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At least 793 records · Page 44

Bottom-Up Meets Top-Down: The Crossroads of Multiscale Chromatin Modeling

Chromatin can be viewed as a hierarchically structured fiber that regulates gene expression. It consists of a complex network of DNA and proteins whose characteristic dynamical modes facilitate compaction and rearrangement in the cell nucleus. These modes stem from chromatin's fundamental unit, the nucleosome, and their effects are propagated across length scales. Understanding the effects of nucleosome dynamics on the chromatin fiber, primarily through post-translational modifications that occur on the histones, is of central importance to epigenetics. Within the last decade, imaging and chromosome conformation capture techniques have revealed a number of structural and statistical features of the packaged chromatin fiber at a hitherto unavailable level of resolution. Such experiments have led to increased efforts to develop polymer models that aim to reproduce, explain, and predict the contact probability scaling and density heterogeneity. At nanometer scales, available models have focused on the role of the nucleosome and epigenetic marks on local chromatin structure. At micrometer scales, existing models have sought to explain scaling laws and density heterogeneity. Less work, however, has been done to reconcile these two approaches: bottom-up and top-down models of chromatin. Here, we highlight the multiscale simulation models that are driving toward an understanding of chromatin structure and function, from the nanometer to the micron scale, and we highlight areas of opportunity and some of the prospects for new frameworks that bridge these two scales. Taken together, experimental and modeling advances over the last few years have established a robust platform for the study of chromatin fiber structure and dynamics, which will be of considerable use to the chromatin community in developing an understanding of the interplay between epigenomic regulation and molecular structure.

59 BASIC BIOLOGICAL SCIENCES↗

Transcriptional Control of hgcAB by an ArsR-Like Regulator in Pseudodesulfovibrio mercurii ND132

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg methylator Pseudodesulfovibrio mercurii ND132, we evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and cotranscribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosylhomocysteine (SAH)- responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR proteins in P. mercurii. Using quantitative PCR (qPCR) and RNA sequencing (RNA-seq) transcriptome analyses, we confirmed this ArsR regulates hgcAB transcription and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations, with significant upregulation of other ArsRregulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to As(V) (but not As(III)) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted rates of Hg methylation by ND132; however, responses varied with culture conditions. Differences in growth and metabolic activity did not account for arsenic impacts on methylation. While arsenic significantly increased hgcAB expression, hgcAB gene and transcript abundance was not a good predictor of Hg methylation rates. Taken together, these results support the idea that Hg and As cycling are linked in P. mercurii ND132. Our results may hold clues to the evolution of hgcAB and the controls on Hg methylation in nature. This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosylhomocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression, which is needed to better predict environmental methylmercury production

RNA-seq↗

Functional diversification within the heme-binding split-barrel family

Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation to reveal a large multifunctional family of putative enzymatic and nonenzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the “FMN-binding split barrel” superfamily and contains separate groups of proteins from prokaryotes, plants, and algae, which bind heme and either catalyze its degradation or function as nonenzymatic heme sensors. In prokaryotes these proteins are often involved in iron assimilation, whereas several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of AtHOZ1 (AT3G03890) or AtHOZ2A (AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, a recent duplication event of a HOZ1 ancestor has resulted in localization of a paralog to the cytosol. Structural characterization of this cytosolic paralog and comparison to published homologous structures suggests conservation of heme-binding sites. This study unifies our understanding of the sequence-structure-function relationships within this multilineage family of heme-binding proteins and presents new molecular players in plant and bacterial heme metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

Heat pipe and surface mass transfer cooling of hypersonic vehicle structures

The problem of determining the feasibility of cooling hypersonic vehicle leading-edge structures exposed to severe aerodynamic surface heating using heat pipe and mass transfer cooling techniques is addressed. A description is presented of a numerical finite-difference-based hypersonic leading-edge cooling model incorporating poststartup liquid metal heat pipe cooling with surface transpiration and film cooling to predict the transient structural temperature distributions and maximum surface temperatures of hypersonic vehicle leading edge. An application of this model to the transient cooling of a typical aerospace plane wing leading-edge section. The results of this application indicated that liquid metal heat pipe cooling alone is insufficient to maintain surface temperatures below an assumed maximum level of 1800 K for about one-third of a typical aerospace plane ascent trajectory through the earth's atmosphere.

Colwell, Gene T.↗

The Use of Steady and Unsteady Detonation Waves for Propulsion Systems

Detonation wave enhanced supersonic combustors such as the Oblique Detonation Wave Engine (ODWE) are attractive propulsion concepts for hypersonic flight. These engines utilize detonation waves to enhance fuel-air mixing and combustion. The benefits of wave combustion systems include shorter and lighter engines which require less cooling and generate lower internal drag. These features allow air-breathing operation at higher Mach numbers than the diffusive burning scramjet delaying the need for rocket engine augmentation. A comprehensive vehicle synthesis code has predicted the aerodynamic characteristics and structural size and weight of a typical single-stage-to-orbit vehicle using an ODWE. Other studies have focused on the use of unsteady or pulsed detonation waves. For low speed applications, pulsed detonation engines (PDE) have advantages in low weight and higher efficiency than turbojets. At hypersonic speeds, the pulsed detonations can be used in conjunction with a scramjet type engine to enhance mixing and provide thrust augmentation.

