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1. Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Author: Céline Foulquier, Antoine Rivière, Mathieu Heulot, Suzanna Dos Reis, Caroline Perdu, Laurence Girbal, Mailys Pinault, Simon Dusséaux, Minyeong Yoo, Philippe Soucaille, and Isabelle Meynial-Salles
Subjects: Science
Abstract: Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.
Published: 2022
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2. Improvement of the Genome Editing Tools Based on 5FC/5FU Counter Selection in Clostridium acetobutylicum

Author: Eglantine Boudignon, Céline Foulquier, and Philippe Soucaille
Subjects: Clostridium acetobutylicum, genome edition, 5FU, PyrR, Biology (General), QH301-705.5
Abstract: Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain’s genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol.
Published: 2023
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3. Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum

Author: James Millard, Alexander Agius, Ying Zhang, Philippe Soucaille, and Nigel Peter Minton
Subjects: Eubacterium limosum, Eubacterium callanderi, toxin–antitoxin systems, RelBE, CRISPR, gene editing, Biology (General), QH301-705.5
Abstract: Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.
Published: 2023
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4. Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources

Author: Guillaume Pregnon, Nigel P. Minton, and Philippe Soucaille
Subjects: Eubacterium limosum, acetogen, adaptive laboratory evolution, genome sequence, methanol, butyric acid, Biology (General), QH301-705.5
Abstract: Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.
Published: 2022
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5. An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors

Author: Celine Foulquier, Ching-Ning Huang, Ngoc-Phuong-Thao Nguyen, Axel Thiel, Tom Wilding-Steel, Julie Soula, Minyeong Yoo, Armin Ehrenreich, Isabelle Meynial-Salles, Wolfgang Liebl, and Philippe Soucaille
Subjects: Clostridium acetobutylicum, Clostridium saccharobutylicum, upp gene, 5-FU, Restrictionless, Markerless, Fuel, TP315-360, Biotechnology, TP248.13-248.65
Abstract: Abstract Background Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. Results The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect. Conclusions The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.
Published: 2019
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6. Physicochemical and metabolic constraints for thermodynamics-based stoichiometric modelling under mesophilic growth conditions.

Author: Claudio Tomi-Andrino, Rupert Norman, Thomas Millat, Philippe Soucaille, Klaus Winzer, David A Barrett, John King, and Dong-Hyun Kim
Subjects: Biology (General), QH301-705.5
Abstract: Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed.
Published: 2021
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7. Reviving the Weizmann process for commercial n-butanol production

Author: Ngoc-Phuong-Thao Nguyen, Céline Raynaud, Isabelle Meynial-Salles, and Philippe Soucaille
Subjects: Science
Abstract: Organic solvent n-butanol is produced mainly by petrochemical method. Here, the authors revive the historical Weizmann process by engineering Clostridium acetobutylicum strain and developing low pressure distillation and high cell density cultures for n-butanol continuous production at high-yield titer and productivity.
Published: 2018
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8. Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Author: Tom Wilding-Steele, Quentin Ramette, Paul Jacottin, and Philippe Soucaille
Subjects: CRISPR/Cas9, Clostridium acetobutylicum, metabolic engineering, genetic tools, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.
Published: 2021
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9. Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

Author: Sangrak Jin, Jiyun Bae, Yoseb Song, Nicole Pearcy, Jongoh Shin, Seulgi Kang, Nigel P. Minton, Philippe Soucaille, and Byung-Kwan Cho
Subjects: acetogenic bacteria, C1 gas fixation, synthetic biology, CRISPR-Cas, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract: Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.
Published: 2020
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10. Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism

Author: Ngoc-Phuong-Thao Nguyen, Sonja Linder, Stefanie K. Flitsch, Bettina Schiel-Bengelsdorf, Peter Dürre, and Philippe Soucaille
Subjects: Microbiology, QR1-502
Abstract: ABSTRACT An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2. A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. IMPORTANCE Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes.
Published: 2016
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11. A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

Author: Minyeong Yoo, Gwenaelle Bestel-Corre, Christian Croux, Antoine Riviere, Isabelle Meynial-Salles, and Philippe Soucaille
Subjects: Microbiology, QR1-502
Abstract: ABSTRACT Engineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol. IMPORTANCE Currently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production of n-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism of C. acetobutylicum and its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-state C. acetobutylicum cells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.
Published: 2015
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12. Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources

Author: Nigel Minton, Guillaume Pregnon, Philippe Soucaille, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Nottingham, UK (UON), UK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC)BB/T010630/1, and ANR-17-COBI-0002,BIOMETCHEM,Une approche de biologie des systèmes et de biologie synthétique pour la production durable de produits chimiques par fermentation anaérobie du méthanol issu de la gazéification de la biomasse(2017)
Subjects: Microbiology (medical), mineral medium, Eubacterium limosum, acetogen, adaptive laboratory evolution, genome sequence, methanol, butyric acid, proteomics, Microbiology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Virology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract: International audience; Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.
Published: 2022

13. Genome Sequence of

Author: Guillaume, Pregnon, Nigel P, Minton, and Philippe, Soucaille
Published: 2022

14. Correction to 'Steady-State Catalytic Wave-Shapes for 2-Electron Reversible Electrocatalysts and Enzymes'

Author: Vincent Fourmond, Carole Baffert, Kateryna Sybirna, Thomas Lautier, Abbas Abou Hamdan, Sébastien Dementin, Philippe Soucaille, Isabelle Meynial-Salles, Hervé Bottin, and Christophe Léger
Subjects: Colloid and Surface Chemistry, General Chemistry, Biochemistry, Catalysis
Published: 2023

15. Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Author: Céline Foulquier, Antoine Rivière, Mathieu Heulot, Suzanna Dos Reis, Caroline Perdu, Laurence Girbal, Mailys Pinault, Simon Dusséaux, Minyeong Yoo, Philippe Soucaille, Isabelle Meynial-Salles, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Nottingham, UK (UON), ANR acetoH2 PNRB 2006 ANR Bio6 BioE-001, ANR-08-BIOE-0012,BioButaFuel,Bioconversion d'hydrolysat de lignocellulose en Butanol, biocarburant de nouvelle génération de haute efficacité, à haut titre et rendement(2008), and ANR-14-CE05-0019,cellutanol,construction d'une souche d'E. coli à cellulosomes pour la conversion de la cellulose en butanol(2014)
Subjects: Clostridium, Multidisciplinary, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV]Life Sciences [q-bio], Butanols, General Physics and Astronomy, Electrons, General Chemistry, NAD, General Biochemistry, Genetics and Molecular Biology, Ferredoxin-NADP Reductase, Fermentation, Ferredoxins, Clostridium acetobutylicum, Oxidoreductases, NADP
Abstract: International audience; Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP(+) reductase and ferredoxin-NAD(+) reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP(+) reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD(+) reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.
Published: 2021

16. Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Author: Paul Jacottin, Tom Wilding-Steele, Quentin Ramette, Philippe Soucaille, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Nottingham, UK (UON), EracoBiotech, ANR-19-COBI-0004,SynConsor4Butanol,Production durable de n-butanol par des consortia artificiels grace à des approches de biologie de synthèse et de biologie des systèmes(2019), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: 0301 basic medicine, Clostridium acetobutylicum, Operon, 030106 microbiology, genetic tools, Computational biology, Biology, Genome, Article, Catalysis, Inorganic Chemistry, Metabolic engineering, lcsh:Chemistry, 03 medical and health sciences, Plasmid, CRISPR-Associated Protein 9, CRISPR, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Physical and Theoretical Chemistry, Cas9, Molecular Biology, CRISPR/Cas9, lcsh:QH301-705.5, Spectroscopy, Gene Editing, Organic Chemistry, General Medicine, biology.organism_classification, Computer Science Applications, Transformation (genetics), 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, CRISPR-Cas Systems, metabolic engineering
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.
Published: 2021

17. Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

Author: Jiyun Bae, Nicole Pearcy, Philippe Soucaille, Yoseb Song, Nigel P. Minton, Seulgi Kang, Jongoh Shin, Byung-Kwan Cho, Sangrak Jin, Korea Advanced Institute of Science and Technology (KAIST), University of Nottingham, UK (UON), Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Innovative Biomaterials Center, Intelligent Synthetic Biology Center, C1 Gas Refinery Program (2018M3D3A1A01055733), National Research Foundation of Korea (NRF) - Ministry of Science and ICT (MSIT) (2018K1A3A1A21044063), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: 0301 basic medicine, 030106 microbiology, C1 gas fixation, Review, Acetates, Natural Gas, Methane, Catalysis, acetogenic bacteria, Metabolic engineering, lcsh:Chemistry, Inorganic Chemistry, 03 medical and health sciences, Synthetic biology, chemistry.chemical_compound, Industrial Microbiology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Physical and Theoretical Chemistry, CRISPR-Cas, lcsh:QH301-705.5, Molecular Biology, Spectroscopy, Clostridium, biology, Chemistry, business.industry, Fossil fuel, Organic Chemistry, Acetogen, General Medicine, biology.organism_classification, Computer Science Applications, 030104 developmental biology, Biodegradation, Environmental, lcsh:Biology (General), lcsh:QD1-999, Carbon dioxide, Biochemical engineering, synthetic biology, business, Genetic Engineering, Carbon monoxide, Syngas
Abstract: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.
Published: 2020

18. Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism

Author: Ngoc-Phuong-Thao Nguyen, Minyeong Yoo, Philippe Soucaille, BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, UK (UON), School of Medicine, Tan Duc e-City, Duc Hoa, Tan Tao University (TTU), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Metabolic Explorer Company, European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Microbiology (medical), biologie des systèmes, Clostridium acetobutylicum, Primary metabolism, approche transcriptomique, Commodity chemicals, Systems biology, fluxomique, Proteomics, Microbiology, Metabolic engineering, Industrial Microbiology, 03 medical and health sciences, proteomics, ingénierie métabolique, Virology, Metabolomics, protéomique, Genetic Association Studies, Fluxomics, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, fungi, systems biology, Gene Expression Regulation, Bacterial, biology.organism_classification, equipment and supplies, clostridium acetobutylicum, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Metabolic regulation, biocarburant, Biofuels, Mutation, biofuel, Biochemical engineering, Genetic Engineering, metabolic engineering, Metabolic Networks and Pathways
Abstract: Clostridium acetobutylicum has received renewed interest worldwide as a promising producer of biofuels and bulk chemicals such as n-butanol, 1,3-propanediol, 1,3-butanediol, isopropanol, and butyrate. To develop commercial processes for the production of bulk chemicals via a metabolic engineering approach it is necessary to better characterize both the primary metabolism and metabolic regulation of C. acetobutylicum. Here, we review the history of the development of omics studies of C. acetobutylicum, summarize the recent application of quantitative/integrated omics approaches to the physiological analysis and metabolic engineering of this bacterium, and provide directions for future studies to address current challenges.
Published: 2020

19. Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum

Author: Christian Croux, Isabelle Meynial-Salles, Philippe Soucaille, Minyeong Yoo, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009)
Subjects: 0301 basic medicine, Butyrate kinase, Analyse des flux métaboliques, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Clostridium acetobutylicum, kinase, Metabolite, Mutant, Bactérie productrice de butyrate, Bioengineering, Butyrate, Biology, fluxomics, Applied Microbiology and Biotechnology, butanol, Phosphate Acetyltransferase, metabolic flexibility, pharmacotherapy, 03 medical and health sciences, chemistry.chemical_compound, chimiothérapie, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, genes, Gene, Alcohol dehydrogenase, system biology, gène, Primary metabolite, Gene Expression Regulation, Bacterial, Phosphotransferases (Carboxyl Group Acceptor), clostridium acetobutylicum, biology.organism_classification, Metabolic Flux Analysis, étude transcriptomique, 030104 developmental biology, Metabolic Engineering, chemistry, Biochemistry, Mutation, biology.protein, Butyric Acid, butyl alcohol, Metabolic Networks and Pathways, Biotechnology
Abstract: Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Delta buk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Delta buk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the Delta buk Delta ptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3 g g-(1)) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.
Published: 2017

20. Engineering of Phosphoserine Aminotransferase Increases the Conversion of l-Homoserine to 4-Hydroxy-2-ketobutyrate in a Glycerol-Independent Pathway of 1,3-Propanediol Production from Glucose

Author: Yujun Zhang, Wanda Dischert, An-Ping Zeng, Philippe Soucaille, Cheng-Wei Ma, Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Biopôle Clermont‐Limagne, METabolic EXplorer S.A, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), BBSRC/EPSRC Synthetic Biology Research Centre School of Life Sciences Centre for Biomolecular Sciences, University of Nottingham, China Scholarship Council, METabolic EXplorer S.A [Saint-Beauzire], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0106 biological sciences, propan-1, Glycerol, phosphoserine aminotransferase, homosérine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Mutant, Homoserine, 3-Propanediol, 01 natural sciences, Applied Microbiology and Biotechnology, 03 medical and health sciences, chemistry.chemical_compound, fermentation discontinue, 010608 biotechnology, propanediol, Escherichia coli, Phosphoserine Aminotransferase, Transaminases, 030304 developmental biology, Alcohol dehydrogenase, 0303 health sciences, biology, Chemistry, Wild type, Substrate (chemistry), protein engineering, General Medicine, Metabolic pathway, Butyrates, Glucose, Biochemistry, Metabolic Engineering, 2-diol, Propylene Glycols, Fermentation, biology.protein, Molecular Medicine, Pyruvate decarboxylase
Abstract: Conference: 12th Metabolic Engineering ConferenceLocation: Munich, GERMANYDate: JUL, 2018; Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4-hydroxy-2-ketobutyrate, a key reaction in producing 1,3-propanediol (1,3-PDO) from glucose in a novel glycerol-independent metabolic pathway. To this end, a computation-based rational approach is used to change the substrate specificity of SerC from l-phosphoserine to l-homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC(R42W/R77W), is successfully improved by 4.2-fold in comparison to the wild type when l-homoserine is used as the substrate, while its activity toward the natural substrate l-phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3-PDO production, the "homoserine to 1,3-PDO" pathway is constructed in E. coli by coexpression of SerC(R42W/R77W) with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L-1 1,3-PDO in fed-batch fermentation, which is 13-fold higher than the wild-type strain and represents an important step forward to realize the promise of the glycerol-independent synthetic pathway for 1,3-PDO production from glucose.
Published: 2019

