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All sources NASA NTRS DOE OSTI

DOE OSTI2022

Sooting tendencies of terpenes and hydrogenated terpenes as sustainable transportation biofuels

Terpenes are a diverse group of molecules that are synthesized by plants and microorganisms through combining units of isoprene (2-methyl-1,3-butadiene). They typically contain rings and methyl branches, which gives them high energy densities and low freezing points and makes them appealing candidates for sustainable transportation biofuels. Between the original biosynthesis and upgrading options such as hydrogenation, they have a large degree of freedom of structures, e.g., different carbon skeletons, positions of double bonds, and functional groups. Therefore, structure-property data is needed to downselect potential fuel candidates. Here, we measured the sooting tendencies of 17 C10 monoterpenes and 7 of their hydrogenated analogues. The hydrogenated compounds were custom synthesized, so the quantities were too small for conventional smoke point measurements. Thus, the sooting tendencies were quantified with yield sooting index (YSI), which is based on the soot yield in a fuel-doped non-premixed methane flame. Derived smoke points (DSPs) were estimated from a correlation between YSI and smoke point for other hydrocarbons. The YSI of terpenes and their derivatives varies widely from 85.6 to 248.5. The YSI follows the trend: terpenes > dihydroterpenes > tetrahydroterpenes. The DSPs of all the tetrahydroterpenes and some dihydroterpenes are higher than that of a Jet-A fuel sample, suggesting that they offer soot reduction benefits. Further, the YSIs depend strongly on molecular structure; for example, α-pinene and β-pinene have identical carbon skeletons and differ only in the position of one carbon-carbon double bond, but the YSI of α-pinene is 34% higher than that of β-pinene. Detailed decomposition analysis via density functional theory (DFT) suggests that compared with β-pinene, α-pinene requires fewer steps to form the first aromatic ring and the process is more thermodynamically favorable. The YSI difference between the pinenes is mainly affected by the identity of the products from the dominant decomposition pathways.

09 BIOMASS FUELS↗

DOE OSTI2025

Plant-microbial interplay for organic nitrogen mediated by functional specificity of root compartments

The organic form of nitrogen (N) is a critical intermediate in mutualistic and competitive root-microbial interactions, mediated by extracellular enzymes. Visualization of the hotspots of organic N and proteolytic activity might be valuable for revealing root functional specificity in N acquisition and transformation at the level of individual roots and compartments. For the first time, we used time-lapse amino-mapping and zymography to co-localize and map the spatial distribution of amino-N and leucine aminopeptidase (LAP) activity in the soil and different root parts of maize (Zea mays L.). Amino-N distribution was mainly associated with seminal roots and root tips, where it overlapped with LAP activity hotspots. In the lateral roots and bulk soil, however, LAP activity was decoupled from amino-N. Distinct functional traits revealed themselves as the highest amino-N content and LAP activity in seminal root tips and as the largest relative extent of the rhizosphere in lateral root tips. Co-localized amino-N and LAP activities highlighted different nutrient acquisition strategies mediated by root-microbe interactions, depending on the root compartment. Seminal roots and their tips appeared to adopt mutualistic strategies, potentially attracting root-associated microorganisms through releasing oligo- and polypeptides. In contrast, lateral roots, with amino-N detected only at their tips, demonstrated stronger N competition, relying on the enzyme activity of the rhizosphere microbial community for N acquisition. These insights emphasized the role of root functional specialization in shaping plant-microbe interactions, offering pathways to enhance nutrient use efficiency.

Maize (Zea mays L.)↗

DOE OSTI2020

Influence of Polymer Type and Carbon Nanotube Properties on Carbon Nanotube/Polymer Nanocomposite Biodegradation

The interaction of anaerobic microorganisms with carbon nanotube/polymer nanocomposites (CNT/PNC) will play a major role in determining their persistence and environmental fate at the end of consumer use when these nano-enabled materials enter landfills and encounter wastewater. Motivated by the need to understand how different parameters (i.e., polymer type, microbial phenotype, CNT characteristics) influence CNT/PNC biodegradation rates, we have used volumetric biogas measurements and kinetic modeling to study biodegradation as a function of polymer type and CNT properties. In one set of experiments, oxidized multiwall carbon nanotubes (O-MWCNTs) with a range of CNT loadings 0 – 5% w/w were incorporated into poly-e-caprolactone (PCL) and polyhydroxyalkanoates (PHA) matrices and subjected to biodegradation by an anaerobic microbial community. For each CNT/PNC, complete polymer biodegradation was ultimately observed, although the rate of biodegradation was inhibited above certain critical CNT loadings dependent upon the polymer type. Higher loadings of pristine MWCNTs were needed to decrease the rate of polymer biodegradation compared to O-MWCNTs, an effect ascribed principally to differences in CNT dispersion within the polymer matrices. Above ertain CNT loadings, a CNT mat of similar shape to the initial PNC was formed after polymer biodegradation, while below this threshold, CNT aggregates fragmented in the media. In situations where biodegradation was rapid, methanogen growth was disproportionately inhibited compared to the overall microbial community. Analysis of the results obtained from this study indicates that the inhibitory effect of CNTs on polymer biodegradation rate is greatest under conditions (i.e., polymer type, microbial phenotype, CNT dispersion) where biodegradation of the neat polymer is slowest. This new insight provides a means to predict the environmental fate, persistence and transformations of CNT-enabled polymer materials.

