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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Global patterns of forest autotrophic carbon fluxes

Carbon (C) fixation, allocation, and metabolism by trees set the basis for energy and material flows in forest ecosystems and define their interactions with Earth’s changing climate. However, we lack a cohesive synthesis on how forest carbon fluxes vary globally with respect to climate and one another. Here, we draw upon 1,319 records from the Global Forest Carbon Database (ForC), representing all major forest types and the nine most significant autotrophic carbon fluxes, to comprehensively explore how C cycling in mature, undisturbed forests varies with latitude and climate on a global scale. We show that, across all flux variables analyzed, C cycling decreases continuously with absolute latitude – a finding that confirms multiple previous studies but contradicts the idea that net primary productivity of temperate forests rivals that of tropical forests. C flux variables generally displayed similar trends across latitude and multiple climate variables, with no differences in allocation detected at this global scale. Temperature variables in general, and mean annual temperature and temperature seasonality in particular, were the best univariate predictors of C flux, explaining 19 - 71% of variation in the C fluxes analyzed. The effects of temperature were modified by moisture availability, with C flux reduced under hot and dry conditions and sometimes under very high precipitation. C fluxes increased with growing season length, but this was never the best univariate predictor. Within the growing season, the influence of climate on C cycling was small but significant for a number of flux variables. These findings clarify how forest C flux varies with latitude and climate on a global scale. In a period of accelerating climatic change, this improved understanding of the fundamental climatic controls on forest C cycling sets a foundation for understanding patterns of change.

Banbury Morgan, Rebecca↗
DOE OSTI2022

Spatiotemporal Metabolic Network Models Reveal Complex Autotroph-Heterotroph Biofilm Interactions Governed by Photon Incidences

Autotroph-heterotroph interactions are ubiquitous in natural environment and play a key role in controlling various essential ecosystem functions, such as production and utilization of organic matter, cycling of nitrogen, sulfur, and other chemical elements. Understanding how these biofilm metabolic interactions are constrained in space and time remains challenging because fully predictive models designed for this purpose are currently limited. Toward filling this gap, here we developed community metabolic network models for two autotroph-heterotroph biofilm consortia (termed UCC-A and UCC-O), which share a suite of common heterotrophic members but have a single distinct photoautotrophic cyanobacterium (Phormidesmis priestleyi str. ANA and Phormidium sp. OSCR) that provides organic carbon and nitrogen sources to support the growth of heterotrophic partners. After determining model parameters by data fitting using the spatiotemporal distributions of microbial abundances, we comparatively analyzed the resulting biofilm models to examine any fundamental differences in microbial interactions between the two consortia under the variation of key environmental variables: CO2 and photon levels. The UCC-A model predicted generally expected responses, i.e., the autotroph population increased in response to elevated levels of CO2 and photon, followed by increase in the heterotroph population. In contrast, the UCC-O model showed somewhat complicated dynamics, e.g., higher photon incidence rates resulted in the increase in autotroph population but decrease in heterotroph population due to the lowered provision of glucose from the autotroph. A further analysis showed that species coexistence was governed by the photon incidences rather than the carbon availability for UCC-O, which was the opposite for UCC-A.

Phalak, Poonam↗
DOE OSTI2020

Extracellular electron uptake by autotrophic microbes: physiological, ecological, and evolutionary implications

Abstract Microbes exchange electrons with their extracellular environment via direct or indirect means. This exchange is bidirectional and supports essential microbial oxidation–reduction processes, such as respiration and photosynthesis. The microbial capacity to use electrons from insoluble electron donors, such as redox-active minerals, poised electrodes, or even other microbial cells is called extracellular electron uptake (EEU). Autotrophs with this capability can thrive in nutrient and soluble electron donor-deficient environments. As primary producers, autotrophic microbes capable of EEU greatly impact microbial ecology and play important roles in matter and energy flow in the biosphere. In this review, we discuss EEU-driven autotrophic metabolisms, their mechanism and physiology, and highlight their ecological, evolutionary, and biotechnological implications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source

ABSTRACT Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures (“py”’) demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c - and b -type cytochromes was observed in “py,” pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells’ photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(−), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet’s history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Thermal acclimation of plant photosynthesis and autotrophic respiration in a northern peatland

