Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10gl−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 0.19h−1. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06h−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 microorganism

Compréhension globale de l'évolution in vivo d'Escherichia coli lors de cultures sous contraintes de rapports NADPH/NADP+ artificiellement élevés

Le métabolisme central de la souche E. coli MG1655 pgi udhA edd qor a été rationnellement modifié afin de produire deux moles de NADPH et deux moles de NADH lors de l oxydation du glucose en acétyl-CoA, alors qu une souche sauvage produit quatre moles de NADH. La conséquence de cette modification est une forte diminution de son taux de croissance sur milieu minimum et glucose. Afin d évaluer les aptitudes de cette souche à s adapter à un tel stress métabolique, son évolution in vivo a été forcée lors de cultures par repiquages successifs sur glucose. Ainsi, après quatre cultures d évolution un clone pur a été réisolé et caractérisé : un taux de croissance multiplié par six par rapport à la souche non évoluée a été mesuré. L analyse par CGS (Séquençage par comparaison de génomes) a permis de corréler l augmentation du taux de croissance à l apparition d une mutation, NuoF*(E183A), dans la sous-unité NuoF du complexe respiratoire I, complexe NADH-dépendant. Des études biochimiques et physiologiques de l impact de cette mutation ont permis de démontrer que le complexe I évolué peut oxyder à la fois le NADPH et le NADH, créant ainsi une nouvelle voie d oxydation du NADPH dans la cellule. L évolution in vivo a ensuite été poursuivie au cours de onze repiquages et un nouveau clone pur a été réisolé et caractérisé : un taux de croissance proche de la souche sauvage et onze fois supérieur à celui de la souche non évoluée a alors été mesuré. L analyse par CGS a permis cette fois de corréler l augmentation du taux de croissance à l apparition de deux mutations : NuoF*(E183A) et d une deuxième dans la sous-unité a de l ARN polymérase, rpoA*. Enfin, une deuxième souche E. coli MG1655 pfkAB udhA edd qor a été construite afin de détourner son métabolisme pour produire cette fois trois moles de NADPH et une mole de NADH lors de l oxydation du glucose en acétyl-CoA. Cette souche étant incapable de se développer en milieu liquide et glucose, une étape de criblage en milieu solide et glucose a permis de sélectionner des clones capables de croître sur glucose. Tous ces clones possédaient soit la mutation NuoF*(E183A), soit une nouvelle mutation NuoF*(E183G), dont la caractérisation biochimique a montré que les deux enzymes évoluées permettent l oxydation du NADPH par la chaîne respiratoire. Le phénomène d évolution in vivo a conduit à la création d une nouvelle fonction pour le NADPH qui n est plus seulement impliqué dans les réactions de synthèse anabolique mais qui peut être utilisé pour produire directement de l énergie catabolique. La compréhension globale du phénomène d évolution a finalement permis la conception de nouvelles souches adaptées pour la production NADPH-dépendante de composés chimiques d intérêtBacterial metabolism is characterized by robustness and plasticity that allow it to adjust too many metabolic perturbations. This present work demonstrates Escherichia coli abilities of evolution and adaptation under stress of NADPH accumulation. We constructed the E. coli MG1655 pgi::FRT udhA::FRT edd::FRT qor::FRT strain where central metabolism has been rationally engineered to produce two mol of NADPH and two mol of NADH during the oxidation of glucose to acetyl-CoA, while a wild-type strain produces 4 mol of NADH per mole of glucose. Consequently, this strain presents a weak growth on glucose mineral medium. So as to evaluate bacterial abilities to overcome such metabolic stress, in vivo evolution of this strain has been forced in laboratory by serial transfer subcultures. After four evolution subcultures, an individual clone has been characterized by a six fold increased growth rate compared to non-evolved strain. CGS (Comparative Genome Sequencing) analysis allowed us to correlate growth improvement with one mutation apparition in respiratory complex: NuoF*(E183A) in NuoF subunit from the NADH dependant complex I. Further biochemical and physiological studies demonstrated that the evolved respiratory complex is able to oxidize both NADH and NADPH, resulting in a new NADPH reoxydation pathway in the cell. In vivo evolution experiments were then continued until eleven subcultures, where a new individual clone has been characterized by an eleven fold increased growth rate compared to non-evolved strain. Additional CGS analysis allowed us to correlate growth improvement with apparition of two mutations: NuoF*(E183A) and another mutation within the RNA polymerase alpha subunit, rpoA*. Thus, a second E. coli MG1655 pfKA::FRT pfKB::FRT udhA::FRT edd::FRT qor::FRT strain has been rationally engineered to produce three mol of NADPH and one mole of NADH per mole of glucose oxidized to acetyl-coA. As this train was unable to growth in liquid glucose mineral medium, we performed a solid-state screening on glucose mineral medium that 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 an E183G mutation, both having NADH and NADPH oxidizing ability. This result highlights need of this new NADPH reoxydation pathway for NADPH accumulating cells. 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. Finally, global understanding of evolution process allowed conception of new engineered strains, well designed for NADPH dependant production of chemicals of interestTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

