Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli.
Acetate
Escherichia coli
Fermentation
Isobutanol
Metabolic engineering
NADH
Redox balance
Journal
Biotechnology for biofuels and bioproducts
ISSN: 2731-3654
Titre abrégé: Biotechnol Biofuels Bioprod
Pays: England
ID NLM: 9918300888906676
Informations de publication
Date de publication:
03 Oct 2023
03 Oct 2023
Historique:
received:
06
03
2023
accepted:
18
09
2023
medline:
4
10
2023
pubmed:
4
10
2023
entrez:
3
10
2023
Statut:
epublish
Résumé
The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs. In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate-product combinations.
Sections du résumé
BACKGROUND
BACKGROUND
The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs.
RESULTS
RESULTS
In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h
CONCLUSIONS
CONCLUSIONS
This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate-product combinations.
Identifiants
pubmed: 37789464
doi: 10.1186/s13068-023-02395-z
pii: 10.1186/s13068-023-02395-z
pmc: PMC10548627
doi:
Types de publication
Journal Article
Langues
eng
Pagination
148Informations de copyright
© 2023. BioMed Central Ltd., part of Springer Nature.
Références
Keasling J, Garcia Martin H, Lee TS, Mukhopadhyay A, Singer SW, Sundstrom E. Microbial production of advanced biofuels. Nat Rev Microbiol. 2021;19:701–15.
pubmed: 34172951
Lee SY, Kim HU, Chae TU, Cho JS, Kim JW, Shin JH, Kim DI, Ko Y-S, Jang WD, Jang Y-S. A comprehensive metabolic map for production of bio-based chemicals. Nat Catal. 2019;2:18–33.
Klamt S, Mahadevan R, Hädicke O. When do two-stage processes outperform one-stage processes? Biotechnol J. 2018;13:1700539.
Raj K, Venayak N, Mahadevan R. Novel two-stage processes for optimal chemical production in microbes. Metab Eng. 2020;62:186–97.
pubmed: 32827703
Burg JM, Cooper CB, Ye Z, Reed BR, Moreb EA, Lynch MD. Large-scale bioprocess competitiveness: the potential of dynamic metabolic control in two-stage fermentations. Curr Opin Chem Eng. 2016;14:121–36.
Harder B-J, Bettenbrock K, Klamt S. Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli. Biotechnol Bioeng. 2018;115:156–64.
pubmed: 28865130
Lempp M, Lubrano P, Bange G, Link H. Metabolism of non-growing bacteria. Biol Chem. 2020;401:1479–85.
pubmed: 32845858
Chubukov V, Desmarais JJ, Wang G, Chan LJG, Baidoo EEK, Petzold CJ, Keasling JD, Mukhopadhyay A. Engineering glucose metabolism of Escherichia coli under nitrogen starvation. NPJ Syst Biol Appl. 2017;3:16035.
pubmed: 28725483
pmcid: 5516864
Boecker S, Zahoor A, Schramm T, Link H, Klamt S. Broadening the scope of enforced ATP wasting as a tool for metabolic engineering in Escherichia coli. Biotechnol J. 2019;14:1800438.
Boecker S, Harder B-J, Kutscha R, Pflügl S, Klamt S. Increasing ATP turnover boosts productivity of 2,3-butanediol synthesis in Escherichia coli. Microb Cell Fact. 2021;20:63.
pubmed: 33750397
pmcid: 7941745
Boecker S, Slaviero G, Schramm T, Szymanski W, Steuer R, Link H, Klamt S. Deciphering the physiological response of Escherichia coli under high ATP demand. Mol Syst Biol. 2021;17:e10504.
pubmed: 34928538
pmcid: 8686765
Zahoor A, Messerschmidt K, Boecker S, Klamt S. ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production. Biotechnol Biofuels. 2020;13:185.
pubmed: 33292464
pmcid: 7654063
Clomburg JM, Gonzalez R. Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol. 2013;31:20–8.
pubmed: 23178075
Boecker S, Espinel-Ríos S, Bettenbrock K, Klamt S. Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink. Metab Eng. 2022;73:50–7.
pubmed: 35636656
Blombach B, Eikmanns BJ. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. Bioeng Bugs. 2011;2:346–50.
pubmed: 22008938
pmcid: 3242789
Atsumi S, Hanai T, Liao JC. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature. 2008;451:86–9.
pubmed: 18172501
Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol. 2010;85:651–7.
pubmed: 19609521
Bastian S, Liu X, Meyerowitz JT, Snow CD, Chen MMY, Arnold FH. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab Eng. 2011;13:345–52.
pubmed: 21515217
Ghosh IN, Martien J, Hebert AS, Zhang Y, Coon JJ, Amador-Noguez D, Landick R. OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate. Metab Eng. 2019;52:324–40.
pubmed: 30594629
Liu X, Bastian S, Snow CD, Brustad EM, Saleski TE, Xu J-H, Meinhold P, Arnold FH. Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. J Biotechnol. 2013;164:188–95.
