Improving isopropanol tolerance and production of Clostridiumbeijerinckii DSM 6423 by random mutagenesisand genome shuffling

• Published in: Applied microbiology and biotechnology Vol. 100; no. 12
• Main Authors: Màté de Gérando, H., Fayolle-Guichard, Françoise, Rudant, L., Millah, S. K., Monot, Frédéric, Lopes Ferreira, Nicolas, Lopez-Contreras, A. M.
• Format: Journal Article
• Language: English
• Published: Springer Verlag 05.06.2016
• Subjects: Chemical Sciences; Genome shuffling; Mutagenesis; IBE fermentation; Clostridium beijerinckii; Isopropanol tolerance
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-016-7302-5

Random mutagenesis and genome shuffling wasapplied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteriaClostridium beijerinckii DSM 6423. Following chemical mutagenesiswith N-methyl-N-nitro-N-nitrosoguanidine (NTG),screening of putatively improved strains was done by submittingthe mutants to toxic levels of inhibitory chemicals or byscreening for their tolerance to isopropanol (>35 g/L). Suicidesubstrates, such as ethyl or methyl bromobutyrate or alcoholdehydrogenase inhibitors like allyl alcohol, were tested and,finally, 36 mutants were isolated. The fermentation profiles ofthese NTG mutant strains were characterized, and the bestperforming mutants were used for consecutive rounds of genomeshuffling. Screening of strains with further enhancementin isopropanol tolerance at each recursive shuffling step wasthen used to spot additionally improved strains. Three highlytolerant strains were finally isolated and able to withstand upto 50 g/L isopropanol on plates. Even if increased tolerance tothe desired end product was not always accompanied byhigher production capabilities, some shuffled strains showedincreased solvent titers compared to the parental strains andthe original C. beijerinckii DSM 6423. This study confirmsthe efficiency of genome shuffling to generate improvedstrains toward a desired phenotype such as alcohol tolerance.This tool also offers the possibility of obtaining improvedstrains of Clostridium species for which targeted genetic engineeringapproaches have not been described yet.