Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production
ABSTRACT Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased bu...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2019-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mBio.02683-18 |
| _version_ | 1818900993811677184 |
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| author | Changhee Cho Seungpyo Hong Hyeon Gi Moon Yu-Sin Jang Dongsup Kim Sang Yup Lee |
| author_facet | Changhee Cho Seungpyo Hong Hyeon Gi Moon Yu-Sin Jang Dongsup Kim Sang Yup Lee |
| author_sort | Changhee Cho |
| collection | DOAJ |
| description | ABSTRACT Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity. IMPORTANCE Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity. |
| first_indexed | 2024-12-19T20:12:41Z |
| format | Article |
| id | doaj.art-28a3def676c448ebab41cf435773928a |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-19T20:12:41Z |
| publishDate | 2019-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-28a3def676c448ebab41cf435773928a2022-12-21T20:07:15ZengAmerican Society for MicrobiologymBio2150-75112019-02-0110110.1128/mBio.02683-18Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol ProductionChanghee Cho0Seungpyo Hong1Hyeon Gi Moon2Yu-Sin Jang3Dongsup Kim4Sang Yup Lee5Metabolic Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of KoreaResearch Division of Food Functionality, Korea Food Research Institute, Wanju-gun, Jeollabuk-do, Republic of KoreaMetabolic Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of KoreaMetabolic Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of KoreaDepartment of Bio and Brain Engineering, KAIST, Daejeon, Republic of KoreaMetabolic Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of KoreaABSTRACT Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity. IMPORTANCE Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.https://journals.asm.org/doi/10.1128/mBio.02683-18Clostridium acetobutylicumaldehyde/alcohol dehydrogenasebutanol selectivitymetabolic engineeringprotein engineering |
| spellingShingle | Changhee Cho Seungpyo Hong Hyeon Gi Moon Yu-Sin Jang Dongsup Kim Sang Yup Lee Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production mBio Clostridium acetobutylicum aldehyde/alcohol dehydrogenase butanol selectivity metabolic engineering protein engineering |
| title | Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production |
| title_full | Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production |
| title_fullStr | Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production |
| title_full_unstemmed | Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production |
| title_short | Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production |
| title_sort | engineering clostridial aldehyde alcohol dehydrogenase for selective butanol production |
| topic | Clostridium acetobutylicum aldehyde/alcohol dehydrogenase butanol selectivity metabolic engineering protein engineering |
| url | https://journals.asm.org/doi/10.1128/mBio.02683-18 |
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