K RE UZ E R. 1999. Diets containing either coconut oil or rumen-protected fat (54 g kg −1 dry matter each) were supplied to Rumen Simulation Technique fermenters filled with faunated and defaunated rumen fluid in a 2 × 2 factorial design. Defaunation immediately reduced methane formation by about 40% with each diet. With coconut oil, methane gradually declined in faunated and defaunated rumen fluid. Finally, the extent of methane suppression was similar, both with coconut oil and with defaunation. Independently of the status of protozoa, the population of methanogens in rumen fluid was significantly reduced by coconut oil. The results suggest that defaunation and coconut oil independently and additively suppress rumen methanogenesis.

The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

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.

No abstract

We study the Moran process as adapted by Lieberman, Hauert and Nowak. This is a model of an evolving population on a graph or digraph where certain individuals, called "mutants" have fitness r and other individuals, called "non-mutants" have fitness 1. We focus on the situation where the mutation is advantageous, in the sense that r > 1. A family of digraphs is said to be strongly amplifying if the extinction probability tends to 0 when the Moran process is run on digraphs in this family. The most-amplifying known family of digraphs is the family of megastars of Galanis et al. We show that this family is optimal, up to logarithmic factors, since every strongly-connected n-vertex digraph has extinction probability Ω(n −1/2 ). Next, we show that there is an infinite family of undirected graphs, called dense incubators, whose extinction probability is O(n −1/3 ). We show that this is optimal, up to constant factors. Finally, we introduce sparse incubators, for varying edge density, and show that the extinction probability of these graphs is O(n/m), where m is the number of edges. Again, we show that this is optimal, up to constant factors. *