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Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1-4

  • Applied Genetics and Molecular Biotechnology
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Abstract

The selective production of acetone and butanol is highly desirable from the viewpoint of biofuel production. We have manipulated the activity level of a hydrogenase for this purpose because hydrogen and solvent production are closely correlated with each other. First, we cloned the hydrogenase gene cluster from Clostridium saccharoperbutylacetonicum strain N1-4 and downregulated its expression using an antisense RNA strategy. The cloned hydrogenase gene cluster contained three adjacent open reading frames, designated hupC, hupB, and hupA. Sequence analysis revealed that HupA could accommodate an H-cluster, which is the catalytic domain of the Fe-hydrogenase. HupB and HupC contained no H-cluster but could accommodate several Fe–S clusters. The hupCBA genes were co-transcribed, and the level of the transcript was maximized in the solventogenic phase. When the antisense RNA of the hupC upstream region (180 bp) was expressed under the bdh (encoding butanol dehydrogenase) promoter, significant reduction of hupC translation was observed, indicating that this antisense RNA is effective in strain N1-4. Production of hydrogen in the antisense transformant increased 3.1-fold. Hydrogen-evolving activity was comparable in both the control and antisense strains, but hydrogen uptake activity significantly decreased in the antisense strain (13% remaining). These results indicate that the HupCBA proteins are involved in hydrogen uptake. Importantly, the level of acetone in the antisense transformant increased 1.6-fold, and butanol production decreased to 75.6% compared to the control strain. Thus, we successfully altered solvent productivity by controlling electron flow in an acetone/butanol-producing Clostridium species.

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Acknowledgment

This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO).

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  1. Kensuke Furukawa

    Present address: Department of Food and Bioscience, Beppu University, Kitaishigaki 82, Beppu, Oita, 874-8501, Japan

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  1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581, Japan

    Shun-ichi Nakayama, Tomoyuki Kosaka, Hanako Hirakawa, Kentaro Matsuura, Sadazo Yoshino & Kensuke Furukawa

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  1. Shun-ichi Nakayama
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Correspondence to Kensuke Furukawa.

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Nakayama, Si., Kosaka, T., Hirakawa, H. et al. Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1-4. Appl Microbiol Biotechnol 78, 483–493 (2008). https://doi.org/10.1007/s00253-007-1323-z

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