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...

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Autores principales:	Changhee Cho, Seungpyo Hong, Hyeon Gi Moon, Yu-Sin Jang, Dongsup Kim, Sang Yup Lee
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Clostridium acetobutylicum aldehyde/alcohol dehydrogenase butanol selectivity metabolic engineering protein engineering Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/28a3def676c448ebab41cf435773928a
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spelling	oai:doaj.org-article:28a3def676c448ebab41cf435773928a2021-11-15T15:55:14ZEngineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production10.1128/mBio.02683-182150-7511https://doaj.org/article/28a3def676c448ebab41cf435773928a2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02683-18https://doaj.org/toc/2150-7511ABSTRACT 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.Changhee ChoSeungpyo HongHyeon Gi MoonYu-Sin JangDongsup KimSang Yup LeeAmerican Society for MicrobiologyarticleClostridium acetobutylicumaldehyde/alcohol dehydrogenasebutanol selectivitymetabolic engineeringprotein engineeringMicrobiologyQR1-502ENmBio, Vol 10, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridium acetobutylicum aldehyde/alcohol dehydrogenase butanol selectivity metabolic engineering protein engineering Microbiology QR1-502
spellingShingle	Clostridium acetobutylicum aldehyde/alcohol dehydrogenase butanol selectivity metabolic engineering protein engineering Microbiology QR1-502 Changhee Cho Seungpyo Hong Hyeon Gi Moon Yu-Sin Jang Dongsup Kim Sang Yup Lee Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production
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.
format	article
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
title	Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production
title_short	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_sort	engineering clostridial aldehyde/alcohol dehydrogenase for selective butanol production
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/28a3def676c448ebab41cf435773928a
work_keys_str_mv	AT changheecho engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction AT seungpyohong engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction AT hyeongimoon engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction AT yusinjang engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction AT dongsupkim engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction AT sangyuplee engineeringclostridialaldehydealcoholdehydrogenaseforselectivebutanolproduction
_version_	1718427203288432640

Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production

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