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Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli

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Abstract

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l−1 with a yield of 1.02 g g−1 total sugars during anaerobic fermentation.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant nos. 20606017 and 21106066), the 973 Program of China (grant no. 2009CB724701), and the PAPD Project of Jiangsu Province.

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Authors and Affiliations

  1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 211816, China

    Rongming Liu, Liya Liang, Kequan Chen, Jiangfeng Ma, Min Jiang, Ping Wei & Pingkai Ouyang

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  1. Rongming Liu
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  2. Liya Liang
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  3. Kequan Chen
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  4. Jiangfeng Ma
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  5. Min Jiang
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  7. Pingkai Ouyang
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Correspondence to Min Jiang.

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Liu, R., Liang, L., Chen, K. et al. Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli . Appl Microbiol Biotechnol 94, 959–968 (2012). https://doi.org/10.1007/s00253-012-3896-4

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  • DOI: https://doi.org/10.1007/s00253-012-3896-4

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