Adelman, Henry G.↗

Synergy and antagonism in a genome-scale model of metabolic hijacking by bacteriophages

Bacteriophage auxiliary metabolic genes (AMGs) alter host metabolism by hijacking reactions, but previous studies mostly inferred their roles from annotations, ignoring system-wide impacts and phage production. Here we integrate AMGs and phage assembly into a genome-scale metabolic model of Prochloroccocus marinus MED4 infected by P-HM2. We show that 17 directly hijacked reactions substantially affect more than 30% of the reactions in MED4 metabolism, including carbon fixation, photosynthesis, and nucleotide synthesis, distinguishing these AMGs as either phage aligned—shifting feasible reaction velocities in accordance with maximal phage production—or phage antialigned. Pareto optimization reveals that phage-aligned reactions alter phage-host growth trade-offs, while phage-antialigned reactions do not. We experimentally validate our predictions of system-level AMG impacts by measuring the N-dependent effect of P-HM2 cp12 expression on growth in a model relative of the genetically intractable MED4, Synechococcus elongatus. We also show that AMGs’ indirect impacts are synergistically and antagonistically coupled, providing systems-level insight into AMG perturbations and highlighting how nontrivial cascading effects shape host metabolism.

Rozum, Jordan C. [Pacific Northwest National Labor↗

Gyrokinetic simulations compared with magnetic fluctuations diagnosed with a Faraday-effect radial interferometer-polarimeter in the DIII-D pedestal

Abstract Experimental data on electromagnetic fluctuations in DIII-D, made available by the Faraday-effect radial interferometer-polarimeter (RIP) diagnostic Chen (2016 Rev. Sci. Instrum. 87 11E108), is examined in comparison with detailed gyrokinetic simulations using gyrokinetic electromagnetic numerical experiment (GENE). The diagnostic has the unique capability of making internal measurements of fluctuating magnetic fields ∫ n e δ B r d R ∫ n e d R . Local linear simulations identify microtearing modes (MTMs) over a substantial range of toroidal mode numbers (peaking at n = 15) with frequencies in good agreement with the experimental data. Local nonlinear simulations reinforce this result by producing a magnetic frequency spectrum in good agreement with that diagnosed by RIP. Simulated heat fluxes are in the range of experimental expectations. However, magnetic fluctuation amplitudes are substantially lower than the experimental expectations. Possible sources of this discrepancy are discussed, notably the fact that the diagnostics are localized at the mid-plane—the poloidal location where the simulations predict the fluctuation amplitudes to be smallest. Despite some discrepancies, several connections between simulations and experiments, combined with general criteria discriminating between potential pedestal instabilities, strongly point to MTMs as the source of the observed magnetic fluctuations.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

In-Flight Personalized Medication Management

Current medication selection for treatment of astronauts during spaceflight missions is primarily dictated by the task of efficiently treating the widest possible range of physiological conditions and illnesses with a limited set of medications. Dosage and recommendations on the combination of drugs are based on the assumption of genetically equal drug sensitivity and unchanged metabolism. To our knowledge, there was no pre-flight drug sensitivity testing on a genetic level for any of the previous manned NASA space missions. Although many of the common, binary drug-drug interactions are, most likely, already considered in the ISS Medical kit composition, multi-drug and multi-drug-gene factors are not incorporated in the medication selection or prescription. Furthermore, due to the physiological changes occurring in microgravity environments, astronauts might be susceptible to potential increased drug toxicity as a result of decreased clearance of numerous drugs. In particular, perturbation of CYP450 enzymes which contribute to the hepatic metabolism of the majority of drugs may have significant effects on therapeutic efficacy and increase treatment-related toxicity5. The genes encoding the CYP450 enzymes are highly variable in humans. Inheritable variations of CYP450 hepatic metabolizer enzymes and transport proteins play a crucial role in the inter-individual variability of drug efficiency and risks of adverse drug reactions5. Additionally, there are some reports that document changes in the levels of production of drug-metabolizing enzymes in microgravity. These data can be extrapolated to provide reasonable assumptions of decreased levels of expression for most CYP450 enzymes in human body during prolonged space travel. If the prescribed medication regiment is not fully effective or causes undesirable side effects, the ability of the astronauts to function and maintain peak performance levels during space flight could be seriously compromised. Therefore, technologies capable of predicting and managing medication side effects, interactions, and toxicity of drugs during spaceflight are needed. We propose to develop and customize for NASAs applications available on the market Personalized Prescribing System (PPS) that would provide a comprehensive, non-invasive solution for safer, targeted medication management for every crew member resulting in safer and more effective treatment and, consequently, better performance. PPS will function as both decision support and record-keeping tool for flight surgeons and astronauts in applying the recommended medications for situations arising in flight. The information on individual drug sensitivity will translate into personalized risk assessment for adverse drug reactions and treatment failures for each drug from the medication kit as well as predefined outcome of any combination of them. Dosage recommendations will also be made individually. The mobile app will facilitate ease of use by crew and medical professionals during training and flight missions.

