No abstract

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

14Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial 15 feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant 16 hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator 17 metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we 18 demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore 19 the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By 20 integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the 21 synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism 22 and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and 23 quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing 24 the formate bioeconomy. 25 26 27 28 Microbial biosynthesis offers an environmentally friendly alternative to fossil-based production. However, the 31 limited availability and questionable sustainability of microbial feedstocks hamper the expansion of 32 biotechnological production and the establishment of a circular carbon economy. The common substrates for 33 microbial bioproduction are plant-based sugars, the utilization of which competes with food supply and 34 necessitates vast land use that negatively impacts the environment. Moreover, alternative feedstocks, such 35 as lignocellulosic biomass, suffer from crucial drawbacks, such as difficult and expensive processing 1 . A 36 fundamental limitation of all photosynthesis-based resources is the low energy conversion efficiency 37 associated with this process, typically below 1% 2,3 . 38Electromicrobial production has gained attention as an alternative route towards sustainable biotechnology 4,5 . 39 This strategy is based on the use of two key feedstocks: CO2-free electricitye.g. from solar, wind, hydro -40 the production of which is rapidly growing, and CO2, a virtually unlimited carbon source, captured either from 41 point sources or directly from air. Some microbes can grow by receiving electrons directly from a cathode; 42 however, low current densities limit the economic viability of this approach 6,7 . A more feasible option is the 43 electrochemical production of small reduced compounds 6 that are subsequently fed to microbes and then 44 converted into value-added chemicals. Among the possible mediator compounds, hydrogen, carbon 45 monoxide, and formate can be produced at high efficiency and rate 8 . Whereas hydrogen and carbon monoxide 46 are gases of low solubility, formate is completely miscible and can be readily introduced to microbial cells 47 without mass transfer limitations and without major safety concerns 9 . Hence, establishing a "formate bio-48 economy" has been proposed as a ro...

Mutualistic exchange of metabolites can play an important role in microbial communities. Under natural environmental conditions, such exchange may be compromised by the dispersal of metabolites and by the presence of non-cooperating microorganisms. Spatial proximity between members during sessile growth on solid surfaces has been shown to promote stabilization of cross-feeding communities against these challenges. Nonetheless, many natural cross-feeding communities are not sessile but rather pelagic and exist in turbulent aquatic environments, where partner proximity is often achieved via direct cell-cell adhesion, and cooperation occurs between physically associated cells. Partner association in aquatic environments could be further enhanced by motility of individual planktonic microorganisms. In this work, we establish a model bipartite cross-feeding community between bacteria and yeast auxotrophs to investigate the impact of direct adhesion between prokaryotic and eukaryotic partners and of bacterial motility in a stirred mutualistic co-culture. We demonstrate that adhesion can provide fitness benefit to the bacterial partner, likely by enabling local metabolite exchange within co-aggregates, and that it counteracts invasion of the community by a non-cooperating cheater strain. In a turbulent environment and at low cell densities, fitness of the bacterial partner and its competitiveness against a non-cooperating strain are further increased by motility that likely facilitates partner encounters and adhesion. These results suggest that, despite their potential fitness costs, direct adhesion between partners and its enhancement by motility may play key roles as stabilization factors for metabolic communities in turbulent aquatic environments.