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

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.