Frank, Benjamin↗

DOE OSTI2020

Microbial potentiometric sensor: A new approach to longstanding challenges

The underlying hypothesis of this study is that simple potentiometric measurements between sensing electrodes and a shared reference electrode - Microbial Potentiometric Sesnor (MPS) system - can be employed in a long-term, continuous mode of operation to resolve the spatial and temporal changes in environmental systems. To address the hypothesis, (1) a conceptual description of the MPS system and its postulated principle of operation are provided; (2) the MPS system performance is documented under controlled laboratory conditions; and (3) the capabilities of the MPS system are documented under quiescent and dynamic field condition. In a laboratory setting, the variability among different MPS signals was insignificant confirming the postulated high accuracy and reproducibility of the sensor performance. It also demonstrated statistically significant correlations with dissolved oxygen (DO) and oxidation-reduction potential (ORP) sensors, while showing capabilities of operating under anoxic and anaerobic conditions. Regardless of their locations in the model wetland system, three MPS sensors functioned without interruption and cleaning for a period >2 years, and thus demonstrating long-term durability of the MPS technology. In real batch-wastewater treatment facility, the deployed MPS system signals were able to describe the organic carbon trends and correlate with each treatment phase in a cycle. Data reproducibility and reliability exceeded the expectations better describing the carbon treatment levels than the DO and ORP sensors (p < 4.4 × 10 –162 vs phase adjusted p < 3.0 × 10 –58 ). While MPS signals correlate with specific parameters that describe the local process or environments, it is more prudent to employ both the magnitude and pattern of a composite signal like the MPS signal describe the change to reflect any shift in the local environment. Finally, when compared to a baseline pattern, this change in signal magnitude and pattern reveals important information that can be employed to tailor and optimize any condition or process which involves microorganisms.

47 OTHER INSTRUMENTATION↗

DOE OSTI2024

Biomolecular budget of persistent, microbial-derived soil organic carbon: The importance of underexplored pools

The details of how soil microorganisms contribute to stable soil organic carbon pools are a pressing knowledge gap with direct implications for soil health and climate mitigation. It is now recognized that microbial necromass contributes substantially to the formation of stable soil carbon. However, the quantification of necromass in soils has largely been limited to model molecules such as aminosugar biomarkers. The abundance and chemical composition of other persistent microbial residues remain unresolved, particularly concerning how these pools may vary with microbial community structure, soil texture, and management practices. We use yearlong soil incubation experiments with an isotopic tracer to quantify the composition of persistent residues derived from microbial communities inhabiting sand or silt dominated soil with annual (corn) or perennial (switchgrass) monocultures. Persistent microbial residues were recovered in diverse soil biomolecular pools including metabolites, proteins, lipids, and mineral-associated organic matter (MAOM). The relative abundances of microbial contributions to necromass pools were consistent across cropping systems and soil textures. The greatest residue accumulation was not recovered in MAOM but in the light density fraction of soil debris that persisted after extraction by chemical fractionation using organic solvents. Necromass abundance was positively correlated with microbial biomass abundance and revealed a possible role of cell wall morphology in enhancing microbial carbon persistence; while gram-negative bacteria accounted for the greatest contribution to microbial-derived carbon by mass at one year, residues from gram-positive Actinobacteria and Firmicutes showed greater durability. Together these results offer a quantitative assessment of the relative importance of diverse molecular classes for generating durable soil carbon.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Global biogeography of fungal and bacterial biomass carbon in topsoil

