Search

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.

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.