21. An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors

Author: Tom Wilding-Steel, Armin Ehrenreich, Isabelle Meynial-Salles, Minyeong Yoo, Julie Soula, Céline Foulquier, Ching-Ning Huang, Axel Thiel, Wolfgang Liebl, Philippe Soucaille, Ngoc-Phuong-Thao Nguyen, Toulouse Biotechnology Institute (TBI), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Tan Tao University (TTU), University of Nottingham, UK (UON), 613802, KBBE Valor Plus, European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0106 biological sciences, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Clostridium acetobutylicum, lcsh:Biotechnology, Mutant, Computational biology, Biotechnologies, Management, Monitoring, Policy and Law, Clostridium saccharobutylicum, upp gene, 5-FU, Restrictionless, Markerless, Gene deletion, Gene replacement, medicine.disease_cause, 01 natural sciences, Applied Microbiology and Biotechnology, Genome, Genetic analysis, lcsh:Fuel, Metabolic engineering, 03 medical and health sciences, lcsh:TP315-360, lcsh:TP248.13-248.65, 010608 biotechnology, medicine, Gene, 0303 health sciences, Mutation, biology, 030306 microbiology, Renewable Energy, Sustainability and the Environment, Chemistry, fungi, equipment and supplies, biology.organism_classification, ddc, General Energy, Functional genomics, Biotechnology
Abstract: Background: Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.Results: The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect.Conclusions: The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.
Published: 2019

22. Physicochemical and metabolic constraints for thermodynamics-based stoichiometric modelling under mesophilic growth conditions

Author: Thomas Millat, Claudio Tomi-Andrino, John R. King, Dong-Hyun Kim, David A. Barrett, Klaus Winzer, Rupert Norman, Philippe Soucaille, University of Nottingham, UK (UON), Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), UK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC)BB/L013940/1, UK Research & Innovation (UKRI)Engineering & Physical Sciences Research Council (EPSRC)BB/L013940/1, University of Nottingham's School of Life Sciences, Maranas, Costas D., and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: Computer science, Gibbs Free Energy, Central carbon metabolism, Biochemistry, Metabolic flux analysis, Metabolites, Centrality, Biology (General), [MATH]Mathematics [math], Free Energy, Protein Metabolism, Carbon Isotopes, 0303 health sciences, Ecology, Physics, Stoichiometry, Flux balance analysis, Chemistry, Metabolic Engineering, Computational Theory and Mathematics, Modeling and Simulation, Physical Sciences, Thermodynamics, Network Analysis, Algorithms, Research Article, Mesophile, Computer and Information Sciences, QH301-705.5, In silico, Models, Biological, Metabolic engineering, Metabolic Networks, 03 medical and health sciences, Cellular and Molecular Neuroscience, Metabolomics, In vivo, Modelling and Simulation, Escherichia coli, Genetics, Computer Simulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ecology, Evolution, Behavior and Systematics, Fluxomics, 030304 developmental biology, Stochastic Processes, 030306 microbiology, Biology and Life Sciences, Experimental data, Metabolic Flux Analysis, Metabolism
Abstract: Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed., Author summary Biotechnology has benefitted from the development of high throughput methods characterising living systems at different levels (e.g. concerning genes or proteins), allowing the industrial production of chemical commodities. Recently, focus has been placed on determining reaction rates (or metabolic fluxes) in the metabolic network of certain microorganisms, in order to identify bottlenecks hindering their exploitation. Two main approaches are commonly used, termed metabolic flux analysis (MFA) and flux balance analysis (FBA), based on measuring and estimating fluxes, respectively. While the influence of thermodynamics in living systems was accepted several decades ago, its application to study biochemical networks has only recently been enabled. In this sense, a multitude of different approaches constraining well-established modelling methods with thermodynamics has been suggested. However, physicochemical parameters are generally not properly adjusted to the experimental conditions, which might affect their predictive capabilities. In this study, we have explored the reliability of currently available tools by investigating the impact of varying said parameters in the simulation of metabolic fluxes and metabolite concentration values. Additionally, our in-depth analysis allowed us to highlight limitations and potential solutions that should be considered in future studies.
Published: 2021

23. Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Author: Philippe Soucaille, Matteo Sensi, Christophe Léger, Isabelle Meynial-Salles, Charles Gauquelin, Carole Baffert, Jochen Blumberger, Laure Saujet, Robert B. Best, David De Sancho, Adam Kubas, Christophe Orain, Hervé Bottin, Vincent Fourmond, Kubas, A, Orain, C, Sancho, D, Saujet, L, Sensi, M, Gauquelin, C, Meynial-Salles, I, Soucaille, P, Bottin, H, Baffert, C, Fourmond, V, Best, R, Blumberger, J, Léger, C, Department of Physics and Astronomy [UCL London], University College of London [London] ( UCL ), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Chemistry [Cambridge, UK], University of Cambridge [UK] ( CAM ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), and Institut de Microbiologie de la Méditerranée ( IMM )
Subjects: Hydrogenase, General Chemical Engineering, Mutant, Nanotechnology, Molecular Dynamics Simulation, Molecular Dynamics, 010402 general chemistry, Electrochemistry, [ CHIM ] Chemical Sciences, 01 natural sciences, Catalysis, Diffusion, Molecular dynamics, Metalloproteins, Site-Directed, Clostridium, CHIM/03 - CHIMICA GENERALE E INORGANICA, chemistry.chemical_classification, biology, 010405 organic chemistry, Chemistry, Mutagenesis, Active site, Electrochemical Techniques, General Chemistry, 0104 chemical sciences, Oxygen, CHIM/02 - CHIMICA FISICA, Enzyme mecanisms, Enzyme, Hydrogen, Mutagenesis, Site-Directed, Oxidation-Reduction, Quantum Theory, Density functional theory, Electrocatalysis, Enzyme mechanisms, Metalloproteins, Molecular dynamics, Density functional theory, Biophysics, biology.protein, Electrocatalysis, Cysteine
Abstract: International audience; FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
Published: 2016

24. Roles of the F-domain in [FeFe] hydrogenase

Author: Isabelle Meynial-Salles, Charles Gauquelin, Isabelle André, Carole Baffert, David Guieysse, Emilien Etienne, Laurence Girbal, Emma Kamionka, Bruno Guigliarelli, Christophe Léger, Vincent Fourmond, Philippe Soucaille, Pierre Richaud, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), National French EPR network (RENARD) [IR3443], EU [1944-32670], Provence Alpes Cote d'Azur (PACA) [DEB 09-621], ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Bioénergie et Microalgues (EBM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergie et Microalgues ( EBM ), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) ( BIAM ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Direction de Recherche Fondamentale (CEA) ( DRF (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Direction de Recherche Fondamentale (CEA) ( DRF (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Hydrogenase, Clostridium acetobutylicum, Stereochemistry, [SDV]Life Sciences [q-bio], Mutant, Mutation, Missense, Biophysics, Electron transfer pathway, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, [ CHIM ] Chemical Sciences, H-cluster, 03 medical and health sciences, Electron transfer, [Fe-Fe] hydrogenase, Bacterial Proteins, Protein Domains, Hyda, Ferredoxin, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, biology, accessory domains, Cell Biology, biology.organism_classification, ferredoxin, Enzyme assay, 0104 chemical sciences, 030104 developmental biology, Enzyme, Amino Acid Substitution, chemistry, biology.protein
Abstract: International audience; The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_Delta DA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H(+)/H-2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.
Published: 2018