Bacteria and fungi, representing two major soil microorganism groups, play an important role in global nutrient biogeochemistry. Biogeographic patterns of bacterial and fungal biomass are of fundamental importance for mechanistically understanding nutrient cycling. We synthesized 1323 data points of phospholipid fatty acid-derived fungal biomass C (FBC), bacterial biomass C (BBC), and fungi:bacteria (F:B) ratio in topsoil, spanning 11 major biomes. The FBC, BBC, and F:B ratio display clear biogeographic patterns along latitude and environmental gradients including mean annual temperature, mean annual precipitation, net primary productivity, root C density, soil temperature, soil moisture, and edaphic factors. At the biome level, tundra has the highest FBC and BBC densities at 3684 (95% confidence interval: 1678–8084) mg kg -1 and 428 (237–774) mg kg -1 , respectively; desert has the lowest FBC and BBC densities at 16.92 (14.4–19.89) mg kg -1 and 6.83 (6.1–7.65) mg kg -1 , respectively. The F:B ratio varies dramatically, ranging from 1.8 (1.6–2.1) in savanna to 8.6 (6.7–11.0) in tundra. An empirical model was developed for the F:B ratio and it is combined with a global dataset of soil microbial biomass C to produce global maps for FBC and BBC in 0–30 cm topsoil. Across the globe, the highest FBC is found in boreal forest and tundra while the highest BBC is in boreal forest and tropical/subtropical forest, the lowest FBC and BBC are in shrub and desert. Global stocks of living microbial biomass C were estimated to be 12.6 (6.6–16.4) Pg C for FBC and 4.3 (0.5–10.3) Pg C for BBC in topsoil. These findings advance our understanding of the global distribution of fungal and bacterial biomass, which facilitates the incorporation of fungi and bacteria into Earth system models. The global maps of bacterial and fungal biomass serve as a benchmark for validating microbial models in simulating the global C cycle under a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Soil microbial EPS resiliency is influenced by carbon source accessibility

The adaptability of soil microbial communities to prolonged periods of drought is influenced by their ability to produce extracellular polymeric substances (EPS) with sufficient water retention properties. Microbial EPSs as water reservoirs during drought have been extensively investigated, but it remains unknown how carbon substrate accessibility to soil microbial communities will affect the chemical properties of the EPS they generate, and whether this in turn will alter their water retention ability. In this work, we observed that the accessibility of carbon substrates influenced microbial community structure and, consequently, the chemical properties of EPS produced by the microbial communities. Further, our results demonstrated that an insoluble carbon substrate (i.e., chitin), stimulated microbial communities to produce EPS with better water retention properties in comparison to a soluble carbon substrate (i.e., N-acetylglucosamine; NAG). In all, this study demonstrates the importance of carbon substrate accessibility by soil microorganisms in regulating the community structure and consequently, the EPS carbon chemistry, which in turn can greatly influence the adaptability of soil microbial communities to drought.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Are enzymes transported in soils by water fluxes?

Transport of extracellular hydrolytic enzymes in soils has always been a subject of doubt. The considerations against its importance are that (i) enzymes benefit their producers the most when they remain in close proximity; and (ii) enzymes are large molecules with low mobility due to high affinity to fine soil particles and organic matter. However, soil mineral colloids (SMC), to which extracellular enzymes also have an affinity and which are known to facilitate transport of a broad variety of chemicals and microorganisms in soils, can serve as vehicles for enzyme transport as well. Since current literature lacks information on enzyme transport in soils, our goal was to determine whether enzymes are transported and, if so, whether they are transported in a free- or in a colloid-associated form. We conducted column transport experiments with four hydrolytic enzymes, namely, β-glucosidase, acid-phosphatase, cellobiohydrolase, and xylosidase, in soils with contrasting textures. The eluents containing enzymes were applied on top of soil columns, while enzyme activities, SMC, and electrical conductivity were measured in the effluents from the columns. Our results provided evidence of joint enzyme transport with soil colloids. The enzymes associated with the coarse SMC (1 μm < Ø) contributed 52–88% of the total enzyme activity in the effluents. The remaining enzyme activity was attributed to the enzymes associated with organic colloids, fine SMC (Ø < 1 μm) and free enzymes in solution. Furthermore, this study suggested a dual effect of ionic strength in the soil suspension on enzyme activity and their release from soils with soil colloids.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Microscale spatial distribution and soil organic matter persistence in top and subsoil

