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Modifying the product pattern of Clostridium acetobutylicum

Physiological effects of disrupting the acetate and acetone formation pathways

  • Applied genetics and molecular biotechnology
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Abstract

Clostridial acetone–butanol–ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.

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Acknowledgements

The authors thank Nigel P. Minton and John T. Heap, University of Nottingham for kindly providing the ClosTron plasmids, and P. Soucaille, Institut nationale des sciences appliquées de Toulouse for kind provision of plasmid pCLF1. Furthermore, experimental support by M. Schmidt and M. Klipp for conducting some of the fermentation experiments is gratefully acknowledged. This study was financially supported by the Süd-Chemie AG, Munich and the German Federal Ministry of Education and Research (grant no. 0315419A).

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  1. Abteilung Mikrobiologie, Institut für Biowissenschaften, Universität Rostock, Albert-Einstein-Str. 3, 18059, Rostock, Germany

    Dörte Lehmann, Hubert Bahl & Tina Lütke-Eversloh

  2. Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, 85350, Freising, Germany

    Daniel Hönicke & Armin Ehrenreich

  3. Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany

    Michael Schmidt & Dirk Weuster-Botz

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  1. Dörte Lehmann
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  2. Daniel Hönicke
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  4. Michael Schmidt
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  7. Tina Lütke-Eversloh
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Correspondence to Tina Lütke-Eversloh.

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Lehmann, D., Hönicke, D., Ehrenreich, A. et al. Modifying the product pattern of Clostridium acetobutylicum . Appl Microbiol Biotechnol 94, 743–754 (2012). https://doi.org/10.1007/s00253-011-3852-8

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