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Engineering a homobutanol fermentation pathway in Escherichia coli EG03

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l−1) under anaerobic condition.

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Acknowledgments

This research was supported by a summer artistry and research grant, biological research incentive fund of Northern Illinois University, a grant from the China National Natural Science Foundation (NSFC31070094), a grant from the Hubei Provincial Natural Science Foundation (2011CDA008) and the Chutian Scholar Program, P.R. China.

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Authors and Affiliations

  1. Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Wuhan, 430068, People’s Republic of China

    Erin Garza, Jinfang Zhao, Yongze Wang & Jinhua Wang

  2. Department of Biological Sciences, Northern Illinois University, DeKalb, IL, 60115, USA

    Erin Garza, Andrew Iverson, Ryan Manow, Chris Finan & Shengde Zhou

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  1. Erin Garza
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  2. Jinfang Zhao
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  3. Yongze Wang
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  4. Jinhua Wang
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  5. Andrew Iverson
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  6. Ryan Manow
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  7. Chris Finan
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  8. Shengde Zhou
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Correspondence to Jinhua Wang or Shengde Zhou.

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Garza, E., Zhao, J., Wang, Y. et al. Engineering a homobutanol fermentation pathway in Escherichia coli EG03. J Ind Microbiol Biotechnol 39, 1101–1107 (2012). https://doi.org/10.1007/s10295-012-1151-8

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  • DOI: https://doi.org/10.1007/s10295-012-1151-8

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