Novak K, Baar J, Freitag P, Pflügl S. Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material. J Ind Microbiol Biotechnol. 2020;47:1117–32.
pubmed: 33068182
Chen C-T, Liao JC. Frontiers in microbial 1-butanol and isobutanol production. FEMS Microbiol Lett. 2016;363:fnw020.
pubmed: 26832641
Maser A, Peebo K, Vilu R, Nahku R. Amino acids are key substrates to Escherichia coli BW25113 for achieving high specific growth rate. Res Microbiol. 2020;171:185–93.
pubmed: 32057959
Trinh CT, Li J, Blanch HW, Clark DS. Redesigning Escherichia coli metabolism for anaerobic production of isobutanol. Appl Environ Microbiol. 2011;77:4894–904.
pubmed: 21642415
pmcid: 3147371
Trinh CT. Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production. Appl Microbiol Biotechnol. 2012;95:1083–94.
pubmed: 22678028
Tanaka S, Lerner SA, Lin EC. Replacement of a phosphoenolpyruvate-dependent phosphotransferase by a nicotinamide adenine dinucleotide-linked dehydrogenase for the utilization of mannitol. J Bacteriol. 1967;93:642–8.
pubmed: 4289962
pmcid: 276489
Hädicke O, Klamt S. EColiCore2: a reference network model of the central metabolism of Escherichia coli and relationships to its genome-scale parent model. Sci Rep. 2017;7:39647.
pubmed: 28045126
pmcid: 5206746
Klamt S, Saez-Rodriguez J, Gilles ED. Structural and functional analysis of cellular networks with Cell NetAnalyzer. BMC Syst Biol. 2007;1:2.
pubmed: 17408509
pmcid: 1847467
von Kamp A, Thiele S, Hädicke O, Klamt S. Use of Cell NetAnalyzer in biotechnology and metabolic engineering. J Biotechnol. 2017;261:221–8.
Quail MA, Haydon DJ, Guest JR. The pdhR–aceEF–lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex. Mol Microbiol. 1994;12:95–104.
pubmed: 8057842
McDowall JS, Murphy BJ, Haumann M, Palmer T, Armstrong FA, Sargent F. Bacterial formate hydrogenlyase complex. Proc Natl Acad Sci USA. 2014;111:E3948–56.
pubmed: 25157147
pmcid: 4183296
Enjalbert B, Millard P, Dinclaux M, Portais J-C, Létisse F. Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway. Sci Rep. 2017;7:42135.
pubmed: 28186174
pmcid: 5301487
Pinhal S, Ropers D, Geiselmann J, de Jong H. Acetate metabolism and the inhibition of bacterial growth by acetate. J Bacteriol. 2019;201:e00147-e219.
pubmed: 30988035
pmcid: 6560135
Orth JD, Conrad TM, Na J, Lerman JA, Nam H, Feist AM, Palsson BØ. A comprehensive genome-scale reconstruction of Escherichia coli metabolism—2011. Mol Syst Biol. 2011;7:535.
pubmed: 21988831
pmcid: 3261703
Atsumi S, Wu TY, Machado IMP, Huang WC, Chen PY, Pellegrini M, Liao JC. Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol Syst Biol. 2010;6:449.
pubmed: 21179021
pmcid: 3018172
Baez A, Cho KM, Liao JC. High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal. Appl Microbiol Biotechnol. 2011;90:1681–90.
pubmed: 21547458
pmcid: 3094657
Brynildsen MP, Liao JC. An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol. 2009;5:277.
pubmed: 19536200
pmcid: 2710865
Sherkhanov S, Korman TP, Chan S, Faham S, Liu H, Sawaya MR, Hsu W-T, Vikram E, Cheng T, Bowie JU. Isobutanol production freed from biological limits using synthetic biochemistry. Nat Commun. 2020;11:4292.
