The design and realization of synthetic pathways for the fixation of carbon dioxide in vitro

The fixation of inorganic carbon and the conversion to organic molecules is a prerequisite for life and the foundation of the carbon cycle on Earth. Since the industrial revolution, this carbon cycle has become inbalanced and consequently the atmospheric carbon dioxide (CO2) concentration is increasing and is a major cause of global warming. On the contrary, atmospheric CO2 can also be considered as an important carbon feedstock of the future. However, human society has not yet come up with a viable solution to convert this inorganic atmospheric CO2 back into reduced carbon compounds and is still relying on natural CO2 fixation. Nature has evolved multiple solutions to reduce CO2 and incorporate it into organic molecules. The involved pathways differ in their cofactor requirements and are often limited to anoxic conditions. Many attempts have been made to improve natural carbon fixation to a more energy efficient process, but showed little success. The emerging field of synthetic biology offers an alternative approach by designing novel pathways for the fixation of CO2. Although, several such artificial pathways have been designed, none of them have been realized so far. This reveals an existing gap between the design and the realization and implementation of such a synthetic CO2 fixation pathway. In this work we designed several synthetic oxygen-tolerant CO2 fixation pathways in a bottom-up approach, by freely combining enzymes from different biological sources. The pathways were designed around an efficient central carboxylase from the family of enoyl-CoA carboxylases/reductases. Some members of this family belong to the most efficient carboxylases known so far, do not accept oxygen as a substrate and only require the ubiquitous NADPH as co-substrate. The theoretical analysis of thermodynamic and energetic properties of the designed pathways for CO2 fixation also showed that they are comparable or even more energy efficient than naturally occurring oxygen-tolerant CO2-fixing pathways. We were able to realize two of these cycles in vitro and investigated their efficiencies for the fixation of inorganic CO2 into organic molecules. We established the Crotonyl-CoA/EThylmalonyl-CoA/Hydroxybutyryl-CoA (CETCH) and HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle in vitro and their CO2 fixation efficiencies were increased in several rounds of optimization. In this process, we energized the systems by ATP- and NADPH-regeneration modules, applied the principle of metabolic proofreading to recycle undesired side products and engineered several enzymes to efficiently catalyze desired reactions. The CETCH cycle in its current version 5.4 is a reaction network of 17 enzymes originating from nine different organisms of all three domains of life. It converts CO2 into organic molecules at a rate of 5 nmol CO2 per minute and mg enzyme. In comparison, the HOPAC cycle in its current version 4.1 comprises 15 enzymes originating from eight different organisms. A stepwise incorporation of 13CO2 into the intermediates of both synthetic pathway confirmed a continuous operation for multiple rounds of conversion. During the development of the synthetic cycles for CO2 fixation, we solved a novel crystal structure of a key enzyme for both pathways, the methylsuccinyl-CoA dehydrogenase. This is a member of the well described family of flavin dependent acyl-CoA dehydrogenases. We elucidated the substrate specificity of the enzyme for (2S)-methylsuccinyl-CoA, which represents a complex substrate amongst the acyl-CoA dehydrogenase family. In summary, this study laid the foundation for the development of artificial pathways for the fixation of CO2 and narrow the gap between theoretical design of synthetic CO2 fixation pathways and their application in vivo. The CETCH and HOPAC cycle expands the solution space beyond the six naturally evolved CO2 fixation pathways by two man-made alternative that are thermodynamically more efficient than the CBB cycle of plants

High-skilled outsiders? Labor market vulnerability, education and welfare state preferences

Recent research has established that employment risk shapes social policy preferences. However, risk is often conceptualized as an alternative measure of the socio-economic status. We show that employment risk and socio-economic status are distinct, crosscutting determinants of social policy preferences. More specifically, we analyze the policy preferences of high-skilled labor market outsiders as a cross-pressured group. We first establish that labor market vulnerability has spread well into the more highly educated segments of the population. We then show that the effect of labor market vulnerability on social policy preferences even increases with higher educational attainment. We conclude that that labor market risk and educational status are not interchangeable and that the high skilled are particularly sensitive to the experience of labor market risk. Thereby, our findings point to a potential cross-class alliance between more highly and lower skilled vulnerable individuals in support of a redistributive and activating welfare state. Thus, they have far-reaching implications for our understanding of both the politicization of insider/outsider divides and the politics of welfare suppor

Rapid growth of HFC-227ea (1,1,1,2,3,3,3-Heptafluoropropane) in the atmosphere

We report the first measurements of 1,1,1,2,3,3,3-heptafluoropropane (HFC-227ea), a substitute for ozone depleting compounds, in remote regions of the atmosphere and present evidence for its rapid growth. Observed mixing ratios ranged from below 0.01 ppt in deep firn air to 0.59 ppt in the northern mid-latitudinal upper troposphere. Firn air samples collected in Greenland were used to reconstruct a history of atmospheric abundance. Year-on-year increases were deduced, with acceleration in the growth rate from 0.026 ppt per year in 2000 to 0.057 ppt per year in 2007. Upper tropospheric air samples provide evidence for a continuing growth until late 2009. Fur- thermore we calculated a stratospheric lifetime of 370 years from measurements of air samples collected on board high altitude aircraft and balloons. Emission estimates were determined from the reconstructed atmospheric trend and suggest that current "bottom-up" estimates of global emissions for 2005 are too high by more than a factor of three

