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Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase produ...

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Autores principales: Omorotionmwan, Bunmi B., Wang, Hengzheng, Baker, Jonathan P., Gizynski, Krzysztof, Yoo, Minyeong, Akaluka, Cynthia, Zhang, Ying, Minton, Nigel P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10312008/
https://www.ncbi.nlm.nih.gov/pubmed/37397966
http://dx.doi.org/10.3389/fbioe.2023.1218099
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author Omorotionmwan, Bunmi B.
Wang, Hengzheng
Baker, Jonathan P.
Gizynski, Krzysztof
Yoo, Minyeong
Akaluka, Cynthia
Zhang, Ying
Minton, Nigel P.
author_facet Omorotionmwan, Bunmi B.
Wang, Hengzheng
Baker, Jonathan P.
Gizynski, Krzysztof
Yoo, Minyeong
Akaluka, Cynthia
Zhang, Ying
Minton, Nigel P.
author_sort Omorotionmwan, Bunmi B.
collection PubMed
description The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::P( bgaL ) promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the P( tcdB ) promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::P( bgaL ) and over 2-fold greater than achieved using the strong P( fdx ) promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.
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spelling pubmed-103120082023-07-01 Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum Omorotionmwan, Bunmi B. Wang, Hengzheng Baker, Jonathan P. Gizynski, Krzysztof Yoo, Minyeong Akaluka, Cynthia Zhang, Ying Minton, Nigel P. Front Bioeng Biotechnol Bioengineering and Biotechnology The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::P( bgaL ) promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the P( tcdB ) promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::P( bgaL ) and over 2-fold greater than achieved using the strong P( fdx ) promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture. Frontiers Media S.A. 2023-06-16 /pmc/articles/PMC10312008/ /pubmed/37397966 http://dx.doi.org/10.3389/fbioe.2023.1218099 Text en Copyright © 2023 Omorotionmwan, Wang, Baker, Gizynski, Yoo, Akaluka, Zhang and Minton. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Omorotionmwan, Bunmi B.
Wang, Hengzheng
Baker, Jonathan P.
Gizynski, Krzysztof
Yoo, Minyeong
Akaluka, Cynthia
Zhang, Ying
Minton, Nigel P.
Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title_full Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title_fullStr Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title_full_unstemmed Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title_short Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum
title_sort chromosomal engineering of inducible isopropanol- butanol-ethanol production in clostridium acetobutylicum
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10312008/
https://www.ncbi.nlm.nih.gov/pubmed/37397966
http://dx.doi.org/10.3389/fbioe.2023.1218099
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