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.
View Article and Find Full Text PDFTetanus vaccination is of major importance for public health in most countries in the world. The World Health Organization indicated that 15,000 tetanus cases were reported in 2018 (Organization, World Health, 2019). Currently, vaccine manufacturers use tetanus toxin produced by Clostridium tetani fermentation in complex media.
View Article and Find Full Text PDFAn amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDFDeveloping 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.
View Article and Find Full Text PDF[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O.
View Article and Find Full Text PDFThe 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_ΔDA 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 potential but not at the lowest potentials, where all enzymes have a similar turnover rate.
View Article and Find Full Text PDFClostridium 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 Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured.
View Article and Find Full Text PDFFeFe hydrogenases are the most efficient H-producing enzymes. However, inactivation by O 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 O diffusion within the enzyme and its reactions at the active site.
View Article and Find Full Text PDFFeFe hydrogenases catalyze H oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe(CO)(CN)(dithiomethylamine)] subcluster covalently attached to a FeS 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.
View Article and Find Full Text PDFBackground: 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.
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.
The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.
View Article and Find Full Text PDFUnlabelled: 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFWhite biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers.
View Article and Find Full Text PDFBackground: Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L.
View Article and Find Full Text PDFNature 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 H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 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 H2 to coordination positions that are normally blocked by intrinsic CO ligands.
View Article and Find Full Text PDFBy analysing the results of experiments carried out with two FeFe hydrogenases and several "channel mutants" of a NiFe hydrogenase, we demonstrate that whether or not hydrogen evolution is significantly inhibited by H2 is not a consequence of active site chemistry, but rather relates to H2 transport within the enzyme.
View Article and Find Full Text PDFClostridium 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 Δcac15ΔuppΔbuk).
View Article and Find Full Text PDFUsing 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.
View Article and Find Full Text PDFDirect electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling.
View Article and Find Full Text PDFA method for in vivo evolution of metabolic pathways in bacteria is described. This method is a powerful tool for synthetic biology type of metabolic design and can lead to the creation of new metabolic pathways or the improvement of existing metabolic enzymes. The proposed strategy also permits to relate the evolved phenotype to the genotype and to analyze evolution phenomenon at the genetic, biochemical, and metabolic levels.
View Article and Find Full Text PDFThe 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.
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