title: Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.
abstract: Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.
authors: Food and Biobased Research Wageningen UR, Wageningen, the Netherlands.|Biotechnology Department, IFP Energies nouvelles, Rueil-Malmaison, France.
authors: H Máté de
authors: HM
authors: Gérando
authors: Biotechnology Department, IFP Energies nouvelles, Rueil-Malmaison, France.
authors: F
authors: F
authors: Fayolle-Guichard
authors: Biotechnology Department, IFP Energies nouvelles, Rueil-Malmaison, France.
authors: L
authors: L
authors: Rudant
authors: Food and Biobased Research Wageningen UR, Wageningen, the Netherlands.
authors: S K
authors: SK
authors: Millah
authors: Biotechnology Department, IFP Energies nouvelles, Rueil-Malmaison, France.
authors: F
authors: F
authors: Monot
authors: Biotechnology Department, IFP Energies nouvelles, Rueil-Malmaison, France. Nicolas.lopes-ferreira@ifpen.fr.
authors: N
authors: N
authors: Lopes Ferreira
authors: Food and Biobased Research Wageningen UR, Wageningen, the Netherlands.
authors: A M
authors: AM
authors: López-Contreras
mesh_terms: D019840:2-Propanol / Q000302:isolation & purification / Q000494:pharmacology*; D019149:Bioreactors; D046970:Clostridium beijerinckii / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D032621:DNA Shuffling; D004361:Drug Tolerance; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods; D016296:Mutagenesis; D012997:Solvents
chemical_list: D012997:Solvents; D019840:2-Propanol
keywords: Clostridium beijerinckii; Genome shuffling; IBE fermentation; Isopropanol tolerance; Mutagenesis