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Increasing levels of CO2 in the atmosphere have led to a growing interest into the various ways nature transforms this greenhouse gas into valuable organic compounds. Crotonyl-CoA carboxylases/reductases (Ccr's) are the most efficient biocatalysts for CO2 fixation because of their oxygen tolerance, their high catalytic rate constants and their high fidelity. The reaction mechanism involving hydride transfer from the NADPH cofactor and addition of CO2 to the reactive enolate, however, is not completely understood. In this study, we use computer simulations in combination with high-level ab initio calculations to trace the free energy landscape along two possible reaction paths: In the direct mechanism hydride transfer is immediately followed by CO2 addition whereas in the C2 mechanism a thermodynamically stable covalent adduct between the substrate and the NADPH cofactor is formed. This C2 adduct, which has been previously characterized experimentally, serves as a stable intermediate avoiding the reduction side reaction of the reactive enolate species and it is able to react with CO2 with similar kinetics as the direct reaction mechanism as confirmed by measured kinetic isotope effects. Thus, our results show that nature's most efficient CO2-fixing enzyme uses the formation of a covalent adduct as a strategy to store the reactive enolate species. The emerging microscopic picture of the CO2-fixing mechanism confirms previous experimental observations and provides new insights into how nature handles highly reactive intermediates to fix this inert greenhouse gas.

Most of our knowledge on microbial physiology and biochemistry is based on studies performed under laboratory conditions. For growing hydrogen-oxidizing anaerobic, autotrophic prokaryotes, an H2:CO2 (80:20, v/v) gas mixture is typically used. However, hydrogen concentrations in natural environments are usually low, but may vary in a wide range. Here we show that the thermophilic anaerobic bacterium Ammonifex degensii balances its autotrophic carbon fixation between two pathways depending on the H2 partial pressure. At 80% H2, favoring ferredoxin reduction, it uses the ferredoxin-dependent Wood-Ljungdahl pathway. In contrast, during growth at 10% H2, it switches to the more ATP-demanding, ferredoxin-independent Calvin-Benson cycle. The study reveals that the H2 redox potential is an important factor influencing the usage of different autotrophic pathways. This type of metabolic adjustment may be widespread in the microbial world.

One-carbon (C1) feedstocks derived from CO2and renewable electricity, such as formate, are promising substrates for sustainable production of chemicals, food and fuels. Energetically more efficient, engineered C1-fixation pathways were proposed to increase biomass yields above their natural counterparts, but have so far not been shown to achieve this. Here, we replace the native ‘energy-inefficient’ Calvin-Benson-Bassham (CBB) cycle inCupriavidus necatorby genomic integration of the synthetic reductive glycine pathway for growth on formate. Our final engineered strain reaches a higher biomass yield than the CBB-cycle-utilizing wild type, showing for the first time that efficiencies found in natural metabolism can be exceeded via a synthetic pathway. This yield increase demonstrates the potential of synthetic metabolism and is an important step towards realizing truly sustainable, economically feasible bio-based production.

In the ‘Fifth Gene Technology Report’, renowned experts provide an overview of current developments and their applications in the dynamically evolving research field of gene and biotechnologies. They examine, among other topics, genetic diagnostics, somatic gene therapy, the development of vaccines, stem cell and organoid research, green gene technology, synthetic biology, gene drives, genome editing, epigenetics and single cell analysis. In addition to reporting on the current state of affairs in this field, the authors also discuss society’s perception of gene technologies and ethical and legal issues relating to them, such as genome edit-ing, cerebral organoids and big data in personalised medicine. Moreover, the interdisciplinary task force ‘Gentechnologiebericht’ (Gene Technology Report) offers recommendations on action that could be taken in relation to the key issues. With contributions by Karla Alex, Sina Bartfeld, Meik Bittkowski, Inge Broer, Lorina Buhr, Stephan Clemens, Wolfgang Van den Daele, Hans-Georg Dederer, Tobias J. Erb, Nina Gasparoni, Heiner Fangerau, Boris Fehse, Jürgen Hampel, Louise Herde, Ferdinand Hucho, Ali Jawaid, Aida Khachatryan, Sarah Kohler, Alma Kolleck, Martin Korte, Cordula Kropp, Alfons Labisch, Markus Lehmkuhl, Melanie Leidecker-Sandmann, Annette Leßmöllmann, Isabelle M. Mansuy, Lilian Marx-Stölting, Andreas Merk, Yannick Milhahn, Fruzsina Molnár-Gábor, Stefan Mundlos, Staffan Müller-Wille, Angela Osterheider, Anja Pichl, Barbara Prainsack, Jens Reich, Marlen Reinschke, Ortwin Renn, Hans-Jörg Rheinberger, Arnold Sauter, Hannah Schickl, Silke Schicktanz, Volker Stollorz, Constanze Störk-Biber, Jochen Taupitz, Jörn Walter, Eva C. Winkler, Martin Zenke and Michael M. Zwick.

