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Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol

Abstract

1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l−1 of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.

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Figure 1: Overview of the Biopathway Predictor network calculation and analysis procedure.
Figure 2: Production of BDO from glucose in engineered E. coli strains.
Figure 3: Flux distribution of ECKh-422 pZS*13S-sucCD-sucD-4hbd/sucA pZE23-002C-Cat2 determined by 13C metabolic flux analysis.
Figure 4: Production of BDO from various carbohydrate sources.
Figure 5: Production of BDO from glucose in 2-l fed-batch fermentation using the OptKnock strain ECKh-422.

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Acknowledgements

Vector backbones were obtained from R. Lutz (Expressys). We thank Florida Crystals, Cargill, Bluefire Ethanol, Gruppo M&G and Verenium for providing crude sugar and biomass hydrolysate, B. Palsson, J. Keasling and G. Church for scientific advice throughout the project, and C. Schilling for critical reading of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

H.Y., R.H. and W.N. cloned and expressed BDO-pathway genes, performed bottle experiments and wrote the manuscript; C.P.-B. constructed the host strain and wrote the manuscript; A.B. conceived the project, performed simulations and wrote the manuscript; J.B. cloned and expressed BDO-pathway genes; J.K. and R.S. performed analytical work; J.D.T. conceived the project and performed simulations; R.E.O. performed simulations and wrote the manuscript; J.E. constructed the host strain and performed bottle experiments; S.T. and H.B.S. performed fermentations; S.A. developed and carried out enzyme assays; T.H.Y. analyzed 13C data for flux analysis; S.Y.L. conceived the project and wrote the manuscript; M.J.B. and S.V.D. conceived and directed the project and wrote the manuscript.

Corresponding author

Correspondence to Stephen Van Dien.

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Competing interests

Support for this work was provided by Genomatica, a for-profit company pursuing commercialization of the 1,4-butanediol process discussed here. All authors except S.Y.L. were employees of Genomatica at the time the work was performed. S.Y.L. is on the scientific advisory board of Genomatica.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Methods (PDF 748 kb)

Supplementary Data 1

List of reactions included in the genome-scale E. coli model used for OptKnock analysis. (XLS 261 kb)

Supplementary Data 2

Metabolite abbreviations and names, also indicating cytosolic or extracellular location. (XLS 89 kb)

Supplementary Data 3

OptKnock results. (XLS 34 kb)

Supplementary Data 4

Set of reaction operators used by the Biopathway Predictor algorithm and corresponding reaction diagrams. (XLSX 1670 kb)

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Yim, H., Haselbeck, R., Niu, W. et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol 7, 445–452 (2011). https://doi.org/10.1038/nchembio.580

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