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DOE OSTI2022

An agent-based modeling tool supporting bioenergy and bio-product community communication regarding cellulosic bioeconomy development

The emergence of the cellulosic bioeconomy requires not only adequate technological, economic, and policy advances, but also effective communication and coordination among the multiple stakeholders in the bioeconomy community. Aiming to facilitate stakeholder communication and collaboration for cellulosic bioeconomy growth, an agent-based model is developed as a community communication tool in this study. The tool simulates the behaviors and interactions of key stakeholders, as well as the responses and interactions between the stakeholders and biophysical, socioeconomic, and environmental conditions. The agent behavior rules are developed and validated based on behavior and economic theories and information attained from a stakeholder focus group study and a land use survey. A pilot study in a Central Illinois watershed shows stakeholder synergies, i.e., positive feedback loops across multiple stakeholders. The modeling tool's role in facilitating community communication is demonstrated via the focus group study. A comparison of policy scenarios suggests that the cellulosic bioeconomy development is fastest under a portfolio of policies combining the views of the multiple stakeholders. Critically, subsidy for small-scale bio-facilities is essential as it rapidly initializes an early market for cellulosic feedstocks and the adoption of Miscanthus. As a result, the communication tool is applied to bringing stakeholders and researchers together to develop shared visions, assess barriers, and explore solution portfolios to initialize and speed up the regional cellulosic bioeconomy.

09 BIOMASS FUELS↗

DOE OSTI2016

Millisecond Pulsed Films Unify the Mechanisms of Cellulose Fragmentation

The mechanism of crystalline cellulose fragmentation has been debated between classical models proposing end-chain or intrachain scission to form short-chain (molten) anhydro-oligomer mixtures and volatile organic compounds. Models developed over the last few decades suggest global kinetics consistent with either mechanism, but validation of the chain-scission mechanism via measured reaction rates of cellulose has remained elusive. To resolve these differences, we introduce a new thermal-pulsing reactor four orders of magnitude faster than conventional thermogravimetic analysis (10 6 vs 10 2 °C/min) to measure the millisecond-resolved evolution of cellulose and its volatile products at 400–550 °C. By comparison of cellulose conversion and furan product formation kinetics, both mechanisms are shown to occur with the transition from chain-end scission to intrachain scission above 467 °C concurrent with liquid formation comprised of short-chain cellulose fragments.

09 BIOMASS FUELS↗

DOE OSTI2017

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Furthermore, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.

09 BIOMASS FUELS↗

DOE OSTI2013

Physical properties of agave cellulose graft polymethyl methacrylate

The grafting polymerization of methyl methacrylate and Agave cellulose was prepared and their structural analysis and morphology were investigated. The grafting reaction was carried out in an aqueous medium using ceric ammonium nitrate as an initiator. The structural analysis of the graft copolymers was carried out by Fourier transform infrared and X-ray diffraction. The graft copolymers were also characterized by field emission scanning electron microscopy (FESEM). An additional peak at 1732 cm{sup −1} which was attributed to the C=O of ester stretching vibration of poly(methyl methacrylate), appeared in the spectrum of grafted Agave cellulose. A slight decrease of crystallinity index upon grafting was found from 0.74 to 0.68 for cellulose and grafted Agave cellulose, respectively. Another evidence of grafting showed in the FESEM observation, where the surface of the grafted cellulose was found to be roughed than the raw one.

36 MATERIALS SCIENCE↗

DOE OSTI2015

Fibril orientation redistribution induced by stretching of cellulose nanofibril hydrogels

The mechanical performance of materials reinforced by cellulose nanofibrils is highly affected by the orientation of these fibrils. This paper investigates the nanofibril orientation distribution of films of partly oriented cellulose nanofibrils. Stripes of hydrogel films were subjected to different amount of strain and, after drying, examined with X-ray diffraction to obtain the orientation of the nanofibrils in the films, caused by the stretching. The cellulose nanofibrils had initially a random in-plane orientation in the hydrogel films and the strain was applied to the films before the nanofibrils bond tightly together, which occurs during drying. The stretching resulted in a reorientation of the nanofibrils in the films, with monotonically increasing orientation towards the load direction with increasing strain. Estimation of nanofibril reorientation by X-ray diffraction enables quantitative comparison of the stretch-induced orientation ability of different cellulose nanofibril systems. The reorientation of nanofibrils as a consequence of an applied strain is also predicted by a geometrical model of deformation of nanofibril hydrogels. Conversely, in high-strain cold-drawing of wet cellulose nanofibril materials, the enhanced orientation is promoted by slipping of the effectively stiff fibrils.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2012

Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear {beta}-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-{beta}-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an ({alpha}/{alpha}){sub 6}-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades {beta}-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Super-resolution imaging illuminates new dynamic behaviors of cellulose synthase

Abstract Confocal imaging has shown that CELLULOSE SYNTHASE (CESA) particles move through the plasma membrane as they synthesize cellulose. However, the resolution limit of confocal microscopy circumscribes what can be discovered about these tiny biosynthetic machines. Here, we applied Structured Illumination Microscopy (SIM), which improves resolution two-fold over confocal or widefield imaging, to explore the dynamic behaviors of CESA particles in living plant cells. SIM imaging reveals that Arabidopsis thaliana CESA particles are more than twice as dense in the plasma membrane as previously estimated, helping explain the dense arrangement of cellulose observed in new wall layers. CESA particles tracked by SIM display minimal variation in velocity, suggesting coordinated control of CESA catalytic activity within single complexes and that CESA complexes might move steadily in tandem to generate larger cellulose fibrils or bundles. SIM data also reveal that CESA particles vary in their overlaps with microtubule tracks and can complete U-turns without changing speed. CESA track patterns can vary widely between neighboring cells of similar shape, implying that cellulose patterning is not the sole determinant of cellular growth anisotropy. Together, these findings highlight SIM as a powerful tool to advance CESA imaging beyond the resolution limit of conventional light microscopy.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

Fusel alcohols↗

DOE OSTI2014

Acetobixan, an Inhibitor of Cellulose Synthesis Identified by Microbial Bioprospecting

In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6 prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14 C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Conifer water use patterns in the East River Watershed, Colorado US, based on stable water isotopes and cellulose isotopes

This data package contains a series of datasets aimed at understanding the seasonal origins of water used by the dominant conifer species, Abies lasiocarpa and Picea engelmannii, in the East River Watershed. There is a distinct difference in the stable isotopic ratio of summer rain and snowpack and thus by tracking the isotopic ratio of the xylem water in trees, it is possible to separate when a tree is using snow vs. summer rain. Following typical protocols, we sampled and extracted water from soils in the "Research Meadow" near Gothic, CO within the East River Watershed through the 2016 growing season. The stable isotopic ratio of these waters were then measured. Using values of the isotopic ratio of precipitation and snowpack from that season, we develop a mixing model to show how the trees transitioned from use of snowpack to summer in late July. We extended the information on the seasonal origins of water from 2016 back until 1980 by using measurements of the isotopic ratio of cellulose from trees in the Research Meadow. The isotopic ratio of tree ring cellulose records a signal both of the Vapor Pressure Deficit at the leaf-atmosphere boundary and also the source water for the trees. We concluded from this extended analysis that the trees underwent transitions between periods where snow was the dominant water source and summer rain was the dominant water source. More details on this analysis can be found in the publication: Persistence and Plasticity in Conifer Water Use Strategies. JGR Biogeoscience. DOI 10.1029/2018JG004845. The dataset specifically includes stable isotope measurements of xylem and soil water for Spruce and Fir trees at the Rocky Mountain Biological Lab's Research Meadow as well as snowpack and precipitation stable isotopes from nearby National Atmospheric Deposition Program sites. Associated meteorological data is included, which is needed to understand the source water for the trees based on the cellulose measurements.This dataset was updated in June 2021 also include intra-ring measurements of the d18O and d13C of cellulose these same species of trees at the Ziegler Reservoir site in Snowmass, CO. These are listed as files: "Interglaciald18OCellulose.csv", "Interglaciald13OCellulose.csv" and "Modernd13CCellulose.csv". The intent of these measurements was to assess whether common conifers (abies and picea) had undergone ecophysiological or ecohydrological changes during the last period in Earth's history was warmer than today, i.e. the last Interglacial (116,000 to 130,000 years ago). This site was chosen because it is a location where subfossil wood from this critical warm period was discovered. We measured the d18O and d13C of the subfossil trees following the same protocol as the modern samples which included chemical extraction of cellulose and isotope ratio mass spectrometry. We found that the trees showed little difference in water use efficiency but utilized more summer rain during the interglacial period. This analysis and detailed methods are provided in the following manuscript: Wetter summers mitigated temperature stress on Rocky Mountain forests during the last interglacial warm period which is in revision at GRL but is available as a preprint here: 10.1002/essoar.10506740.2