Background: Thioesters of coenzyme A participate in 5% of all enzymatic reactions and at least one third of all cellular carbon is typically metabolized through a CoA thioester. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5 – 10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance these metabolites explains the high interest to trace and quantify them in microbial cells.Results: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach detected CoA thioesters down to the level of 40 attomole and exhibited high precision and reproducibility for all microbes as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA-spectra. A succinyl-CoA synthase defective mutant of C. glutamicum, exhibited an unaffected level of succinyl-CoA, which indicated a complete compensation of the l-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the microbe. S. albus revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use on single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

Das Kapitel fasst zunächst den aktuellen Sachstand zusammen und bietet einen Ausblick auf die zukünftige Bedeutung von Organoiden für Forschung und Gesundheitsversorgung, wobei auch die Rechtslage und ethische Diskussionen berücksichtigt werden. Daran anschließend werden Handlungsempfehlungen für die Politik abgeleitet.

Polymer/fullerene bulk heterojunction solar cells consisting of PDDTP:PCBM blends have been investigated and optimized. The structural properties of the photoactive layers were studied by GID-XRD, which revealed a pronounced response of photovoltaic parameters on the polymer crystallinity. In addition to P3HT, another donor polymer was found that develops crystalline domains upon annealing. We found a clear correlation between annealing time, power conversion efficiency, and polymer crystallinity. The PDDTP crystallization ability is a promising property. This is a first step toward the synthesis of low-bandgap polymers, which likewise show the ability to form crystalline domains and may therefore lead to a substantial increase in efficiency.

In order to develop nanocarbon photoprobes for the DNA molecule estimation in fluids, we have constructed ones using hydrated C60 and C60 derivative aggregates predicting their integration by ds-, ss-DNA molecules in aqueous suspensions. Their photoresponse under UV–vis excitation depends from electronic features of biomolecular surrounding – natural ds-, ss-DNA molecules – was studied by UV–vis absorption and photoluminescence spectroscopy. We detected features in absorption spectra at 275–380 and 235–290 nm ranges and in photoluminescence spectra at 490–635 and 525–635 nm ranges, caused by the absorption and photoluminescence at interface between the C60, C60 derivative cores and the ds-, ss-DNA molecules, respectively. Spectrum analysis is based on models of electrostatic and/or hydrophobic interactions of C60, C60 derivatives with charged phosphate groups and bases of the ds-, ss-DNA molecules in aqueous suspensions.

The evolution of nanomorphology within thin solid-state films of poly(3-alkylthiophene):[6,6]-phenyl-C61 butyric acid methyl ester (P3AT:PCBM) blends during the film formation and subsequent thermal annealing is reported. In detail, the influence of the P3AT's alkyl side chain length on the polymer/fullerene phase separation is discussed. Butyl, hexyl, octyl, decyl, and dodecyl side groups are investigated. All of the P3ATs used were regioregular. To elucidate the nanomorphology, atomic force microscopy (AFM), X-ray diffraction, and optical spectroscopy are applied. Furthermore, photovoltaic devices of each of the different P3ATs have been constructed, characterized, and correlated with the nanostructure of the blends. It is proposed that the thermal-annealing step, commonly applied to these P3AT:PCBM blend films, controls two main issues at the same time: a) the crystallization of P3AT and b) the phase separation and diffusion of PCBM. The results show that PCBM diffusion is the main limiting process for reaching high device performances.

