicillium digitatum, which utilises a-ketoglutarate (AKG) and arginine in a reaction catalysed by the ethylene-forming enzyme (EFE). This project aims to utilise an augmented directed evolution approach, incorporating systems biology, omics data mining and computational biology; to evolve the EFE enzyme improving both solubility and enzyme activity, thus allowing for the substantial productivity improvements required in manufacturing green ethylene. Directed evolution requires a high throughput screen, this project will address this by developing an ethylene biosensor. The transcriptome of the host strain will be evaluated in the presence and absence of ethylene. Transcriptional activation in response to ethylene will be accessed via RNA Seq and used as a proportional expression response to ethylene concentration. Selected candidates will then be used to regulate expression of an essential gene, thus establishing a growth screen in response to ethylene concentration, capable of screening libraries of up to 108-12 variants. In addition, expression of GFP transcribed by the ethylene sensitive promoter candidates will be assessed as a reporter to enable FACS-based screen of up to 108 enzyme variants. We will also access known ethylene biosensors utilising the EnvZ/OmpR two component system in E.coli. Finally the project will aim to elucidate a definitive protein structure for EFE, which is currently lacking. By combining the respective strengths of Nottingham University SBRC and the NREL, there is a real opportunity to make significant progress towards new bacterial-based routes to chemicals and fuels from biomass.