ABSTRACT. Microbial production of acetate from CO2 is catalyzed by the ecophysiologically as well as biotechnologically important group of acetogenic bacteria. CO2 is reduced by the Wood-Ljungdahl pathway (WLP), a two branched, linear pathway in which one CO2 is reduced to a methyl group and another to a carbonyl group that are combined by the key enzyme, the CODH/ACS to acetyl-CoA, the precursor of acetate. The carbon reduction pathway to acetate is not coupled to net ATP formation but nevertheless it allows acetogens to make a living. Net ATP synthesis is catalyzed by a respiratory chain that is hooked up to the carbon reduction pathway. The respiratory chains are different in different acetogens and are either of the Rnf- or Ech-type that are going to be discussed. Both respiratory chains use reduced ferredoxin as electron donor that is reduced with hydrogen as reductant by electron bifurcating hydrogenases. The concept in electron bifurcation in saving cellular energy will be discussed. Of critical importance is the nature of the electron carriers involved in the carbon reduction pathway: any use of reduced ferredoxin will reduce the amount of ATP synthesized. I will describe how different acetogens solved the problem of reducing CO2 to formic acid, the first step in the methyl branch of the WLP, without using reduced ferredoxin as reductant. Some use a hydrogen-dependent CO2 reductase, others an electron-bifurcating formate dehydrogenase. Acetogens are phylogenetically very different but have in common the basic chemistry of acetogenesis. However, they differ in (i) the respiratory chain used for ATP synthesis, (ii) the nature of the electron carriers used in the WLP and finally (iii) the amount of ATP synthesized. This is the more important since acetogens operate at the thermodynamic limit of life and the production of many value-added compounds from H2 + CO2 is thermodynamically restricted. At the end, I will present the entire biochemistry and bioenergetics of acetogenesis from H2 + CO2 or CO in model acetogens such as Acetobacterium woodii, Thermoanaerobacter kivui, Eubacterium limosum, Sporomusa ovata, Clostridium aceticum and Clostridium autoethanogenum and outline ways to improve the energetics for producing ATP-intensive valued-added products from H2 + CO2. "

ABSTRACT. Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

ABSTRACT. Clostridia exhibit highly diverse biochemical capabilities and hold enormous potential to produce various chemicals from renewable resources. A crucial step in unlocking this potential is the establishment of robust genetic tools, particularly systems enabling markerless gene editing. However, this availability of efficient markerless gene editing tools for a given strain often represents a major bottleneck. We refer to the development of effective markerless gene editing tools as the 'domestication' of a strain. After performing this domestication process for several Clostridia, a common workflow as well as recurring challenges have become apparent. In this talk, I will present general insights gained from working with multiple Clostridia species, with a particular focus on the domestication of Clostridium cellulovorans and Clostridium kluyveri. Additionally, I will discuss results from applying the developed gene editing systems in these organisms.

ABSTRACT. Synthesis gas conversion to products (“fermentation”) has been established with mesophilic bacteria including Acetobacterium woodii, Clostridium authoethanogenum and Clostridium ljungdahlii, and commercialized by the company LanzaTech. Alternatively, gas fermentation may be carried out at at higher temperatures, as the advantages of higher turnover rates and lower cooling costs may balance the low gas solubilities. The thermophilic acetogen Thermoanaerobacter kivui within the Clostridial order Thermoanaerobacterales (Topt 66°C) utilizes H2+CO2 or sugars as substrates (td <1.5 h) and has been adapted to thrive on CO, with acetate as metabolic product. Towards understanding its physiology and the development of an industrially-relevant platform strain, we developed a genetic system that allows for genome integration and plasmid-based protein overproduction. Recently, we added a reporter gene assay and some inducible promoters to the toolset. With collaboration partners from the Universities of Frankfurt (Prof. Volker Müller) and Göttingen (Dr. Anja Poehlein, Prof. Rolf Daniel), we studied the metabolism of T. kivui and its regulation during growth on different substrates, revealing surprises in the redox and energy metabolism. With regards to its bioenergetics, T. kivui is now the best understood thermophilic acetogen and serves as a model organism to better understand acetogenesis at high temperatures. Based on these fundamental findings, we then engineered several strains of T. kivui for ethanol production from synthesis gas. We are currently evaluating the potential of genetically modified strains and developing novel and improved methods toward a better understanding and application of T. kivui for conversion of synthesis gas at high temperature.