Changes in the atmospheric CH4 gradient between Greenland and Antarctica during the Holocene

High-resolution records of atmospheric methane over the last 11,500 years have been obtained from two Antarctic ice cores (D47 and Byrd) and a Greenland core (Greenland Ice Core Project). These cores show similar trapping conditions for trace gases in the ice combined with a comparable sampling resolution; this together with a good relative chronology, provided by unequivocal CH4 features, allows a direct comparison of the synchronized Greenland and Antarctic records, and it reveals significant changes in the interpolar difference of CH4 mixing ratio with time. On the average, over the full Holocene records, we find an interpolar difference of 44±7 ppbv. A minimum difference of 33±7 ppbv is observed from 7 to 5 kyr B.P. whereas the maximum gradient (50±3 ppbv) took place from 5 to 2.5 kyr B.P. A gradient of 44±4 ppbv is observed during the early Holocene (11.5 to 9.5 kyr B.P). We use a three-box model to translate the measured differences into quantitative contributions of methane sources in the tropics and the middle to high latitudes of the northern hemisphere. The model results support the previous interpretation that past natural CH4 sources mainly lay in tropical regions, but it also suggests that boreal regions provided a significant contribution to the CH4 budget especially at the start of the Holocene. The growing extent of peat bogs in boreal regions would also have counterbalanced the drying of the tropics over the second half of the Holocene. Finally, our model results suggest a large source increase in tropical regions from the late Holocene to the last millennium, which may partly be caused by anthropogenic emissions

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases

Clinical outcomes of patients with estimated low or intermediate surgical risk undergoing transcatheter aortic valve implantation

Aims Transcatheter aortic valve implantation (TAVI) is an established treatment alternative to surgical aortic valve replacement in high-risk and inoperable patients and outcomes among patients with estimated low or intermediate risk remain to be determined. The aim of this study was to assess clinical outcomes among patients with estimated low or intermediate surgical risk undergoing TAVI. Methods and results Between August 2007 and October 2011, 389 consecutive patients underwent TAVI and were categorized according to the Society of Thoracic Surgeons (STS) score into low (STS 8%; n = 94, 24.2%) groups for the purpose of this study. Significant differences were found between the groups (low risk vs. intermediate risk vs. high risk) for age (78.2 ± 6.7 vs. 82.7 ± 5.7 vs. 83.7 ± 4.9, P < 0.001), body mass index (28.1 ± 6.1 vs. 26.5 ± 4.9 vs. 24.4 ± 4.6, P < 0.001), chronic renal failure (34 vs. 67 vs. 90%, P < 0.001), all-cause mortality at 30 days (2.4 vs. 3.9 vs. 14.9%, P = 0.001), and all-cause mortality at 1 year (10.1 vs. 16.1 vs. 34.5%, P = 0.0003). No differences were observed with regards to cerebrovascular accidents and myocardial infarction during 1-year follow-up. Conclusion In contemporary practice, TAVI is not limited to inoperable or STS-defined high-risk patients and should be guided by the decision of an interdisciplinary Heart Team. Compared with patients at calculated high risk, well-selected patients with STS-defined intermediate or low risk appear to have favourable clinical outcome

The response of atmospheric nitrous oxide to climate variations during the last glacial period

Detailed insight into natural variations of the greenhouse gas nitrous oxide (N₂O) in response to changes in the Earth's climate system is provided by new measurements along the ice core of the North Greenland Ice Core Project (NGRIP). The presented record reaches from the early Holocene back into the previous interglacial with a mean time resolution of about 75 years. Between 11 and 120 kyr BP, atmospheric N₂O concentrations react substantially to the last glacial-interglacial transition (Termination 1) and millennial time scale climate variations of the last glacial period. For long-lasting Dansgaard/Oeschger (DO) events, the N₂O increase precedes Greenland temperature change by several hundred years with an increase rate of about 0.8-1.3 ppbv/century, which accelerates to about 3.8-10.7 ppbv/century at the time of the rapid warming in Greenland. Within each bundle of DO events, the new record further reveals particularly low N₂O concentrations at the approximate time of Heinrich events. This suggests that the response of marine and/or terrestrial N₂O emissions on a global scale are different for stadials with and without Heinrich events.Keywords: N2O, Variability, Lifetimes, Ocean circulation, Late pleistocene, Millennial scale, Monsoon, Record, North Atlantic, Ice coresKeywords: N2O, Variability, Lifetimes, Ocean circulation, Late pleistocene, Millennial scale, Monsoon, Record, North Atlantic, Ice core