title: Overcoming substrate limitations for improved production of ethylene in E. coli.
abstract: BACKGROUND
  Ethylene is an important industrial compound for the production of a wide variety of plastics and chemicals. At present, ethylene production involves steam cracking of a fossil-based feedstock, representing the highest CO2-emitting process in the chemical industry. Biological ethylene production can be achieved via expression of a single protein, the ethylene-forming enzyme (EFE), found in some bacteria and fungi; it has the potential to provide a sustainable alternative to steam cracking, provided that significant increases in productivity can be achieved. A key barrier is determining factors that influence the availability of substrates for the EFE reaction in potential microbial hosts. In the presence of O2, EFE catalyzes ethylene formation from the substrates α-ketoglutarate (AKG) and arginine. The concentrations of AKG, a key TCA cycle intermediate, and arginine are tightly controlled by an intricate regulatory system that coordinates carbon and nitrogen metabolism. Therefore, reliably predicting which genetic changes will ultimately lead to increased AKG and arginine availability is challenging.
  
  
  RESULTS
  We systematically explored the effects of media composition (rich versus defined), gene copy number, and the addition of exogenous substrates and other metabolites on the formation of ethylene in Escherichia coli expressing EFE. Guided by these results, we tested a number of genetic modifications predicted to improve substrate supply and ethylene production, including knockout of competing pathways and overexpression of key enzymes. Several such modifications led to higher AKG levels and higher ethylene productivity, with the best performing strain more than doubling ethylene productivity (from 81 ± 3 to 188 ± 13 nmol/OD600/mL).
  
  
  CONCLUSIONS
  Both EFE activity and substrate supply can be limiting factors in ethylene production. Targeted modifications in central carbon metabolism, such as overexpression of isocitrate dehydrogenase, and deletion of glutamate synthase or the transcription regulator ArgR, can effectively enhance substrate supply and ethylene productivity. These results not only provide insight into the intricate regulatory network of the TCA cycle, but also guide future pathway and genome-scale engineering efforts to further boost ethylene productivity.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA ; Renewable and Sustainable Energy Institute, University of Colorado Boulder, Boulder, CO 80309 USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Lynch
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA ; Renewable and Sustainable Energy Institute, University of Colorado Boulder, Boulder, CO 80309 USA.
authors.forename: Carrie
authors.initials: C
authors.lastname: Eckert
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Jianping
authors.initials: J
authors.lastname: Yu
authors.affiliation: Renewable and Sustainable Energy Institute, University of Colorado Boulder, Boulder, CO 80309 USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Gill
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Pin-Ching
authors.initials: PC
authors.lastname: Maness
keywords: Arginine; E. coli; Ethylene; Ethylene-forming enzyme; α-ketoglutarate