The spatial distribution of organic substrates and microscale soil heterogeneity significantly influence organic matter (OM) persistence as constraints on OM accessibility to microorganisms. However, it is unclear how changes in OM spatial heterogeneity driven by factors such as soil depth affect the relative importance of substrate spatial distribution on OM persistence. This work evaluated the decomposition and persistence of 13 C and 15 N labeled water-extractable OM inputs over 50 days as either hotspot (i.e., pelleted in 1–2 mm-size pieces) or distributed (i.e., added as OM < 0.07 μm suspended in water) forms in topsoil (0–0.2 m) and subsoil (0.8–0.9 m) samples of an Andisol. We observed greater persistence of added C in the subsoil with distributed OM inputs relative to hotspot OM, indicated by a 17% reduction in cumulative mineralization of the added C and a 10% higher conversion to mineral-associated OM. A lower substrate availability potentially reduced mineralization due to OM dispersion throughout the soil. NanoSIMS (nanoscale secondary ion mass spectrometry) analysis identified organo-mineral associations on cross-sectioned aggregate interiors in the subsoil. On the other hand, in the topsoil, we did not observe significant differences in the persistence of OM, suggesting that the large amounts of particulate OM already present in the soil outweighed the influence of added OM spatial distribution. Here, we demonstrated under laboratory conditions that the spatial distribution of fresh OM input alone significantly affected the decomposition and persistence of OM inputs in the subsoil. On the other hand, spatial distribution seems to play a lower role in topsoils rich in particulate OM. The divergence in the influence of OM spatial distribution between the top and subsoil is likely driven by differences in soil mineralogy and OM composition.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Higher levels of mixed-linkage (1,3;1,4)-β-glucan in transgenic grasses may impact soil C processing

Carbohydrates, including mixed-linkage glucan (MLG), in grass cell walls make them a valuable potential feedstock for biofuel production. Hence, the development of transgenic grasses with elevated levels of MLG is being actively pursued worldwide. Changes in chemical and physical root characteristics of MLG-overproducing transgenic plants can affect processing of the root-derived carbon (C) by soil microorganisms, impacting soil C cycling. Here, this study is the first attempt to elucidate the impact of MLG-related genetic modifications on root traits, root decomposition, and soil C processing. We explored four genotypes of Brachypodium ( Brachypodium distachyon ): a wildtype, a loss-of-function mutant with low MLG, an MLG overexpressing line, and a line lacking MLG hydrolase (with high MLG), incubating their roots in soils of two contrasting vegetation histories: monoculture switchgrass and polyculture restored prairie. The four genotypes exhibited contrasting root MLG and soluble sugar concentrations, and different growth phenotypes. Roots with the highest MLG content resulted in a ∼55 % increase in microbial biomass C compared to wildtype in both soils. However, the genotype effects on C mineralization rates were influenced by the vegetation history, with significant effects observed only in the soil from switchgrass but not prairie origin. While further work is required to understand the full impact of MLG-overproducing plants on soil C accrual, our findings suggest that their influence on soil C processes cannot be discounted.

Brachypodium distachyon↗

DOE OSTI2025

Lipids represent a dynamic, yet stable pool of microbially-derived soil carbon

There is an emerging consensus that microorganisms are a primary source of persistent, slow-cycling soil organic matter (SOM), however which microbial residues contribute to SOM and what controls their accumulation remains unresolved. Lipids are a commonly overlooked biomolecular pool that could contribute significantly to stable SOM. While current estimates for phospholipid degradation in soils are rapid, lipids are structurally heterogeneous molecules that could turnover at different rates. Through a year-long soil incubation and compound-specific, stable isotope probing (SIP)-lipidomics, we were able to rigorously track the persistence of lipid compounds in silty and sandy switchgrass bioenergy crop soils. Here, we assessed the influence of lipid structure on soil lipid accrual and degradation as moderated by soil texture, since mineral association is presumed to be the primary mechanism for lipid persistence. After rapid incorporation of 13 C glucose into microbial biomass, we found 13 C label was retained broadly across chemically diverse lipid classes, even after one year. 13 C-labeled lipid profiles varied significantly with soil texture; however, we found no difference between sandy and silty soils in lipid retention, suggesting soil texture may only play a minor role in modulating lipid persistence. Only two lipid subclasses were found to be persistent (i.e., retention of 13 C label without significant degradation or production): phosphatidylinositol lipids and hydroxyceramide lipids, both of which are negatively charged, possibly facilitating stabilization by mineral complexation. However, several other subclasses displayed substantial ongoing production. In particular, the accumulation of triacylglycerol lipids across soil textures suggests that storage lipids may be an important component of SOC, highlighting a potential target for management strategies to promote C retention by lipid accrual. Overall, the retention for over a year of 13 C label in microbial intact lipid biomarkers reveals the importance of efficient biomass production and turnover when considering microbial C contributions to SOM.