Peatlands contain one-third of global soil carbon (C), but the responses of peatland ecosystems to long-term warming are not well understood. Here, we pursue an emergent understanding of warming effects on ecosystem C fluxes at peatlands by constraining a process-oriented model, the terrestrial ECOsystem model, with observational data from a long-term warming experiment at the Spruce and Peatland Responses Under Changing Environments site. Model-based assessments show that ecosystem-level photosynthesis and autotrophic respiration exhibited significant thermal acclimation, with temperature sensitivities being linearly decreased with warming. Using the thermal-acclimated parameter values, simulated gross primary production, net primary production, and plant autotrophic respiration (R a ), were all lower than those simulated with non-thermal acclimated parameter values. In contrast, ecosystem respiration simulated with thermal acclimated parameter values was higher than that simulated with non-thermal acclimated parameter values. Net ecosystem CO 2 exchange was much higher after constraining model parameters with observational data from the warming treatments, releasing C at a rate of 28.3 g C m -2 yr -1 °C -1 . Our data-model integration study suggests that peatlands are likely to release more C than previously estimated. Earth system models may overestimate C uptake by peatlands under warming if physiological thermal acclimation of plants is not incorporated. Thus, it is critical to consider the long-term physiological thermal acclimation of plants in the models to better predict global C dynamics under future climate and their feedback to climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Functional variability in specific root respiration translates to autotrophic differences in soil respiration in a temperate deciduous forest

CO 2 release from forest soils (R s ) is a prominent flux in the global carbon cycle. Rs is derived from roots (autotrophic respiration, R a ) and microbial (heterotrophic) respiration and is highly dynamic, as it depends on edaphic and environmental conditions as well as root functional traits and microbial community composition. It is unclear how root functional traits affect root and microbial respiration rates; however, their consideration may help parse out the relative contributions of root and microbial respiration to R s . At a temperate forest site, root systems of 3–4 functional root orders and their surrounding surface soil were carefully excavated and placed into custom trays designed to repeatedly measure R s in situ on eight temperate tree species that varied in their root functional strategies and mycorrhizal affinity. R s was measured bi-weekly to monthly for nearly one year using a custom chamber attached to a gas exchange system. R s varied over time, ranging from 0.3 to 12 µmol m -2 s -1 . Comparable root systems of the same species were excised from the soil and specific root respiration rates (R r ) were measured. Rr ranged from 2.5 to 9.0 nmol g -1 s -1 and was negatively correlated with root tissue density and positively related to root tissue nitrogen concentration. Using R r to estimate R a , we estimate that R a accounts for <10%, on average 2–3%, of R s for individual root systems (averaging 1.2 g dry biomass) housed in surrounding soil (average 1.3 kg dry mass) in situ; thus, Ra was roughly 20 times greater than Rh per unit mass. The contribution of R a peaked in the fall and coincided with leaf senescence of the forest canopy. A soil-sterilizing experimental treatment designed to help isolate R a in situ reduced bacterial biomass and shifted fungal community composition, but there was no reduction in Rs of the in-situ root-soil tray systems. The relative R a to R s ratio increased with root functional strategies characterized by greater specific root length and tip abundance, but also to greater root tissue density. The ratio of R a to R s also increased with warmer soil temperatures and decreased slightly with increasing soil moisture. We discuss how incorporating root functional traits as modulators of the autotrophic contribution to R s could be considered when modeling total soil CO 2 efflux from forests.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments

To facilitate the synthesis of biomass from CO 2 , autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multi-subunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found.

carbon fixation↗
DOE OSTI2022

Trace metal contents of autotrophic flagellates from contrasting open‐ocean ecosystems

Abstract Nano‐ and picophytoplankton are a major component of open‐ocean ecosystems and one of the main plankton functional types in biogeochemical models, yet little is known about their trace metal contents. In cultures of the picoeukaryote Ostreococcus lucimarinus , iron limitation reduced iron quotas by 68%, a fraction of the plasticity known in diatoms. In contrast, a commonly co‐occurring cyanobacterium, Prochlorococcus , showed variable iron contents with iron availability in culture. Synchrotron X‐ray fluorescence was used to measure single‐cell metal (Mn, Fe, Co, Ni, Zn) quotas of autotrophic flagellates (1.4–16.8‐ μ m diameter) collected from four ocean regions. Iron quotas were tightly constrained and showed little response to iron availability, similar to cultured Ostreococcus . Zinc quotas also did not vary with zinc availability but appeared to vary with phosphorus availability. These results suggest that macronutrient and metal availability may be equally important for controlling metal contents of small eukaryotic open‐ocean phytoplankton.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET derived 3D segmented volumes as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the “dark complex,” modulate metabolic flux at a conserved regulatory node of the Calvin–Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4.

