Membrane-anchored HDCR nanowires drive hydrogen-powered CO2 fixation.

Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive1. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO2 fixation-hydrogen-dependent CO2 reductase (HDCR)2,3-which directly converts H2 and CO2 into formic acid. HDCR reduces CO2 with a higher activity than any other known biological or chemical catalyst4,5, and it has therefore gained considerable interest in two areas of global relevance: hydrogen storage and combating climate change by capturing atmospheric CO2. However, the mechanistic basis of the high catalytic turnover rate of HDCR has remained unknown. Here we use cryo-electron microscopy to reveal the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui. The minimum repeating unit is a hexamer that consists of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of hydrogenase Fe-S subunits, one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize through their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we show that filamentation and rapid electron transfer through the filament enhance the activity of HDCR. To investigate the structure of HDCR in situ, we imaged T. kivui cells with cryo-electron tomography and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance the stability and connectivity of HDCR to form a specialized metabolic subcompartment within the cell.

Capture of carbon dioxide and hydrogen by engineered Escherichia coli: hydrogen-dependent CO2 reduction to formate.

In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel-based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage. KEY POINTS: • A functional HDCR enzyme complex was heterologously produced in E. coli. • The metabolic bottlenecks for HDCR production were identified. • HDCR enabled E. coli cell to capture and store H2 and CO2 in the form of formate.

Formate-driven H2 production by whole cells of Thermoanaerobacter kivui.

BACKGROUND: In times of global warming there is an urgent need to replace fossil fuel-based energy vectors by less carbon dioxide (CO2)-emitting alternatives. One attractive option is the use of molecular hydrogen (H2) since its combustion emits water (H2O) and not CO2. Therefore, H2 is regarded as a non-polluting fuel. The ways to produce H2 can be diverse, but steam reformation of conventional fossil fuel sources is still the main producer of H2 gas up to date. Biohydrogen production via microbes could be an alternative, environmentally friendly and renewable way of future H2 production, especially when the flexible and inexpensive C1 compound formate is used as substrate. RESULTS: In this study, the versatile compound formate was used as substrate to drive H2 production by whole cells of the thermophilic acetogenic bacterium Thermoanaerobacter kivui which harbors a highly active hydrogen-dependent CO2 reductase (HDCR) to oxidize formate to H2 and CO2 and vice versa. Under optimized reaction conditions, T. kivui cells demonstrated the highest H2 production rates (qH2 = 685 mmol g-1 h-1) which were so far reported in the literature for wild-type organisms. Additionally, high yields (Y(H2/formate)) of 0.86 mol mol-1 and a hydrogen evolution rate (HER) of 999 mmol L-1 h-1 were observed. Finally, stirred-tank bioreactor experiments demonstrated the upscaling feasibility of the applied whole cell system and indicated the importance of pH control for the reaction of formate-driven H2 production. CONCLUSIONS: The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst for the oxidation of formate to H2 (and CO2). The existing genetic tool box of acetogenic bacteria bears further potential to optimize biohydrogen production in future and to contribute to a future sustainable formate/H2 bio-economy.

Whole-cell biocatalysis for hydrogen storage and syngas conversion to formate using a thermophilic acetogen.

BACKGROUND: In times of global climate change, the conversion and capturing of inorganic CO2 have gained increased attention because of its great potential as sustainable feedstock in the production of biofuels and biochemicals. CO2 is not only the substrate for the production of value-added chemicals in CO2-based bioprocesses, it can also be directly hydrated to formic acid, a so-called liquid organic hydrogen carrier (LOHC), by chemical and biological catalysts. Recently, a new group of enzymes were discovered in the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which catalyze the direct hydrogenation of CO2 to formic acid with exceptional high rates, the hydrogen-dependent CO2 reductases (HDCRs). Since these enzymes are promising biocatalysts for the capturing of CO2 and the storage of molecular hydrogen in form of formic acid, we designed a whole-cell approach for T. kivui to take advantage of using whole cells from a thermophilic organism as H2/CO2 storage platform. Additionally, T. kivui cells were used as microbial cell factories for the production of formic acid from syngas. RESULTS: This study demonstrates the efficient whole-cell biocatalysis for the conversion of H2 + CO2 to formic acid in the presence of bicarbonate by T. kivui. Interestingly, the addition of KHCO3 not only stimulated formate formation dramatically but it also completely abolished unwanted side product formation (acetate) under these conditions and bicarbonate was shown to inhibit the membrane-bound ATP synthase. Cell suspensions reached specific formate production rates of 234 mmol gprotein -1 h-1 (152 mmol gCDW -1 h-1), the highest rates ever reported in closed-batch conditions. The volumetric formate production rate was 270 mmol L-1 h-1 at 4 mg mL-1. Additionally, this study is the first demonstration that syngas can be converted exclusively to formate using an acetogenic bacterium and high titers up to 130 mM of formate were reached. CONCLUSIONS: The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst which makes this organism a promising candidate for future biotechnological applications in hydrogen storage, CO2 capturing and syngas conversion to formate.

Revealing formate production from carbon monoxide in wild type and mutants of Rnf- and Ech-containing acetogens, Acetobacterium woodii and Thermoanaerobacter kivui.

Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2 -dependent CO2 reductase and furthermore belong to different classes of 'Rnf'- and 'Ech-acetogens'. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood-Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g-1  h-1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.

Hydrogenation of CO2 at ambient pressure catalyzed by a highly active thermostable biocatalyst.

BACKGROUND: Replacing fossil fuels as energy carrier requires alternatives that combine sustainable production, high volumetric energy density, easy and fast refueling for mobile applications, and preferably low risk of hazard. Molecular hydrogen (H2) has been considered as promising alternative; however, practical application is struggling because of the low volumetric energy density and the explosion hazard when stored in large amounts. One way to overcome these limitations is the transient conversion of H2 into other chemicals with increased volumetric energy density and lower risk hazard, for example so-called liquid organic hydrogen carriers such as formic acid/formate that is obtained by hydrogenation of CO2. Many homogenous and heterogenous chemical catalysts have been described in the past years, however, often requiring high pressures and temperatures. Recently, the first biocatalyst for this reaction has been described opening the route to a biotechnological alternative for this conversion. RESULTS: The hydrogen-dependent CO2 reductase (HDCR) is a highly active biocatalyst for storing H2 in the form of formic acid/formate by reversibly catalyzing the hydrogenation of CO2. We report the identification, isolation, and characterization of the first thermostable HDCR operating at temperatures up to 70 °C. The enzyme was isolated from the thermophilic acetogenic bacterium Thermoanaerobacter kivui and displays exceptionally high activities in both reaction directions, substantially exceeding known chemical catalysts. CO2 hydrogenation is catalyzed at mild conditions with a turnover frequency of 9,556,000 h-1 (specific activity of 900 µmol formate min-1 mg-1) and the reverse reaction, H2 + CO2 release from formate, is catalyzed with a turnover frequency of 9,892,000 h-1 (930 µmol H2 min-1 mg-1). The HDCR of T. kivui consists of a [FeFe] hydrogenase subunit putatively coupled to a tungsten-dependent CO2 reductase/formate dehydrogenase subunit by an array of iron-sulfur clusters. CONCLUSIONS: The discovery of the first thermostable HDCR provides a promising biological alternative for a chemically challenging reaction and might serve as model for the better understanding of catalysts able to efficiently reduce CO2. The catalytic activity for reversible CO2 hydrogenation of this enzyme is the highest activity known for bio- and chemical catalysts and requiring only ambient temperatures and pressures. The thermostability provides more flexibility regarding the process parameters for a biotechnological application.