pubmed: 32855421
pmcid: 7453195
Liu J, Qi H, Wang C, Wen J. Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli. Biotechnol Biofuels. 2015;8:108.
pubmed: 26236397
pmcid: 4522091
Noda S, Mori Y, Oyama S, Kondo A, Araki M, Shirai T. Reconstruction of metabolic pathway for isobutanol production in Escherichia coli. Microb Cell Fact. 2019;18:124.
pubmed: 31319852
pmcid: 6637570
Shi A, Zhu X, Lu J, Zhang X, Ma Y. Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab Eng. 2013;16:1–10.
pubmed: 23246519
Liu Z, Liu P, Xiao D, Zhang X. Improving isobutanol production in metabolically engineered Escherichia coli by co-producing ethanol and modulation of pentose phosphate pathway. J Ind Microbiol Biotechnol. 2016;43:851–60.
pubmed: 26946319
Liang S, Chen H, Liu J, Wen J. Rational design of a synthetic Entner-Doudoroff pathway for enhancing glucose transformation to isobutanol in Escherichia coli. J Ind Microbiol Biotechnol. 2018;45:187–99.
pubmed: 29380153
Song H-S, Seo H-M, Jeon J-M, Moon Y-M, Hong JW, Hong YG, Bhatia SK, Ahn J, Lee H, Kim W, et al. Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli. Biotechnol Bioeng. 2018;115:1971–8.
pubmed: 29663332
Kiefer D, Merkel M, Lilge L, Henkel M, Hausmann R. From acetate to bio-based products: underexploited potential for industrial biotechnology. Trends Biotechnol. 2021;39:397–411.
pubmed: 33036784
Gong G, Wu B, Liu L, Li J, Zhu Q, He M, Hu G. Metabolic engineering using acetate as a promising building block for the production of bio-based chemicals. Eng Microbiol. 2022;2:100036.
Basan M, Hui S, Okano H, Zhang Z, Shen Y, Williamson JR, Hwa T. Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature. 2015;528:99–104.
pubmed: 26632588
pmcid: 4843128
Yi C, Song W, Zhang Y, Qiu X. Liquid-liquid extraction of biobased isobutanol from an aqueous solution. J Chem Eng Data. 2019;64:2350–6.
Omidali M, Raisi A, Aroujalian A. Separation and purification of isobutanol from dilute aqueous solutions by a hybrid hydrophobic/hydrophilic pervaporation process. Chem Eng Process. 2014;77:22–9.
Fu C, Li Z, Zhang Y, Yi C, Xie S. Assessment of extraction options for a next-generation biofuel: recovery of bio-isobutanol from aqueous solutions. Eng Life Sci. 2021;21:653–65.
pubmed: 34690636
pmcid: 8518583
Outram V, Lalander C-A, Lee JGM, Davies ET, Harvey AP. Applied in situ product recovery in ABE fermentation. Biotechnol Prog. 2017;33:563–79.
pubmed: 28188696
pmcid: 5485034
Nguyen N-P-T, Raynaud C, Meynial-Salles I, Soucaille P. Reviving the Weizmann process for commercial n-butanol production. Nat Commun. 2018;9:3682.
pubmed: 30206218
pmcid: 6134114
Tashiro Y, Desai SH, Atsumi S. Two-dimensional isobutyl acetate production pathways to improve carbon yield. Nat Commun. 2015;6:7488.
pubmed: 26108471
Guadalupe Medina V, Almering MJH, van Maris AJA, Pronk JT. Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor. Appl Environ Microbiol. 2010;76:190–5.
pubmed: 19915031
Erian AM, Gibisch M, Pflügl S. Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis. Microb Cell Fact. 2018;17:190.
pubmed: 30501633
pmcid: 6267845
Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000;97:6640–5.
pubmed: 10829079
pmcid: 18686
Zeppenfeld T, Larisch C, Lengeler Joseph W, Jahreis K. Glucose transporter mutants of Escherichia coli K-12 with changes in substrate recognition of IICB
pubmed: 10913077
pmcid: 94615
Petras D, Phelan VV, Acharya D, Allen AE, Aron AT, Bandeira N, Bowen BP, Belle-Oudry D, Boecker S, Cummings DA, et al. GNPS Dashboard: collaborative exploration of mass spectrometry data in the web browser. Nat Methods. 2022;19:134–6.
pubmed: 34862502
pmcid: 8831450