Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Sophie Vaud; Nicole Pearcy; Marko Hanževački; Alexander M.W. Van Hagen; Salah Abdelrazig; Laudina Safo; Muhammad Ehsaan; Magdalene Jonczyk; Thomas Millat; Sean Craig; Edward Spence; James Fothergill; Rajesh Reddy Bommareddy; Pierre Yves Colin; Jamie Twycross; Paul A. Dalby; Nigel P. Minton; Christof M. Jäger; Dong Hyun Kim; Jianping Yu; Pin Ching Maness; Sean Lynch; Carrie A. Eckert; Alex Conradie; Samantha J. Bryan

doi:10.1016/j.ymben.2021.07.001

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Sophie Vaud, Nicole Pearcy, Marko Hanževački, Alexander M.W. Van Hagen, Salah Abdelrazig, Laudina Safo, Muhammad Ehsaan, Magdalene Jonczyk, Thomas Millat, Sean Craig, Edward Spence, James Fothergill, Rajesh Reddy Bommareddy, Pierre Yves Colin, Jamie Twycross, Paul A. Dalby, Nigel P. Minton, Christof M. Jäger, Dong Hyun Kim, Jianping YuPin Ching Maness, Sean Lynch, Carrie A. Eckert, Alex Conradie, Samantha J. Bryan

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO₂ contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

Original language	English
Pages (from-to)	308-320
Number of pages	13
Journal	Metabolic Engineering
Volume	67
DOIs	https://doi.org/10.1016/j.ymben.2021.07.001
State	Published - Sep 2021
Externally published	Yes

Funding

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC); grant number BB/L013940/1 ) and the Engineering and Physical Sciences Research Council (EPSRC) under the same grant number and the Green Chemicals Beacon of Excellence , University of Nottingham . This work was supported in part by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (Grant Number DE-SC008812 ). The metabolomic analysis demonstrated that the L-Arg concentration had increased in U3-26 EFE, while the level of AKG decreased, although not significantly, which supports the idea that the stoichiometry of the minor and major reactions of the EFE enzyme was altered in U3-26 EFE. Acetate is produced as a by-product of L-Arg biosynthesis, therefore the decrease in acetate concentration in U3-26 EFE also supports reduced L-Arg demand. Furthermore, metabolomic analysis confirmed there was a reduction in both P5C and guanidine in U3-26 EFE, which supports the notion that L-Arg is in a much less favorable position for hydroxylation in the minor reaction. The in vitro P5C assays also demonstrated that there was a statistically significant reduction in P5C in U3-26 EFE compared to MG1655 pGEM p15 efe and ?proB pGEM p15 efe (p ? 0.05), supporting the notion that the SNPs in U3-26 EFE have altered the stoichiometric balance of the ethylene-forming reaction in favor of the major reaction (Supplementary Fig. 8). Interestingly expression of the wild-type EFE in the U3-26 background also resulted in a significant decrease in P5C in vitro compared to MG1655 pGEM p15 efe and ?proB pGEM p15 efe (p ? 0.05). This could be due to substrate availability in the U3-26 background. Furthermore, the proline concentration surprisingly increased in U3-26 EFE despite the reduction in P5C, however the flux response analysis predicts that P5C is directed towards glutamate thus increasing L-Arg biosynthesis and therefore available flux towards proline may have increased with reduced L-Arg demand.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC); grant number BB/L013940/1) and the Engineering and Physical Sciences Research Council (EPSRC) under the same grant number and the Green Chemicals Beacon of Excellence, University of Nottingham. This work was supported in part by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (Grant Number DE-SC008812).

