Genome-Scale Design and Engineering Approach Towards Optimizing Ethylene Production in E. coli

This project aims to apply rational design and state-of-the-art synthetic and systems biology tools to optimize E. coli for sustained production of biofuels. At present, global ethylene production involves steam cracking of a fossil-based feedstock, representing the largest CO₂-emitting process in the chemical industry. Biological ethylene production has the potential to provide a sustainable alternative. Chassis biofuel strains, optimized for production based on predictive design and systems biology knowledge, will serve as the framework for high throughput genome re-design.

Herein, we focus on the construction of an E. coli prototype chassis strain for the production of ethylene and the development of a selection strategy for gene-to-trait mapping at single nucleotide resolution to identify key factors for this optimization process. The expression of a single gene found in some bacteria and fungi, ethylene-forming enzyme (efe), catalyzes ethylene formation. Construction of the first generation chassis strain is based on E. coli MG1655 as the host and the efe gene from Pseudomonas syringae (Ps). EFE has been postulated to catalyze ethylene production according to the equation (1):

3 a-ketoglutarate + Arginine + 3O₂ ® 2C₂H₄ + Succinate + 7CO₂ + 3H₂O + guanidine + P5C*

*P5C:L-D¹-pyrroline-5-carboxylate

The two key substrates α-ketoglutarate (AKG) and arginine are tightly controlled by an intricate regulatory network that coordinates carbon and nitrogen metabolism. We conducted genetic modifications to rewire central carbon metabolic flux and improved ethylene production by 2.3-fold (1). This chassis strain will serve as the framework to guide genome-scale redesign and optimization to further boost ethylene production using CRISPR enabled trackable genome engineering (2). Succinate is a byproduct of the EFE reaction. We generated a succinate auxotroph in E. coli and showed that it must rely on an active heterologous EFE pathway yielding succinate to afford growth. Work is also ongoing to construct high-throughput sensors to screen for AKG and ethylene, in situ. As such, current work from our groups at the National Renewable Energy Laboratory and the University of Colorado at Boulder seeks to improve ethylene production by combining traditional metabolic engineering strategies with synthetic biology-enabled evolutionary approaches involving the high-throughput construction of genome-scale libraries. Coupled with novel screens and selections, these methods will identify strains with increased production of key intermediates and/or ethylene.

References

1. Eckert, C., W. Xu, W. Xiong, S. Lynch, J. Ungerer, L. Tao, R. Gill, P. C. Maness, and J. Yu. 2014. Ethylene-forming enzyme and bioethylene production. Biotech. Biofuels 7:33-43.
2. Lynch, S., C. Eckert, J. Yu, R. Gill, and P. C. Maness. 2016. Overcoming substrate limitations for improved production of ethylene in E. coli. Biotech. Biofuels 9:3, DOI 10.1186/s13068-015-0413-x
3. Garst, A. D., Bassalo, M. C., Pines, G., Lynch, S. A., Halweg-Edwards, A. L., Liu, R., Liang, L., Wang, Z., Zeitoun, R., Alexander, W. G., Gill, R. T. 2016. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat. Biotechnol, DOI 10.1038/nbt.3718

Funding Source: A Platform for Genome-scale Design, Redesign, and Optimization of Bacterial Systems; Project grant number (DE-SC008812) and FWP number (ERWER44) for NREL.