Optimizing designer metabolisms in vitro: Biological carbon fixation requires several enzymes to turn CO ₂ into biomass. Although this pathway evolved in plants, algae, and microorganisms over billions of years, many reactions and enzymes could aid in the production of desired chemical products instead of biomass. Schwander et al. constructed an optimized synthetic carbon fixation pathway in vitro by using 17 enzymes—including three engineered enzymes—from nine different organisms across all three domains of life (see the Perspective by Gong and Li). The pathway is up to five times more efficient than the in vivo rates of the most common natural carbon fixation pathway. Further optimization of this and other metabolic pathways by using similar approaches may lead to a host of biotechnological applications. Science , this issue p. 900 ; see also p. 830

Significance: CO ₂ fixation is the most important biosynthesis process on Earth, enabling autotrophic organisms to synthesize their entire biomass from inorganic carbon at the expense of energy generated by photo- or chemotrophic processes. In the present study we demonstrate an autotrophy pathway that represents the most energy-efficient mechanism for fixing inorganic carbon in the presence of oxygen. This novel variant of the hydroxypropionate/hydroxybutyrate cycle appears to be common in a ubiquitous and abundant group of microorganisms that can thrive in nutrient-limited environments. This discovery offers a biochemical explanation for the remarkable ecological success of the ammonia-oxidizing archaea in extremely nutrient-limited environments typical of most of the open ocean.

Hybrid approach catches light: Plant chloroplasts enclose two major photosynthetic processes: light reactions, which generate the energy carriers adenosine triphosphate and reduced nicotinamide dinucleotide phosphate (NADPH), and dark reactions, which use these molecules to fix carbon dioxide and build biomass. Miller et al. appropriated natural components, thylakoid membranes from spinach, for the light reactions and showed that these could be coupled to a synthetic enzymatic cycle that fixes carbon dioxide within water-in-oil droplets. The composition of the droplets could be tuned and optimized and the metabolic activity monitored in real time by NADPH fluorescence (see the Perspective by Gaut and Adamala). These chloroplast-mimicking droplets bring together natural and synthetic components in a small space and are amenable to further functionalization to perform complex biosynthetic tasks. Science , this issue p. 649 ; see also p. 587

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.

Nature has evolved eight different pathways for the capture and conversion of CO ₂ , including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO ₂ -fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO ₂ fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning–guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO ₂ into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.

Darwinian evolution has given rise to all the enzymes that enable life on Earth. Mimicking natural selection, scientists have learned to tailor these biocatalysts through recursive cycles of mutation, selection and amplification, often relying on screening large protein libraries to productively modulate the complex interplay between protein structure, dynamics and function. Here we show that by removing destabilizing mutations at the library design stage and taking advantage of recent advances in gene synthesis, we can accelerate the evolution of a computationally designed enzyme. In only five rounds of evolution, we generated a Kemp eliminase—an enzymatic model system for proton transfer from carbon—that accelerates the proton abstraction step >10⁸-fold over the uncatalyzed reaction. Recombining the resulting variant with a previously evolved Kemp eliminase HG3.17, which exhibits similar activity but differs by 29 substitutions, allowed us to chart the topography of the designer enzyme’s fitness landscape, highlighting that a given protein scaffold can accommodate several, equally viable solutions to a specific catalytic problem.