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σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Abstract : the solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. this strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ 54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and Rt-qpCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ 54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052. In the context of worldwide energy transition, research for alternatives to fossil fuels has become a priority. In particular, the replacement of petrochemistry by a low carbon emission industry has been a major challenge as our global consumption of petrochemicals keeps on increasing 1. The valorization of plant biomass to synthesize ethanol by microbial fermentation has already been pioneered for biofuel production 2 and could therefore be applied to bio-based chemistry 3. A few strains from the Clostridium genus are naturally able to produce isopropanol and butanol 4,5 , two compounds that could be used as biochemical and biofuel, respectively. However, those organisms are not producing these metabolites in quantities compatible with an economically viable industrial process 6. However, with the increasing availability of efficient genetic tools in Clostridia 7 , metabolic engineering approaches could be undertaken to enhance solvent productivity. Clostridium beijerinckii DSM 6423 (NRRL B-593) is the only natural isopropanol-butanol producing strain whose genome and transcriptome have been investigated 8. It may therefore be the best candidate for genetic engineering, although its particular physiology is still poorly understood. For this purpose, gaining additional knowledge on metabolism regulation in this strain would greatly benefit future optimization efforts. In particular, identifying the molecular effectors controlling solvent production may provide valuable insights to define adequate genetic engineering strategies. As no genetic toolbox was available for this particular strain, Máté de Gerando and coworkers performed random mutagenesis coupled with genome shuffling to increase isopropanol productivity by selecting isopropanol-tolerant strains 9. In this work, random mutagenesis followed by allyl alcohol selection also generated an interesting mutant, further referred to as AA mutant. This strain mainly produces acids, shows no isopropanol production and a strongly attenuated butanol synthesis capacity. These results are consistent with those obtained in Clostridium acetobutylicum DSM 1792, in which mutants obtained in the presence of allyl alcohol-precursor of the highly toxic acrolein molecule in the reaction catalyzed by alcohol dehydrogenases-permitted the selection of butanol-deficient strains 10. Nevertheless, in both cases the key mutated genes causing these phenotypes have not been clearly identified.
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Submitted on : Tuesday, June 18, 2019 - 10:53:00 AM
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Rémi Hocq, Maxime Bouilloux-Lafont, Nicolas Lopes Ferreira, François Wasels. σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii. Scientific Reports, Nature Publishing Group, 2019, 9 (1), pp.7228. ⟨10.1038/s41598-019-43822-2⟩. ⟨hal-02158584⟩



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