compound-specific↗

DOE OSTI2026

Cryo-electron microscopy structure of a zinc uptake ABC transporter

Zn2+ is an essential micronutrient required for many biological processes. Microorganisms employ high-affinity Zn2+ ABC transporters to take up zinc from the environment when it is scarce, yet the mechanism of zinc uptake and its regulation remain poorly understood. Here, we report the cryo-electron microscopy structure of the Zn2+ ABC transporter complex ZnuB-ZnuC from Escherichia coli. It contains two ZnuB transport and two ZnuC regulatory subunits. The ZnuB homodimer is in an outward-facing, closed conformation with a large hydrophilic cavity at the dimer interface. Each ZnuC subunit contains an N-terminal nucleotide-binding domain and a C-terminal zinc-sensing domain (ZSD). Zn2+ binding to the ZSD locks the transporter in a closed state, whereas under low-Zn2+ conditions, the disorder of ZSD permits ATP-driven zinc uptake. Zn2+ ABC transporters are ubiquitously utilized by pathogenic bacteria to compete with hosts for Zn2+. These findings reveal potential therapeutic targets for disrupting Zn2+ homeostasis in antibiotic-resistant pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

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 OSTI2024

Microbe of the month: Methanococcus maripaludis

Methanococcus maripaludis was first isolated in 1983 from the anoxic sediment of a salt marsh in South Carolina, USA. M. maripaludis grows by using H 2 or formate to reduce CO 2 to CH 4 and is an autotroph that assimilates CO 2 via the archaeal version of the Wood–Ljungdahl pathway. In some strains, supplementation of growth medium with acetate or propionate can increase biomass yield. Furthermore, this microorganism is strictly anaerobic, has a pleomorphic coccoid shape, and forms translucent, pale yellow, smooth colonies within 3 days on agar plates. The optimum temperature for growth is 38°C with optimal pH between 6.8 and 7.2.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Synthesis and Function of Complex Sphingolipid Glycosylation

Glycosylinositol phosphorylceramides (GIPCs) constitute an important class of plasma-membrane lipids in plants. The complex glycan headgroups of GIPCs vary between plant species and tissues. Recent studies show that the structure of the glycan headgroup is important for plant development, abiotic stress tolerance, and interactions with pathogenic and symbiotic microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Enabling microbial electrolysis cell scale-up via electrochemistry-, hydrodynamic-, and microbial ecology-informed framework

Microbial electrolysis cells (MECs) can produce green hydrogen while removing organic contaminants from liquid waste streams by leveraging the metabolic activity of electroactive microorganisms. Despite their potential in a sustainable, circular economy, large-scale MECs that can treat relevant volumes of wastewater have failed to deliver performance proportional to their lab-scale counterparts. The reason behind this lower performance at scale remains unclear. Here, in this study, we developed a combined electrochemistry-, hydrodynamic-, and microbial ecology-informed framework to analyze and optimize MEC performance during scale-up, enabling accurate quantification of major limitations and the identification of strategies to overcome them, ultimately facilitating equivalent performance at scale. Applying this framework to the scale-up of a zero-gap MEC from 9 cm 2 electrode area to 100 cm 2 electrode area, resulted in similar maximum current densities in a 100 cm 2 MEC (21.7 ± 1.1 A/m 2 ) compared to a 9 cm 2 system (25.1 ± 2.7 A/m 2 ), as well as equivalent hydrogen production rates of 69.3 L/L-d (100 cm 2 ) and 67.7 ± 2.4 L/L-d (9 cm 2 ). COMSOL flow dynamics simulations were used to scale up the reactor configuration without negatively affecting electrolyte velocity and distribution in the cell, minimizing the increase in internal resistances during scale-up (11.7 ± 0.5 mΩm 2 at 9 cm 2 ; 19.7 ± 1.3 mΩm 2 at 100 cm 2 ). Microbial community structures were assessed at both scales using high-throughput sequencing, highlighting the differences of populations across electrode dimensions and operational parameters. The framework presented here accelerates the development of effective strategies toward the scale-up of MECs by furthering the understanding of how electrochemical, hydrodynamic, and microbial ecology parameters change as the reactor dimension is increased. Ultimately, this approach contributes to advancing electrochemical biotechnology toward practical deployment in energy-efficient wastewater treatment systems.

Flow path↗

DOE OSTI2020

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. Finally, we anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Specifically, we engineered Pseudomonas putida, a robust microorganism capable of catabolizing aromatic compounds, organic acids, and D-glucose, to utilize D-xylose and L-arabinose by tuning D-xylose transport and pentose phosphate pathway flux. Distinct L-arabinose pathways enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as corn stover hydrolysate. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose, hemicellulose, and lignin, demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.

Elmore, Joshua R.↗