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J Biosci Bioeng. 2019 Mar;127(3):301-308. doi: 10.1016/j.jbiosc.2018.08.013. Epub 2018 Oct 25.

Metabolome analysis revealed the knockout of glyoxylate shunt as an effective strategy for improvement of 1-butanol production in transgenic Escherichia coli.

Author information

1
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: katsuaki_nitta@bio.eng.osaka-u.ac.jp.
2
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Microbiology, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, College, 4031 Laguna, Philippines. Electronic address: walavina@up.edu.ph.
3
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA. Electronic address: sppontrelli@gmail.com.
4
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA. Electronic address: liaoj@ucla.edu.
5
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: sastia_putri@bio.eng.osaka-u.ac.jp.
6
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: fukusaki@bio.eng.osaka-u.ac.jp.

Abstract

High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli.

KEYWORDS:

1-Butanol; Escherichia coli; Glyoxylate shunt; Metabolomics; Strain improvement

PMID:
30482596
DOI:
10.1016/j.jbiosc.2018.08.013
[Indexed for MEDLINE]

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