Johnson, Connah G.↗
DOE OSTI2023

Prospects for engineering Ralstonia eutropha and Zymomonas mobilis for the autotrophic production of 2,3-butanediol from CO 2 and H 2

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO 2 emissions make CO 2 an attractive feedstock for manufacturing chemicals. Moreover, H 2 is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water (scH 2 O) gasification enable sustainable production of molecular hydrogen (H 2 ). We review the recent advances in engineering Ralstonia eutropha, the representative species of "Knallgas" bacteria, for utilizing CO 2 and H 2 to autotrophically produce 2,3-butanediol (2,3-BDO). This assessment is focused on state-of-the-art approaches for splitting H 2 to supply energy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO 2 fixation. Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO 2 and H 2 -utilizing microorganisms, exemplified by Zymomonas mobilis.

42 ENGINEERING↗
DOE OSTI2021

A decreasing carbon allocation to belowground autotrophic respiration in global forest ecosystems

Belowground autotrophic respiration (RAsoil) depends on carbohydrates from photosynthesis flowing to roots and rhizospheres, and is one of the most important but least understood components in forest carbon cycling. Carbon allocation plays an important role in forest carbon cycling and reflects forest adaptation to changing environmental conditions. However, carbon allocation to RAsoil has not been fully examined at the global scale. To fill this knowledge gap, first, the spatio-temporal patterns of RAsoil from 1981 to 2017 were predicted by a Random Forest (RF) algorithm using the most updated Global Soil Respiration Database (v5) with global environmental variables; second, carbon allocation from photosynthesis to RAsoil (CAB), was calculated as the ratio of RAsoil to gross primary production; and its temporal and spatial patterns were assessed in global forest ecosystems. . Globally, mean RAsoil from forests was 8.9 ± 0.08 Pg C yr-1 (mean ± standard deviation) from 1981 to 2017 with strong spatial variabilities. Temporally, RAsoil increased at a rate of 0.0059 Pg C yr-2, paralleling broader soil respiration changes and indicating increasing carbon respired by roots. Mean CAB was 0.243 ± 0.016 and decreased over time. The temporal trend of CAB varied greatly in space, reflecting uneven responses of CAB to environmental changes. This study is the first attempt to predict global CAB and analyze its temporal and spatial patterns. With the linkage of carbon use efficiency, the developed CAB offers an completely independent approach to quantify global aboveground autotropic respiration spatially and temporally, which could provide crucial insights into carbon flux partition and global carbon cycling under climate change.

Tang, Xiaolu↗
DOE OSTI2025

Thiohalorhabdus methylotropha sp. nov., an extremely halophilic autotrophic methylotiotroph from hypersaline lakes

So far, there have been no reports of trimethylamine (TMA)-utilizing extremely halophilic microorganisms in hypersaline habitats. Our aerobic enrichments at 4 M total Na + with 5 mM TMA inoculated with surface sediments from hypersaline soda (at pH 9.5) or chloride-sulfate (at pH 7) lakes in southwestern Siberia were successful only for the latter. The initial enrichment included both bacteria and haloarchaea but only the bacterial component was able to grow as a pure culture with TMA. Strain Cl-TMA forms a new-species lineage within the genus Thiohalorhabdus which includes extremely halophilic and obligate lithoautotrophic sulfur-oxidizing gammaproteobacteria. Cl-TMA can grow methyloautotrophically utilizing TMA, dimethylamine (DMA) and methanol (MeOH) as the electron donors or chemolithoautotrophically with thiosulfate. Mixotrophic growth was also observed with the three methyl compounds and thiosulfate. Carbon is assimilated autotrophically via the Calvin-Benson-Basham pathway. Unlike the type species of Thiohalorhabdus, T. denitrificans , Cl-TMA was incapable of anaerobic growth via denitrification. The isolate belongs to extreme halophiles growing between 2.5 and 5 M NaCl with an optimum at 3–3.5 M. Genome analysis identified two gene clusters coding for PQQ-dependent methanol dehydrogenases (MxaFI and XoxF), four homologues of the formaldehyde activating enzymes (Faes), a TMA/DMA oxidation locus, and two cluster of genes encoding an N-methylglutamate dehydrogenase pathway (NMGP) for methylamine oxidation. The first steps of C 1 -subtrate conversions are followed by the tetrahydrofolate (THF)-linked and tetrahydromethanopterin (H4MPT)-linked formaldehyde oxidation pathways and two formate dehydrogenases. All of those signatures of methylotrophy were absent in T. denitrificans . In contrast, genes for two key sulfur oxidation enzymes, thiosulfate dehydrogenase TsdAB and sulfide dehydrogenase FccAB, that are present in the type species are missing in Cl-TMA. Thiosulfate is oxidized to sulfate by a combination of an incomplete Sox cycle and an sHdr system. Strain Cl-TMA T (JCM 35977 = UQM 41915) is proposed to be classified as Thiohalorhabdus methylotrophus sp. nov.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advances in systems metabolic engineering of autotrophic carbon oxide-fixing biocatalysts towards a circular economy

