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.

Keywords: Arginine; E. coli; Ethylene; Ethylene-forming enzyme; α-ketoglutarate.

Fig. 1

a Putative metabolic scheme for ethylene production in E. coli via the Ethylene-Forming Enzyme (EFE) and the formation of glutamate from α-ketoglutarate (AKG); genes responsible for the catalytic steps or regulation relevant to this work indicated in red (knockout) and green (overexpression). b Ethylene production, headspace O₂, and growth over time in LB media for E. coli (MG1655) harboring the pUC-Plac-efe-Flag plasmid revealing ethylene production to peak at an optical density between 0.2 and 0.3. P5C: L-Δ¹-pyrroline-5-carboxylate. Gene abbreviations: icd (isocitrate dehydrogenase), gdhA (glutamate dehydrogenase), gltBD (glutamate synthase), argR (transcriptional regulator of arginine biosynthesis), sucA (2-oxoglutarate decarboxylase)

Fig. 2

a Peak production of ethylene from high-, medium-, and low-copy plasmids in LB media, M9 (0.2 % glucose) and MOPS (0.2 % glucose) minimal media. b Western blots of EFE-Flag proteins expressed in E. coli (MG1655) from high-, medium-, and low-copy plasmids in different media. In each instance a significant amount of expressed protein remained insoluble. W whole cell; S soluble; P lysed cell pellet

Fig. 3

Peak production of ethylene from the wild-type E. coli in MOPS minimal media (0.2 % glucose) supplemented with the indicated nutrient at the following concentrations: glutamate (50 mM), glutamine (5 mM), AKG (2 mM), arginine (3 mM), proline (1 mM), and FeCl₂ (80 µM)

Fig. 4

Peak Ethylene production from pUC-P_psbA-efe-Flag in wild-type E. coli (MG1655) and each modified strain grown at 30 °C in MOPS minimal media (0.2 % glucose) with the modified genes displayed in red on the accompanying pathway figure

Fig. 5

a Intracellular AKG levels in wild-type E. coli (MG1655) and each modified strain grown at 30 °C in MOPS minimal media (0.2 % glucose). b Ethylene production and growth as a function of time for the ΔargR/ΔgltBD strain with EFE expressed from the pUC-P_psbA-efe-Flag plasmid