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

International audienceThe 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

Synthetic biology on acetogenic bacteria for highly efficient conversion of c1 gases to biochemicals

© 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

Chromosomal integration of the pSOL1 megaplasmid of Clostridium acetobutylicum for continuous and stable advanced biofuels production

Biofuel production by Clostridium acetobutylicum is compromised by strain degeneration due to loss of its pSOL1 megaplasmid. Here we used engineering biology to stably integrate pSOL1 into the chromosome together with a synthetic isopropanol pathway. In a membrane bioreactor continuously fed with glucose mineral medium, the final strain produced advanced biofuels, n-butanol and isopropanol, at high yield (0.31 g g−1), titre (15.4 g l−1) and productivity (15.5 g l−1 h−1) without degeneration

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

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

© 2021 Tomi-Andrino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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

Le métabolisme des polyosides chez Clostridium acetobutylicum (étude fonctionnelle du cellulosome)

Clostridium acetobutylicum est une bactérie sporulante, anaérobie stricte et capable de produire des solvants (acétone et butanol). Les résultats du séquençage du génome C. acetobutylicum ont révélé la présence d'un cluster de gènes codant potentiellement pour un cellulosome de structure semblable à celui de C. cellulolyticum. Ce cluster, contient dans l'ordre : cipA (protéine charpente CipA), celA (Cel48A), celB (Cel5B), celC (Cel9C), orfX (protéine non enzymatique Orfxp), celD (Cel5D), celE (Cel9E), celF (Cel9F), celG (cel5G) et celH (Cel9H). La présence d'un tel cluster est surprenante dans la mesure où C. acetobutylicum est incapable d'utiliser la cellulose comme source de carbone. La caractérisation biochimique de ce cellulosome (SDS PAGE, séquençage N terminal et Western blotting) a été entreprise. Les résultats ont permis de mettre en évidence la présence d'au moins 4 protéines cellulosomales (CipA, Cel48A, Cel9C et Cel9X) capables de s'assembler au sein d'un complexe cellulolytique de haut poids moléculaire. La caractérisation biochimique de ces composants a été effectuée.Clostridium acetobutylicum is a strictly anaerobic spore forming bacteria able to produce solvents (acetone and butanol). Analysis of the genome nucleotide sequence revealed the presence of a cellulosomal gene cluster similar to that of C. cellulolyticum. This cluster contains the following genes: cipA (scaffolding protein CipA), celA (Cel48A), celB (Cel5B), celC (Cel9C), orfX (Orfxp), celD (Cel5D), celE (Cel9E), celF (Cel9F), celG (cel5G) and celH (Cel9H). As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome is surprising. Biochemical evidence for the expression of a cellulosomal complex (SDS PAGE, N-terminal sequencing and Western blotting) was investigated. Results revealed that at least 4 major cellulosomal proteins are present (CipA, Cel48A, Cel9C and Cel9X) and are able to be associated into a large cellulolytic complex. Biochemical characterization of these components was done.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Improvement of the Genome Editing Tools Based on 5FC/5FU Counter Selection in <i>Clostridium acetobutylicum</i>

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