High levels of anthropogenic CO 2 emissions are driving the warming of global climate. If this pattern of increasing emissions does not change, it will cause further climate change with severe consequences for the human population. On top of this, the increasing accumulation of solid waste within the linear economy model is threatening global biosustainability. The magnitude of these challenges requires several approaches to capture and utilize waste carbon and establish a circular economy. Microbial gas fermentation presents an exciting opportunity to capture carbon oxides from gaseous and solid waste streams with high feedstock flexibility and selectivity. Here we discuss available microbial systems and review in detail the metabolism of both anaerobic acetogens and aerobic hydrogenotrophs and their ability to utilize C1 waste feedstocks. More specifically, we provide an overview of the systems-level understanding of metabolism, key metabolic pathways, scale-up opportunities and commercial successes, and the most recent technological advances in strain and process engineering. Finally, we also discuss in detail the gaps and opportunities to advance the understanding of these autotrophic biocatalysts for the efficient and economically viable production of bioproducts from recycled carbon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Permafrost Active Layer Microbes From Ny Ålesund, Svalbard (79°N) Show Autotrophic and Heterotrophic Metabolisms With Diverse Carbon-Degrading Enzymes

The active layer of permafrost in Ny Ålesund, Svalbard (79°N) around the Bayelva River in the Leirhaugen glacier moraine is measured as a small net carbon sink at the brink of becoming a carbon source. In many permafrost-dominating ecosystems, microbes in the active layers have been shown to drive organic matter degradation and greenhouse gas production, creating positive feedback on climate change. However, the microbial metabolisms linking the environmental geochemical processes and the populations that perform them have not been fully characterized. In this paper, we present geochemical, enzymatic, and isotopic data paired with 10 Pseudomonas sp. cultures and metagenomic libraries of two active layer soil cores (BPF1 and BPF2) from Ny Ålesund, Svalbard, (79°N). Relative to BPF1, BPF2 had statistically higher C/N ratios (15 ± 1 for BPF1 vs. 29 ± 10 for BPF2; n = 30, p < 10 –5 ), statistically lower organic carbon (2% ± 0.6% for BPF1 vs. 1.6% ± 0.4% for BPF2, p < 0.02), statistically lower nitrogen (0.1% ± 0.03% for BPF1 vs. 0.07% ± 0.02% for BPF2, p < 10 –6 ). The d 13 C values for inorganic carbon did not correlate with those of organic carbon in BPF2, suggesting lower heterotrophic respiration. An increase in the δ 13 C of inorganic carbon with depth either reflects an autotrophic signal or mixing between a heterotrophic source at the surface and a lithotrophic source at depth. Potential enzyme activity of xylosidase and N-acetyl-β-D-glucosaminidase increases twofold at 15°C, relative to 25°C, indicating cold adaptation in the cultures and bulk soil. Potential enzyme activity of leucine aminopeptidase across soils and cultures was two orders of magnitude higher than other tested enzymes, implying that organisms use leucine as a nitrogen and carbon source in this nutrient-limited environment. Besides demonstrating large variability in carbon compositions of permafrost active layer soils only ∼84 m apart, results suggest that the Svalbard active layer microbes are often limited by organic carbon or nitrogen availability and have adaptations to the current environment, and metabolic flexibility to adapt to the warming climate.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13 C and 2 H metabolic network mapping

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13 C and 2 H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO 2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis , and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO 2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria

Abstract Carbon-negative synthesis of biochemical products has the potential to mitigate global CO 2 emissions. An attractive route to do this is the reverse β-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL −1 ), as well as hexanoic acid (3.06 ± 0.03 gL −1 ) and 1-hexanol (1.0 ± 0.1 gL −1 ) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL −1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