25. Reviving the Weizmann process for commercial n-butanol production

Author: Philippe Soucaille, Céline Raynaud, Ngoc-Phuong-Thao Nguyen, Isabelle Meynial-Salles, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), UMR5504, Centre National de la Recherche Scientifique (CNRS), School of Medicine, Tan Tao University (TTU), Biopôle Clermont-Limagne, IMAXIO, BBSRC EPSRC Synthetic Biological Research Center SBRC, School of Life Science, University of Nottingham, European Community [PEOPLE-ITN-2008-237942], Metabolic Explorer Company, Soucaille, Philippe, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Toulouse Biotechnology Institute (TBI)
Subjects: 0301 basic medicine, Clostridium acetobutylicum, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Science, General Physics and Astronomy, Biotechnologies, Wet-milling, Article, General Biochemistry, Genetics and Molecular Biology, butanol, law.invention, 03 medical and health sciences, chemistry.chemical_compound, law, n-Butanol, Glucose syrup, lcsh:Science, Distillation, Multidisciplinary, biology, Butanol, General Chemistry, Pulp and paper industry, biology.organism_classification, equipment and supplies, clostridium acetobutylicum, 030104 developmental biology, fermentation continue, chemistry, Yield (chemistry), 8. Economic growth, Fermentation, lcsh:Q, lipids (amino acids, peptides, and proteins), butyl alcohol
Abstract: Developing a commercial process for the biological production of n-butanol is challenging as it needs to combine high titer, yield, and productivities. Here we engineer Clostridium acetobutylicum to stably and continuously produce n-butanol on a mineral media with glucose as sole carbon source. We further design a continuous process for fermentation of high concentration glucose syrup using in situ extraction of alcohols by distillation under low pressure and high cell density cultures to increase the titer, yield, and productivity of n-butanol production to the level of 550 g/L, 0.35 g/g, and 14 g/L/hr, respectively. This process provides a mean to produce n-butanol at performance levels comparable to that of corn wet milling ethanol plants using yeast as a biocatalyst. It may hold the potential to be scaled-up at pilot and industrial levels for the commercial production of n-butanol., Organic solvent n-butanol is produced mainly by petrochemical method. Here, the authors revive the historical Weizmann process by engineering Clostridium acetobutylicum strain and developing low pressure distillation and high cell density cultures for n-butanol continuous production at high-yield titer and productivity.
Published: 2018

26. Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Author: Kateryna Sybirna, Carole Baffert, Pierre Ezanno, Luca De Gioia, Isabelle Meynial-Salles, Maurizio Bruschi, Hervé Bottin, Philippe Soucaille, Jochen Blumberger, C Greco, Po-hung Wang, Christophe Léger, Vincent Fourmond, and Marco Montefiori
Subjects: Hydrogenase, Flexibility (anatomy), medicine.anatomical_structure, Chemistry, Stereochemistry, General Chemical Engineering, medicine, Cluster (physics), General Chemistry, Oxidative phosphorylation
Published: 2019

27. Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases

Author: Carole Baffert, Claudio Greco, Luca Bertini, Souvik Roy, Charles Gauquelin, Isabelle Meynial-Salles, Matteo Sensi, Vincent Artero, Hervé Bottin, Luca De Gioia, Marc Fontecave, Laure Saujet, Christophe Léger, Philippe Soucaille, Vincent Fourmond, Giorgio Caserta, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Department of Earth and Environmental Sciences [Milano], Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Laboratoire de Chimie des Processus Biologiques (LCPB), Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF)-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Institut de Biologie et de Technologies de Saclay (IBITECS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Earth and Environmental Sciences, Università degli Studi di Milano-Bicocca [Milano], Laboratoire de Chimie des Processus Biologiques ( LCPB ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Collège de France ( CdF ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Laboratoire de Chimie et Biologie des Métaux ( LCBM - UMR 5249 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Collège de France - Chaire Chimie des processus biologiques, Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), CNRS, Aix Marseille Universite, INSA, CEA, ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), ANR-14-CE05-0010,HEROS,Hydrogénases résistantes à l'Oxygène(2014), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-11-IDEX-0001,Amidex,INITIATIVE D'EXCELLENCE AIX MARSEILLE UNIVERSITE(2011), Chaire Chimie des processus biologiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Sensi, M, Baffert, C, Greco, C, Caserta, G, Gauquelin, C, Saujet, L, Fontecave, M, Roy, S, Artero, V, Soucaille, P, Meynial Salles, I, Bottin, H, DE GIOIA, L, Fourmond, V, Léger, C, and Bertini, L
Subjects: Hydrogenase, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, [ CHIM ] Chemical Sciences, Catalysis, [ CHIM.CATA ] Chemical Sciences/Catalysis, Colloid and Surface Chemistry, Cluster (physics), [CHIM]Chemical Sciences, Reactivity (chemistry), Hydrogen, hydrogenase, biology, 010405 organic chemistry, Chemistry, Active site, General Chemistry, Time-dependent density functional theory, [CHIM.CATA]Chemical Sciences/Catalysis, 0104 chemical sciences, [ PHYS.PHYS.PHYS-CHEM-PH ] Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Covalent bond, Excited state, biology.protein
Abstract: International audience; FeFe hydrogenases catalyze H-2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe-2(CO)(3)(CN)(2)(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the Use of hydrogenase for the photoproduction of H-2. Here, we explore in great detail the reactivity of the excited states of the H-duster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This could be used for probing other, aspects of the photoreactivity of the H-cluster.
Published: 2016

28. Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum

Author: Philippe Soucaille, Maria Gonzalez-Pajuelo, Christian Croux, Florence Saint-Prix, Ngoc-Phuong-Thao Nguyen, Isabelle Meynial-Salles, Céline Raynaud, Jieun Lee, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), College of Life Sciences and Biotechnology, Korea University, Metabolic Explorer, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Korea University [Seoul], and Soucaille, Philippe
Subjects: 0301 basic medicine, Clostridium acetobutylicum, FLP, Mutant, génomique fonctionnelle, Management, Monitoring, Policy and Law, 7. Clean energy, Applied Microbiology and Biotechnology, Gene replacement, upp gene, Metabolic engineering, 03 medical and health sciences, ingénierie métabolique, Plasmid, resistance gene, Recombinase, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, 5-FU, Génie des procédés, Gene, Genetics, biology, Gene deletion, Renewable Energy, Sustainability and the Environment, Research, Microbiology and Parasitology, séquence d'adn, upp, gène de résistance, biology.organism_classification, Cac824I, Microbiologie et Parasitologie, gene deletion, gene replacement, FRT, Restriction enzyme, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, 030104 developmental biology, General Energy, Process Engineering, Biochemistry, biocarburant, bactérie anaérobie, biofuel, metabolic engineering, Functional genomics, Biotechnology
Abstract: Background: Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.Results: The method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLS R ) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the “marker-less” genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate.Conclusions: The restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.
Published: 2016