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca ) fused with endocellulase Cel5A (from T. fusca ) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli , purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

09 BIOMASS FUELS↗

DOE OSTI2015

Non‐invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy

ABSTRACT Cellulose microfibrils represent the major scaffold of plant cell walls. Different packing and orientation of the microfibrils at the microscopic scale determines the macroscopic properties of cell walls and thus affect their functions with a profound effect on plant survival. We developed a polarized Raman microspectroscopic method to determine cellulose microfibril orientation within rice plant cell walls. Employing an array of point measurements as well as area imaging and subsequent Matlab‐assisted data processing, we were able to characterize the distribution of cellulose microfibril orientation in terms of director angle and anisotropy magnitude. Using this approach we detected differences between wild type rice plants and the rice brittle culm mutant, which shows a more disordered cellulose microfibril arrangement, and differences between different tissues of a wild type rice plant. This novel non‐invasive Raman imaging approach allows for quantitative assessment of cellulose fiber orientation in cell walls of herbaceous plants, an important advancement in cell wall characterization. Biotechnol. Bioeng. 2016;113: 82–90. © 2015 Wiley Periodicals, Inc.

Sun, Lan↗

DOE OSTI2021

Engineering Clostridium cellulovorans for highly selective n ‐butanol production from cellulose in consolidated bioprocessing

Abstract Cellulosic n ‐butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans , a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adh E2 from C. acetobutylicum for n ‐butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl‐CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adh E2 would enhance n ‐butanol production. In this study, thiolase ( thl A CA ) from C. acetobutylicum and 3‐hydroxybutyryl‐CoA dehydrogenase ( hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adh E2 to increase the flux from acetyl‐CoA to butyryl‐CoA. In addition, ferredoxin‐NAD(P) + oxidoreductase ( fnr ), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl‐CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n ‐butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adh E2‐ fnr CA ‐ thl A CA ‐ hbd CT produced n ‐butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n ‐butanol production from cellulosic biomass in consolidated bioprocessing.

Bao, Teng↗

DOE OSTI2023

Xylan-cellulose core structure of oat water-extractable β-glucan macromolecule: Insight into interactions and organization of the cell wall complex

Water-extractable β-glucan with high molar mass (HM) determines health benefits of oat food. Oat β-glucan was extracted by a standardized in vitro digestion method and co-existing water-extractable polysaccharide (WEP) fraction and its HM-arabinoxylan (HM-AX) subfraction were isolated to identify their highly acid-resistant subunit and investigate molecular interactions between constituent polymers. The WEP and HM-AX samples consisted of arabinoxylans (AXs) (74 and 76 %, respectively), however, cellulose constituted the secondary component (6.6 and 12.8 %, respectively). Multi-detection HPSEC along with specific enzymatic hydrolysis of AXs revealed the presence of the HM-xylan domain (16 and 34 %, respectively) built of numerous single- and multi-component populations with random coil and rod-like conformations, which were embedded in a xylan matrix with spherical conformation and controlled the macromolecular shape. Unlike single-component populations, the multi-component ones were resistant to hydrolytic action of AX-hydrolyzing enzymes and represented the subunits that anchor matrix polysaccharides onto cellulose surface. These results indicate that water-extractable β-glucan macromolecule comprises as integral element a cellulose core with two linking populations, HM-xylan and low molar mass glucomannan, which are surrounded by a feruloylated AX-arabinan-arabinogalactan composite and next laminated by β-glucan matrix. Here, the stiff cellulose-xylan backbone is the basis of HM β-glucan organization, controlled by its cellulose-like segments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

09 BIOMASS FUELS↗

DOE OSTI2012

Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

Chauvigne-Hines, Lacie M↗

DOE OSTI2019

Function-driven single-cell genomics uncovers cellulose-degrading bacteria from the rare biosphere

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Molecular organization of the E. coli cellulose synthase macrocomplex

Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA–B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA’s periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA’s regulatory PilZ domain. As a result, the macrocomplex is a fascinating example of cellulose synthase specification.

59 BASIC BIOLOGICAL SCIENCES↗