Funders	Funder number
Green Chemicals Beacon of Excellence , University of Nottingham
Green Chemicals Beacon of Excellence, University of Nottingham
U.S. Department of Energy
Office of Science
Biological and Environmental Research	DE-SC008812
Engineering and Physical Sciences Research Council
Biotechnology and Biological Sciences Research Council	BB/L013940/1

Keywords

Adaptive evolution
Directed evolution
Fermentation
Metabolic engineering
Systems biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1016/j.ymben.2021.07.001

Cite this

Vaud, S., Pearcy, N., Hanževački, M., Van Hagen, A. M. W., Abdelrazig, S., Safo, L., Ehsaan, M., Jonczyk, M., Millat, T., Craig, S., Spence, E., Fothergill, J., Bommareddy, R. R., Colin, P. Y., Twycross, J., Dalby, P. A., Minton, N. P., Jäger, C. M., Kim, D. H., ... Bryan, S. J. (2021). Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution. Metabolic Engineering, 67, 308-320. https://doi.org/10.1016/j.ymben.2021.07.001

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title = "Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution",

abstract = "Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.",

keywords = "Adaptive evolution, Directed evolution, Fermentation, Metabolic engineering, Systems biology",

author = "Sophie Vaud and Nicole Pearcy and Marko Han{\v z}eva{\v c}ki and {Van Hagen}, {Alexander M.W.} and Salah Abdelrazig and Laudina Safo and Muhammad Ehsaan and Magdalene Jonczyk and Thomas Millat and Sean Craig and Edward Spence and James Fothergill and Bommareddy, {Rajesh Reddy} and Colin, {Pierre Yves} and Jamie Twycross and Dalby, {Paul A.} and Minton, {Nigel P.} and J{\"a}ger, {Christof M.} and Kim, {Dong Hyun} and Jianping Yu and Maness, {Pin Ching} and Sean Lynch and Eckert, {Carrie A.} and Alex Conradie and Bryan, {Samantha J.}",

note = "Publisher Copyright: {\textcopyright} 2021 The Authors",

year = "2021",

month = sep,

doi = "10.1016/j.ymben.2021.07.001",

language = "English",

volume = "67",

pages = "308--320",

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Vaud, S, Pearcy, N, Hanževački, M, Van Hagen, AMW, Abdelrazig, S, Safo, L, Ehsaan, M, Jonczyk, M, Millat, T, Craig, S, Spence, E, Fothergill, J, Bommareddy, RR, Colin, PY, Twycross, J, Dalby, PA, Minton, NP, Jäger, CM, Kim, DH, Yu, J, Maness, PC, Lynch, S, Eckert, CA, Conradie, A & Bryan, SJ 2021, 'Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution', Metabolic Engineering, vol. 67, pp. 308-320. https://doi.org/10.1016/j.ymben.2021.07.001

TY - JOUR

T1 - Engineering improved ethylene production

T2 - Leveraging systems biology and adaptive laboratory evolution

AU - Vaud, Sophie

AU - Pearcy, Nicole

AU - Hanževački, Marko

AU - Van Hagen, Alexander M.W.

AU - Abdelrazig, Salah

AU - Safo, Laudina

AU - Ehsaan, Muhammad

AU - Jonczyk, Magdalene

AU - Millat, Thomas

AU - Craig, Sean

AU - Spence, Edward

AU - Fothergill, James

AU - Bommareddy, Rajesh Reddy

AU - Colin, Pierre Yves

AU - Twycross, Jamie

AU - Dalby, Paul A.

AU - Minton, Nigel P.

AU - Jäger, Christof M.

AU - Kim, Dong Hyun

AU - Yu, Jianping

AU - Maness, Pin Ching

AU - Lynch, Sean

AU - Eckert, Carrie A.

AU - Conradie, Alex

AU - Bryan, Samantha J.

PY - 2021/9

Y1 - 2021/9

N2 - Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

AB - Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

KW - Adaptive evolution

KW - Directed evolution

KW - Fermentation

KW - Metabolic engineering

KW - Systems biology

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U2 - 10.1016/j.ymben.2021.07.001

DO - 10.1016/j.ymben.2021.07.001

M3 - Article

C2 - 34245888

AN - SCOPUS:85111223211

SN - 1096-7176

VL - 67

SP - 308

EP - 320

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Abstract

Funding

Keywords

UN SDGs

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