29. Elucidation of the roles of adhE1 and adhE2 in the primary metabolism of Clostridium acetobutylicum by combining in-frame gene deletion and a quantitative system-scale approach

Author: Minyeong Yoo, Isabelle Meynial-Salles, Philippe Soucaille, Christian Croux, Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Institut National Polytechnique (Toulouse) (Toulouse INP), European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Université de Toulouse (UT)
Subjects: 0301 basic medicine, Clostridium acetobutylicum, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, 030106 microbiology, Mutant, Chemostat, Biotechnologies, Management, Monitoring, Policy and Law, Biology, Applied Microbiology and Biotechnology, Transcriptome, 03 medical and health sciences, chemistry.chemical_compound, Gene expression, AdhE, Gene, chemistry.chemical_classification, Butanol, System-scale analysis, Renewable Energy, Sustainability and the Environment, Research, biology.organism_classification, equipment and supplies, General Energy, Enzyme, chemistry, Biochemistry, lipids (amino acids, peptides, and proteins), Biotechnology
Abstract: Background Clostridium acetobutylicum possesses two homologous adhE genes, adhE1 and adhE2, which have been proposed to be responsible for butanol production in solventogenic and alcohologenic cultures, respectively. To investigate their contributions in detail, in-frame deletion mutants of each gene were constructed and subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic, and alcohologenic chemostat cultures. Results Under solventogenesis, compared to the control strain, only ΔadhE1 mutant exhibited significant changes showing decreased butanol production and transcriptional expression changes in numerous genes. In particular, adhE2 was over expressed (126-fold); thus, AdhE2 can partially replace AdhE1 for butanol production (more than 30 % of the in vivo butanol flux) under solventogenesis. Under alcohologenesis, only ΔadhE2 mutant exhibited striking changes in gene expression and metabolic fluxes, and butanol production was completely lost. Therefore, it was demonstrated that AdhE2 is essential for butanol production and thus metabolic fluxes were redirected toward butyrate formation. Under acidogenesis, metabolic fluxes were not significantly changed in both mutants except the complete loss of butanol formation in ΔadhE2, but numerous changes in gene expression were observed. Furthermore, most of the significantly up- or down-regulated genes under this condition showed the same pattern of change in both mutants. Conclusions This quantitative system-scale analysis confirms the proposed roles of AdhE1 and AdhE2 in butanol formation that AdhE1 is the key enzyme under solventogenesis, whereas AdhE2 is the key enzyme for butanol formation under acidogenesis and alcohologenesis. Our study also highlights the metabolic flexibility of C. acetobutylicum to genetic alterations of its primary metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0507-0) contains supplementary material, which is available to authorized users.
Published: 2016

30. A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

Author: Antoine Riviere, Isabelle Meynial-Salles, Philippe Soucaille, Minyeong Yoo, Christian Croux, Gwénaëlle Bestel-Corre, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Metab Explorer, Partenaires INRAE, European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), and Soucaille, Philippe
Subjects: [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Clostridium acetobutylicum, Hydrogenase, Proteome, Systems biology, Molecular Sequence Data, Biotechnologies, Biology, Microbiology, Metabolic engineering, Enzyme activator, Virology, Metabolic flux analysis, Primary (chemistry), Gene Expression Profiling, Systems Biology, caractérisation, Sequence Analysis, DNA, clostridium acetobutylicum, biology.organism_classification, QR1-502, Metabolic Flux Analysis, Biochemistry, Metabolic Networks and Pathways, Research Article, analyse du métabolisme
Abstract: Engineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol., IMPORTANCE Currently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production of n-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism of C. acetobutylicum and its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-state C. acetobutylicum cells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.
Published: 2015

31. Mechanism of O

Author: Adam, Kubas, Christophe, Orain, David, De Sancho, Laure, Saujet, Matteo, Sensi, Charles, Gauquelin, Isabelle, Meynial-Salles, Philippe, Soucaille, Hervé, Bottin, Carole, Baffert, Vincent, Fourmond, Robert B, Best, Jochen, Blumberger, and Christophe, Léger
Subjects: Clostridium, Diffusion, Oxygen, Hydrogenase, Mutagenesis, Site-Directed, Quantum Theory, Electrochemical Techniques, Molecular Dynamics Simulation, Oxidation-Reduction, Catalysis, Article, Hydrogen
Abstract: FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of the highly reactive OH radical and hydroxylated cysteine, consistent with recent crystallographic evidence. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species at prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
Published: 2015

32. Molecular Characterization of the Glycerol-Oxidative Pathway of Clostridium butyricum VPI 1718

Author: Christian Croux, Céline Raynaud, Jieun Lee, Philippe Soucaille, Isabelle Meynial-Salles, Patricia Sarcabal, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, UMR 5504, Centre National de la Recherche Scientifique (CNRS), Korea University, Centre National de la Recherche Scientifique, Agence de l'Environment et de la Maitrise de l'Energie [94N80_0168], European Committee [FAIR-CT96-1912, QLK5-CT1999-01364], Ministry of Education, Science, and Technology [2010-0394-000], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Toulouse Biotechnology Institute (TBI), Korea University [Seoul], and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Glycerol, DOWN-REGULATION, Enzyme complex, Transcription, Genetic, Operon, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Dihydroxyacetone, Genetics and Molecular Biology, ELECTRON FLOW, SIGNAL-TRANSDUCTION SYSTEMS, ANTISENSE RNA, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, DEHYDROGENASE, Bacterial Proteins, [SDV.IDA]Life Sciences [q-bio]/Food engineering, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Amino Acid Sequence, Cloning, Molecular, Kinase activity, Molecular Biology, Clostridium butyricum, 030304 developmental biology, PHOSPHORYL DONOR, 0303 health sciences, CITROBACTER-FREUNDII, Base Sequence, biology, 030306 microbiology, DIHYDROXYACETONE KINASE, Gene Expression Regulation, Bacterial, biology.organism_classification, Molecular biology, Complementation, chemistry, Biochemistry, ESCHERICHIA-COLI, Multigene Family, Glycerol dehydrogenase, Heterologous expression, KLEBSIELLA-PNEUMONIAE, Oxidation-Reduction
Abstract: The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR , encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.
Published: 2011

33. Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity

Author: Philippe Soucaille, Clément Auriol, Gwénaëlle Bestel-Corre, Isabelle Meynial-Salles, Jean-Baptiste Claude, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Institut National de la Recherche Agronomique (INRA), UMR 5504, Centre National de la Recherche Scientifique (CNRS), Metab Explorer, Partenaires INRAE, Association Nationale de la Recherche et de la Technologie/Delegation Regionale a la Recherche et a la Technologie and METabolic Explorer (Clermont-Ferrand, France), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), and Toulouse Biotechnology Institute (TBI)
Subjects: Models, Molecular, [SDV]Life Sciences [q-bio], medicine.disease_cause, PATHWAY, Quinone Reductases, [SDV.IDA]Life Sciences [q-bio]/Food engineering, chemistry.chemical_classification, 0303 health sciences, Mutation, adaptive evolution, Multidisciplinary, Strain (chemistry), complex I, Escherichia coli Proteins, TRANSHYDROGENASE, Biological Sciences, Adaptation, Physiological, Aerobiosis, LONG-TERM EXPERIMENT, NADPH metabolism, Phenotype, NUCLEOTIDES, Biochemistry, GROWTH, Oxidation-Reduction, OXIDOREDUCTASE COMPLEX-I, METABOLISM, Biology, Cofactor, 03 medical and health sciences, Stress, Physiological, Escherichia coli, medicine, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, 030304 developmental biology, Binding Sites, 030306 microbiology, Catabolism, NADH DEHYDROGENASE FRAGMENT, NAD, Protein Structure, Tertiary, Kinetics, Glucose, Enzyme, Amino Acid Substitution, chemistry, Biocatalysis, biology.protein, CHAIN, NAD+ kinase, Directed Molecular Evolution, Genome, Bacterial, NADP, SYSTEM
Abstract: Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy.
Published: 2011

34. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase

Author: Marc Rousset, Christophe Léger, Bruno Guigliarelli, Sébastien Dementin, Thomas Lautier, Patrick Bertrand, Christine Cavazza, Juan C. Fontecilla-Camps, Fanny Leroux, Pierre-Pol Liebgott, Isabelle Meynial-Salles, Carole Baffert, Bénédicte Burlat, Pierre Ceccaldi, Philippe Soucaille, Vincent Fourmond, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Microbiologie de la Méditerranée (IMM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Azzopardi, Laure, and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Hydrogenase, Diffusion, Inorganic chemistry, Molecular Conformation, chemistry.chemical_element, Molecular Dynamics Simulation, Crystallography, X-Ray, Oxygen, Catalytic Domain, Electrochemistry, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acids, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Carbon Monoxide, Electron Spin Resonance Spectroscopy, Substrate (chemistry), Cell Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Amino acid, Kinetics, chemistry, Biophysics, Desulfovibrio, Hydrogen
Abstract: In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.
Published: 2009

35. Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

Author: Thomas Happe, Philippe Soucaille, Alexey Silakov, Laurence Girbal, Gregory von Abendroth, Christian Croux, and Sven T. Stripp
Subjects: 0303 health sciences, Hydrogenase, Clostridium acetobutylicum, biology, Renewable Energy, Sustainability and the Environment, Chemistry, Energy Engineering and Power Technology, Chlamydomonas reinhardtii, 010402 general chemistry, Condensed Matter Physics, biology.organism_classification, 01 natural sciences, Redox, 0104 chemical sciences, law.invention, 03 medical and health sciences, Fuel Technology, Biochemistry, law, Yield (chemistry), Recombinant DNA, Homologous recombination, Gene, 030304 developmental biology
Abstract: It was previously shown that Clostridium acetobutylicum is capable to over-express various [FeFe] hydrogenases although the protein yield was low. In this study we report on doubling the yield of the clostridial hydrogenase by replacing the native gene hydA1Ca with a recombinant one via homologous recombination. The purified protein HydA1Ca shows an unexpected high specific activity (up to 2257 μmol H2 min−1 mg−1) for hydrogen evolution. Furthermore, the highly active green algal hydrogenase HydA1Cr from Chlamydomonas reinhardtii was heterologously expressed in C. acetobutylicum, and purified with increased yield (1 mg protein per liter of cells) and high activity (625 μmol H2 min−1 mg−1). EPR studies demonstrate intact H-clusters for homologously and heterologously expressed [FeFe] hydrogenases in the CO-inhibited oxidized redox state, and prove the high efficiency of the C. acetobutylicum expression system.
Published: 2008

36. Electrochemical measurements of the kinetics of inhibition of two FeFe hydrogenases by O2 demonstrate that the reaction is partly reversible

Author: Isabelle Meynial-Salles, Carole Baffert, Hervé Bottin, Charles Gauquelin, Vincent Fourmond, Laure Saujet, Philippe Soucaille, Christophe Léger, Christophe Orain, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Photocatalyse et Biohydrogène (LPB), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Photocatalyse et Biohydrogène ( LPB ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène ( 2012 ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), CNRS, Aix Marseille Universite, INSA, CEA, Agence Nationale de la Recherche ANR-12-BS08-0014 ANR-14-CE05-0010, A*Midex foundation of Aix- Marseille University ANR-11-IDEX- 0001-02, Region Provence Alpes Cote d'Azur (PACA), Pole de compétitivité Capenergie, ANR-14-CE05-0010,HEROS,Hydrogénases résistantes à l'Oxygène(2014), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Iron-Sulfur Proteins, Models, Molecular, Clostridium acetobutylicum, Hydrogenase, Inorganic chemistry, Kinetics, Chlamydomonas reinhardtii, chemistry.chemical_element, Electrochemistry, Biochemistry, Oxygen, [ CHIM ] Chemical Sciences, Catalysis, Colloid and Surface Chemistry, Protein structure, Catalytic Domain, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, hydrogenase, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, chemistry.chemical_classification, biology, General Chemistry, biology.organism_classification, Combinatorial chemistry, Enzyme, chemistry, electrochemistry
Abstract: International audience; The mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al, Nat. Chem. 4 336 (2014)] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect.
Published: 2015

37. Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

Author: Jean-Paul Schwitzguébel, Paul Péringer, Laurence Girbal, Philippe Soucaille, and Christophe Collet
Subjects: Pyruvate decarboxylation, Pyruvate Synthase, Lactose, Dehydrogenase, Biochemistry, Microbiology, chemistry.chemical_compound, Adenosine Triphosphate, Lactate dehydrogenase, Genetics, Glycolysis, Biomass, Molecular Biology, Acetic Acid, Clostridium, Acetate kinase, Ethanol, L-Lactate Dehydrogenase, Acetate Kinase, Chemistry, General Medicine, Metabolism, Carbon Dioxide, NAD, beta-Galactosidase, Adenosine Diphosphate, Fermentation, Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+), Hydrogen
Abstract: The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l(-1)) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h(-1). Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h(-1). The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms.
Published: 2006

38. New Tool for Metabolic Pathway Engineering in Escherichia coli : One-Step Method To Modulate Expression of Chromosomal Genes

Author: Isabelle Meynial-Salles, Philippe Soucaille, and Marguerite A. Cervin
Subjects: Operator (biology), Molecular Sequence Data, Biology, Applied Microbiology and Biotechnology, Start codon, Gene expression, Escherichia coli, Coding region, Genomic library, Cloning, Molecular, Promoter Regions, Genetic, Gene, Gene Library, Recombination, Genetic, Genetics, Regulation of gene expression, Base Sequence, Ecology, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Chromosomes, Bacterial, Physiology and Biotechnology, Ribosomal binding site, Genetic Engineering, Food Science, Biotechnology
Abstract: A simple and highly efficient method was developed to produce a library of Escherichia coli clones that express a particular chromosomal gene at a wide range of expression levels. The basic strategy was to replace all or part of the upstream region of a coding sequence containing the elements involved in its expression (promoter, operator, gene coding for a regulator, ribosome binding site, and start codon) with a PCR-generated library of expression cassettes.
Published: 2005

39. CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Author: Emilien Etienne, Claudio Greco, Philippe Soucaille, Kateryna Sybirna, Christophe Léger, Luca Bertini, Thomas Lautier, Patrick Bertrand, Hervé Bottin, Luca De Gioia, Pierre Ezanno, Isabelle Meynial-Salles, Carole Baffert, Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Milano [Milano] (UNIMI), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR, Pole de competitivite Capenergies, European Commission [SolarH2 212508], Università degli Studi di Milano = University of Milan (UNIMI), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Baffert, C, Bertini, L, Lautier, T, Greco, C, Sybirna, K, Ezanno, P, Etienne, E, Philippe Soucaille, P, Bertrand, P, Bottin, H, Meynial Salles, I, DE GIOIA, L, and Leger, C
Subjects: Hydrogenase, Stereochemistry, In silico, [SDV]Life Sciences [q-bio], Chlamydomonas reinhardtii, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, CHLAMYDOMONAS-REINHARDTII, CARBON-MONOXIDE BINDING, chemistry.chemical_compound, Colloid and Surface Chemistry, ONLY HYDROGENASE, Catalytic Domain, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Electrochemistry, Cluster (physics), Protein Film Voltammetry, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Density Functional theory, CO binding, CHIM/03 - CHIMICA GENERALE E INORGANICA, Carbon Monoxide, ANALOGS, biology, 010405 organic chemistry, ACTIVE-SITE, Active site, General Chemistry, biology.organism_classification, 0104 chemical sciences, CHIM/02 - CHIMICA FISICA, chemistry, Iron-hydrogenasi, Protein film voltammetry, biology.protein, Quantum Theory, Carbon monoxide binding, CLOSTRIDIUM-PASTEURIANUM, Oxidation-Reduction, ENZYMES, Carbon monoxide
Abstract: International audience; Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H-2 binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe-2 subsite.
Published: 2011

40. Characterization of the cellulolytic complex (cellulosome) ofClostridium acetobutylicum

Author: Fabrice Sabathé, Anne Belaich, and Philippe Soucaille
Subjects: Cohesin domain, Clostridium acetobutylicum, Sequence analysis, Blotting, Western, Molecular Sequence Data, Cellulosomes, Clostridium cellulolyticum, Microbiology, Substrate Specificity, Cellulosome, Clostridium, Bacterial Proteins, Cellulase, Sequence Analysis, Protein, Gene cluster, Genetics, Amino Acid Sequence, Cellulose, Molecular Biology, Phylogeny, Sequence Homology, Amino Acid, biology, Membrane Proteins, Gene Expression Regulation, Bacterial, biology.organism_classification, Biochemistry, Multigene Family, Carrier Proteins
Abstract: A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.
Published: 2002

41. amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824

Author: Philippe Soucaille
Subjects: Genetics, Molecular Biology, Microbiology
Published: 2002

42. amyP, a reporter gene to study strain degeneration inClostridium acetobutylicumATCC 824

Author: Fabrice Sabathé, Emmanuel Cornillot, Christian Croux, and Philippe Soucaille
Subjects: Clostridium acetobutylicum, Glycoside Hydrolases, Molecular Sequence Data, Chemostat, Biology, Microbiology, Open Reading Frames, Bacterial Proteins, Species Specificity, Genes, Reporter, Transcription (biology), Sequence Homology, Nucleic Acid, Genetics, Extracellular, Glycoside hydrolase, Amino Acid Sequence, Cloning, Molecular, Molecular Biology, Gene, Clostridium, chemistry.chemical_classification, Reporter gene, Sequence Homology, Amino Acid, biology.organism_classification, Enzyme, Oligodeoxyribonucleotides, chemistry, Biochemistry, Sequence Alignment
Abstract: Clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus alpha-amylases. Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures. These results are in agreement with the extracellular alpha-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon.
Published: 2002

43. Molecular Characterization and Transcriptional Analysis of adhE2 , the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

Author: Isabelle Meynial-Salles, Laurence Girbal, Xinghong Yang, Philippe Soucaille, Christian Croux, and Lisa Fontaine
Subjects: Clostridium acetobutylicum, Operon, Butanol, Mutant, Alcohol oxidoreductase, Biology, biology.organism_classification, Microbiology, Molecular biology, chemistry.chemical_compound, Plasmid, Clostridium, Biochemistry, chemistry, biology.protein, Molecular Biology, Alcohol dehydrogenase
Abstract: The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum , a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep -tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2 , are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.
Published: 2002

44. The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Author: Isabelle Meynial-Salles, Jochen Blumberger, Vincent Fourmond, Carole Baffert, Marco Montefiori, Luca De Gioia, Pierre Ezanno, Claudio Greco, Philippe Soucaille, Kateryna Sybirna, Po-hung Wang, Hervé Bottin, Christophe Léger, Maurizio Bruschi, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Department of Earth and Environmental Sciences [Milano], Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Department of Physics and Astronomy [UCL London], University College of London [London] (UCL), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Department of Biotechnologies and Biosciences, Centre National de la Recherche Scientifique, Aix-Marseille Universite, Agence Nationale de la Recherche [ANR-12-BS08-0014, ANR-2010-BIOE-004], Ministero dell'Istruzione, dell'Universita e della Ricerca [Prin 2010M2JARJ], Ministry of Education, Republic of China (Taiwan), Engineering and Physical Sciences Research Council [EP/J015571/1, EP/F067496], Royal Society, Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), University of Milano-Bicocca, Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial Salles, I, Soucaille, P, Blumberger, J, Bottin, H, DE GIOIA, L, Léger, C, Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Earth and Environmental Sciences ( DEES ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), University College of London [London] ( UCL ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), and Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS )
Subjects: Iron-Sulfur Proteins, Hydrogenase, Coordination sphere, Protein Conformation, General Chemical Engineering, Phenylalanine, Oxidative phosphorylation, Hydrogenase mimic, Photochemistry, Electrocatalyst, [ CHIM ] Chemical Sciences, Catalysis, Oxidizing agent, [CHIM]Chemical Sciences, chemistry.chemical_classification, General Chemistry, Combinatorial chemistry, Kinetics, Enzyme, chemistry, Mutation, Enzyme mechanisms, Tyrosine, hydrogenases, hydrogen, density functional theory, molecular dynamics, Electrocatalysis, Oxidation-Reduction, Hydrogen
Abstract: Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H 2. In FeFe hydrogenases, H 2 oxidation occurs at the H-cluster, and catalysis involves H 2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H 2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O 2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H 2 oxidation. © 2014 Macmillan Publishers Limited.
Published: 2014

