Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance
N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, l...
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Frontiers Media S.A.
2020-11-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2020.598392/full |
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| author | Maryna Vasylkivska Barbora Branska Karel Sedlar Katerina Jureckova Ivo Provaznik Petra Patakova |
| author_facet | Maryna Vasylkivska Barbora Branska Karel Sedlar Katerina Jureckova Ivo Provaznik Petra Patakova |
| author_sort | Maryna Vasylkivska |
| collection | DOAJ |
| description | N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering. |
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| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-20T05:39:02Z |
| publishDate | 2020-11-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-fc00549fe2e34c559cdd4003539185512022-12-21T19:51:31ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-11-01810.3389/fbioe.2020.598392598392Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol ToleranceMaryna Vasylkivska0Barbora Branska1Karel Sedlar2Katerina Jureckova3Ivo Provaznik4Petra Patakova5Department of Biotechnology, University of Chemistry and Technology, Prague, Prague, CzechiaDepartment of Biotechnology, University of Chemistry and Technology, Prague, Prague, CzechiaDepartment of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, CzechiaDepartment of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, CzechiaDepartment of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, CzechiaDepartment of Biotechnology, University of Chemistry and Technology, Prague, Prague, CzechiaN-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.https://www.frontiersin.org/articles/10.3389/fbioe.2020.598392/fullbutanol tolerancerandom chemical mutagenesissolventogenic Clostridium speciesgenome sequencebutanol efflux |
| spellingShingle | Maryna Vasylkivska Barbora Branska Karel Sedlar Katerina Jureckova Ivo Provaznik Petra Patakova Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance Frontiers in Bioengineering and Biotechnology butanol tolerance random chemical mutagenesis solventogenic Clostridium species genome sequence butanol efflux |
| title | Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance |
| title_full | Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance |
| title_fullStr | Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance |
| title_full_unstemmed | Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance |
| title_short | Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance |
| title_sort | phenotypic and genomic analysis of clostridium beijerinckii nrrl b 598 mutants with increased butanol tolerance |
| topic | butanol tolerance random chemical mutagenesis solventogenic Clostridium species genome sequence butanol efflux |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2020.598392/full |
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