Composite films of P3HT/PCBM-(poly[3-2,5-diyl]/[6,6]-phenyl C61 butyric acid methyl ester) are widely used as an active layer in plastic solar cells. We have studied the influence of thermal annealing on nano-structural and optical properties of thin spin-coated P3HT/PCBM-films. Their structural properties were studied by X-ray diffraction in grazing incidence geometry. It was found that the crystallinity of the investigated films is drastically increased upon annealing. Furthermore, the anisotropic dielectric function of such films was determined by spectroscopic ellipsometry. Significant changes were observed both in parallel and perpendicular components of the dielectric function after annealing. These changes were attributed to the formation of crystalline domains upon annealing.

The photovoltaic action of plastic solar cells based on P3HT/PCBM- (poly[3-hexylthiophene-2,5-diyl]/[6,6]-phenyl C 61 butyric acid methyl ester)-composites depends strongly on structure of the active layer. In this work, the impact of P3HT-crystallinity on optical absorption of P3HT/PCBM-films was investigated. We observed, that both P3HT-crystallinity and optical absorption of thermally annealed films are increased in comparison to the not annealed ones. The highest crystallinity was achieved for films annealed at 125 °C. Further increase of annealing temperature leads to decrease of P3HT crystallinity. It is shown that the absorption coefficient of the films at low photon energies is proportional to the area under the X-ray-diffraction peak, which is a measure for the degree of film crystallinity.

We investigate thin poly(3-hexylthiophene-2,5-diyl)/[6,6]-phenyl C61 butyric acid methyl ester (P3HT/PCBM) films, which are widely used as active layers in plastic solar cells. Their structural properties are studied by grazing-incidence X-ray diffraction (XRD). The size and the orientation of crystalline P3HT nanodomains within the films are determined. PCBM crystallites are not detected in thin films by XRD. Upon annealing, the P3HT crystallinity increases, leading to an increase in the optical absorption and spectral photocurrent in the low-photon-energy region. As a consequence, the efficiency of P3HT/PCBM solar cells is significantly increased. A direct relation between efficiency and P3HT crystallinity is demonstrated.

We have investigated the structural and optical properties of P3OT and P3OT/fullerene thin films in view of their application as active layer in plastic solar cells. Films of these materials were prepared by spin coating from toluene solutions onto silicon substrates. Their optical properties were studied by spectroscopic ellipsometry, which provides the anisotropic dielectric function of the films. Moreover, structural properties were studied using X-ray diffraction. A close correlation between the results obtained by both methods could be found. Especially, the strong optical anisotropy of the films can be explained in terms of a preferable orientation of the polymer chains parallel to the substrate. The effect of the optical anisotropy on the performance of optoelectronic devices is discussed.

Composite films of P3HT/PCBM (poly[3-hexylthiophene-2,5-diyl]/[6,6]-phenyl C61 butyric acid methyl ester) are widely used as active layer in plastic solar cells. It was shown that the annealing of the active layer leads to increased optical absorption and therefore to improved performance of solar cells. However the origin of the improved optical absorption is not clear yet. In this work, structural and optical properties of such films were studied by X-ray diffraction and spectroscopic ellipsometry, respectively. The influence of the annealing temperature on film crystallinity and on optical absorption was studied. A direct correlation between optical absorption and film crystallinity was demonstrated, i.e. increased crystallinity leads to an increased optical absorption. The highest crystallinity and the highest optical absorption were observed for films annealed at 125 °C.

We report on nanomorphology evolution within different poly[3-alkylthiophene]/phenyl C61-butyric acid methyl ester (P3AT/PCBM) blends upon film formation and subsequent thermal annealing. A correlation between achievable solar cell performance and corresponding nanomorphology is drawn in conclusion.