45. FeFe hydrogenase reductive inactivation and implication for catalysis

Author: Isabelle Meynial-Salles, Christophe Léger, Viviane Hajj, Philippe Soucaille, Carole Baffert, Vincent Fourmond, Kateryna Sybirna, Hervé Bottin, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), CNRS, AMU, CEA, INSA, ANR [ANR-2012-BS08-0014, ANR-2010-BIOE-004], Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), and Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS )
Subjects: chemistry.chemical_classification, Hydrogenase, biology, 010405 organic chemistry, Renewable Energy, Sustainability and the Environment, Chemistry, Active site, Oxidative phosphorylation, 010402 general chemistry, Photochemistry, [ CHIM ] Chemical Sciences, 01 natural sciences, Pollution, 0104 chemical sciences, Catalysis, Enzyme, Nuclear Energy and Engineering, Catalytic cycle, biology.protein, Environmental Chemistry, [CHIM]Chemical Sciences
Abstract: International audience; We show that FeFe hydrogenases inactivate at low potential, in a complex process that is mostly reversible. A form of the enzyme that is produced slowly and reversibly under reductive conditions has no proton activity under reductive conditions, although it can still oxidize H2 under oxidative conditions. This suggests that the so-called “super-reduced” state of the active site H-cluster is not part of the normal catalytic cycle. We also discuss our findings in relation to the optimization of H2-photoproduction devices based on FeFe hydrogenases that receive electrons from low potential photosensitizers.
Published: 2014

46. Genome Sequence and Comparative Analysis of the Solvent-Producing BacteriumClostridium acetobutylicum

Author: H M Lee, Fabrice Sabathé, George N. Bennett, Roman L. Tatusov, G Breton, Jean-Yves F. Dubois, J Hitti, R Gibson, Michael J. Daly, J Nölling, Lynn Doucette-Stamm, Yuri I. Wolf, M. V. Omelchenko, D Qiu, Eugene V. Koonin, Douglas Smith, Kira S. Makarova, Qiandong Zeng, and Philippe Soucaille
Subjects: Clostridium acetobutylicum, Molecular Sequence Data, Genetics and Molecular Biology, Sequence alignment, Bacillus subtilis, Biology, Models, Biological, Microbiology, Genome, Bacterial Proteins, Operon, Amino Acid Sequence, Molecular Biology, Gene, Conserved Sequence, Phylogeny, Genomic organization, Clostridium, Genetics, Whole genome sequencing, Base Sequence, Sequence Homology, Amino Acid, Chromosomes, Bacterial, biology.organism_classification, Enzymes, Genes, Bacterial, Horizontal gene transfer, Solvents, Sequence Alignment, Genome, Bacterial, Plasmids
Abstract: The genome sequence of the solvent-producing bacteriumClostridium acetobutylicumATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison ofC. acetobutylicumtoBacillus subtilisreveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, theC. acetobutylicumgenome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not withB. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome ofC. acetobutylicumcomprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified inB. subtilisare missing inC. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
Published: 2001

47. Regulation of Carbon and Electron Flow in Clostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

Author: Laurence Girbal, José Carlos Andrade, Kerstin Ahrens, Sylvie Saint-Amans, and Philippe Soucaille
Subjects: Glycerol, Hydrogenase, Physiology and Metabolism, Glycerol dehydratase, Electrons, Dehydrogenase, Microbiology, Phosphates, Electron Transport, chemistry.chemical_compound, Molecular Biology, Clostridium butyricum, Clostridium, biology, Chemiosmosis, Proton-Motive Force, Gene Expression Regulation, Bacterial, Metabolism, Hydrogen-Ion Concentration, biology.organism_classification, Carbon, Culture Media, Glucose, chemistry, Biochemistry, Propylene Glycols, Glycerol dehydrogenase
Abstract: The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H + ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
Published: 2001

48. Regulation of solvent production in Clostridium acetobutylicum

Author: Laurence Girbal and Philippe Soucaille
Subjects: Clostridium acetobutylicum, biology, Butanol, Bioengineering, equipment and supplies, biology.organism_classification, Chemical synthesis, Solvent, chemistry.chemical_compound, Biochemistry, chemistry, Acetone, Fermentation, Clostridiaceae, Bacteria, Biotechnology
Abstract: The production of acetone and butanol by Clostridium acetobutylicum was once one of the largest fermentation processes but, once it was no longer competitive with chemical synthesis, it was discontinued. However, the combined efforts of several laboratories have increased our knowledge of the molecular basis of solvent production and this, combined with a better understanding of the regulation of the genes responsible for solvent formation, should enable a more pragmatic approach to the construction of C. acetobutylicum strains producing high yields of specific metabolites in a selective manner.
Published: 1998

49. Steady-State Catalytic Wave-Shapes for 2-Electron Reversible Electrocatalysts and Enzymes

Author: Vincent Fourmond, Christophe Léger, Hervé Bottin, Philippe Soucaille, Abbas Abou Hamdan, Kateryna Sybirna, Thomas Lautier, Sébastien Dementin, Isabelle Meynial-Salles, Carole Baffert, Bioénergétique et Ingénierie des Protéines (BIP ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, CNRS, CEA, Aix-Marseille Univ, ANR [ANR-12-BS08-0014- 01], City of Marseilles, Region Provence Alpes Cote d'Azur (PACA), Pole de competitivite Capenergie, Region PACA, Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Université Paul Sabatier - Toulouse 3 ( UPS ), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay
Subjects: Iron-Sulfur Proteins, Models, Molecular, Hydrogenase, ANAEROBIC INACTIVATION, [SDV]Life Sciences [q-bio], NIFE HYDROGENASE, Electrons, 02 engineering and technology, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, Redox, Catalysis, Electron transfer, Colloid and Surface Chemistry, OXYGEN TOLERANCE, REDOX ENZYMES, ELECTRON-TRANSPORT, DIRECT ELECTROCHEMISTRY, H-CLUSTER, biology, [ SDV ] Life Sciences [q-bio], Chemistry, Active site, CARBON ELECTRODES, General Chemistry, Electrochemical Techniques, 021001 nanoscience & nanotechnology, Electron transport chain, 0104 chemical sciences, SUCCINATE-DEHYDROGENASE, Catalytic cycle, Intramolecular force, biology.protein, Biocatalysis, Clostridium acetobutylicum, 0210 nano-technology, Oxidation-Reduction, Chlamydomonas reinhardtii, FE-ONLY HYDROGENASES
Abstract: Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts.
Published: 2013

50. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture

Author: Simon Dusséaux, Isabelle Meynial-Salles, Philippe Soucaille, Christian Croux, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), BIOCORE European Project from the seventh framework program [FP7-241566], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0106 biological sciences, PH, Butanols, [SDV]Life Sciences [q-bio], medicine.disease_cause, 01 natural sciences, 7. Clean energy, Applied Microbiology and Biotechnology, 2-Propanol, chemistry.chemical_compound, 0303 health sciences, FERMENTATION, biology, Hydrogen-Ion Concentration, BEIJERINCKII, 2 STRAINS, Biochemistry, Biofuel, ESCHERICHIA-COLI, Clostridium acetobutylicum, INACTIVATION, Biotechnology, Plasmids, EXPRESSION, Biofuel production, Bioengineering, Metabolic engineering, 03 medical and health sciences, DEHYDROGENASE, 010608 biotechnology, Operon, parasitic diseases, medicine, Escherichia coli, Chromatography, Ethanol, 030306 microbiology, ISOPROPANOL, Butanol, Synthetic isopropanol pathway, BUTYLICUM, biology.organism_classification, body regions, chemistry, Yield (chemistry), Biofuels, Fermentation
Abstract: Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C acetobutylicum ATCC 824 Delta cac15 Delta upp Delta buk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridia! species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate](int) and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium. (C) 2013 Elsevier Inc. All rights reserved.
Published: 2013

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