Carbon dioxide (CO) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO in vitro. The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO into organic molecules at a rate of 5 nanomoles of CO per minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO fixation pathways, thereby opening the way for in vitro and in vivo applications.
One of the biggest challenges to realize a circular carbon economy is the synthesis of complex carbon compounds from one-carbon (C1) building blocks. Since the natural solution space of C1−C1 condensations is limited to highly complex enzymes, the development of more simple and robust biocatalysts may facilitate the engineering of C1 assimilation routes. Thiamine diphosphatedependent enzymes harbor great potential for this task, due to their ability to create C−C bonds. Here, we employed structure-guided iterative saturation mutagenesis to convert oxalyl-CoA decarboxylase (OXC) from Methylobacterium extorquens into a glycolyl-CoA synthase (GCS) that allows for the direct condensation of the two C1 units formyl-CoA and formaldehyde. A quadruple variant MeOXC4 showed a 100 000-fold switch between OXC and GCS activities, a 200-fold increase in the GCS activity compared to the wild type, and formaldehyde affinity that is comparable to natural formaldehyde-converting enzymes. Notably, MeOCX4 outcompetes all other natural and engineered enzymes for C1−C1 condensations by more than 40-fold in catalytic efficiency and is highly soluble in Escherichia coli. In addition to the increased GCS activity, MeOXC4 showed up to 300-fold higher activity than the wild type toward a broad range of carbonyl acceptor substrates. When applied in vivo, MeOXC4 enables the production of glycolate from formaldehyde, overcoming the current bottleneck of C1− C1 condensation in whole-cell bioconversions and paving the way toward synthetic C1 assimilation routes in vivo.
Here we describe the capabilities of laser-ablation coupled to inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) for high-speed, high-resolution, quantitative three-dimensional (3D) multielemental imaging. The basic operating principles of this instrumental setup and a verification of 3D quantitative elemental imaging are provided. To demonstrate the potential of 3D LA-ICP-TOFMS imaging, high-resolution multielement images of a cesium-infiltrated Opalinus clay rock were recorded using LA with a laser-spot diameter of 5 μm coupled to ICP-TOFMS. Quantification of elements ablated from each individual laser pulse was carried out by 100% mass normalization, and the 3D elemental concentration images generated match well with the expected distribution of elements. After laser-ablation imaging, the sample surface morphology was investigated using confocal microscopy, which showed substantial surface roughness and evidence of matrix-dependent ablation yields. Depth assignment based on ablation yields from heterogeneous materials, such as Opalinus clay rock, will remain a challenge for 3D LA-ICPMS imaging. Nevertheless, this study demonstrates quantitative 3D multielemental imaging of geological samples at a considerably higher image-acquisition speed than previously reported, while also offering high spatial resolution and simultaneous multielemental detection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.