Personalized Medication↗

Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process

The neurons of the neocortex are generated over a 6 day neuronogenetic interval that comprises 11 cell cycles. During these 11 cell cycles, the length of cell cycle increases and the proportion of cells that exits (Q) versus re-enters (P) the cell cycle changes systematically. At the same time, the fate of the neurons produced at each of the 11 cell cycles appears to be specified at least in terms of their laminar destination. As a first step towards determining the causal interrelationships of the proliferative process with the process of laminar specification, we present a two-pronged approach. This consists of (i) a mathematical model that integrates the output of the proliferative process with the laminar fate of the output and predicts the effects of induced changes in Q and P during the neuronogenetic interval on the developing and mature cortex and (ii) an experimental system that allows the manipulation of Q and P in vivo. Here we show that the predictions of the model and the results of the experiments agree. The results indicate that events affecting the output of the proliferative population affect both the number of neurons produced and their specification with regard to their laminar fate.

Evaluation Studies↗

Climate adaptation and sustainability in switchgrass: exploring plant-microbe-soil interactions across continental scale environmental gradients

Less carbon-intensive energy sources are needed to reduce greenhouse gas emissions and their predicted role in climate change. There is growing interest in the potential of biofuels for meeting this need. A critical question is whether large-scale biofuel production can be sustainable over the time scales needed to mitigate our carbon debt from fossil fuel consumption. The carbon balance and ultimately the sustainability of biofuel feedstock production is the result of complex climate-coupled interactions between carbon fixation, sequestration, and release through combustion. Similarly, the long-term productivity of biofuels depends on the environmental factors limiting plant growth. These factors are often related to soil resources which involve complex interactions at the plant-microbe-soil interface impacting their availability and cycling. Our collaborative project addressed sustainable switchgrass (Panicum virgatum) production by exploring Plant Systems, Plant-Microbiome Interactions, and Ecosystem Processes through the integrating lens of Multi-Scale Modeling. Our research was based on detailed characterization of genetically diverse switchgrass genotypes planted in common gardens across a continental latitudinal gradient. The underlying theme of our Plant Systems research was the use of locally adapted plant material to explore plant function, to understand the mechanistic basis of environmental interactions, and to discover the plant genes important for adaptation and sustainability in the face of climate change. Our Plant-Microbiome Interaction project characterized the microbial communities associated with switchgrass using genomic tools. Our Ecosystem Processes research focused on carbon cycle responses at the ecosystem level using stand level plantings. Finally, our Multi-Scale Modeling helped to define conditions of a sustainable biofuel system and identify key tradeoffs between genetic diversity, productivity, and ecosystem services. Genome-wide association analyses were used to identify alleles that contribute to successful establishment and biomass production across North America. Together, our work provided a baseline analyses of the potential of switchgrass as a biofuel feedstock. Our project resulted in a number of successful outcomes. First, we were successful in collecting switchgrass germplasm across the species range, propagating the material, and establishing common garden experiments across the species range. In collaboration with DOE JGI, we successfully assembled the first tetraploid switchgrass genome and published this resource with an analyses of the genetic basis local adaptation from our gardens (Lowry et al. 2019, Lovell et al. 2021). The gardens were used to characterize the genetic architecture for a number of important plant phenotypes. Our project also conducted extensive sampling and sequencing to characterize the bacterial and fungal associates of switchgrass roots and leaves. We showed that host genotype, location, and harvesting practices can play a role in microbiome assembly (Singer et al. 2019 & 2022, Van Wallendael et al. 2020 & 2022, Edwards et al. 2023). Our ecosystem processes work created baseline dataset of carbon and nutrient cycling in realistic stand plantings of switchgrass. Data from this experiment provided new insight into the role of plant traits, phenology, and local environments in ecosystem processes like soil respiration, net-ecosystem exchange, and dynamics of soil and plant nutrients (Ricketts et al. 2023). Finally, our crop modelling experiments help to characterize the sensitivity of common modeling frameworks to parameters, identify key limiters of productivity across large geographic scales, and leverage patterns of local adaptation in prediction. Ultimately, these studies help to identify critical plant-microbe-soil traits that may be manipulated, through breeding or agronomic management, to improve the sustainability of biofuel feedstocks.

09 BIOMASS FUELS↗

Rethinking Suicide Thi4 Thiazole Synthases: Comparative Genomic Insights and Pilot Functional Evidence

Suicide thiazole synthases (Thi4) are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD + , glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. Comparative genomic analysis shows that prokaryotic Thi4 genes often cluster on the chromosomal regions encoding ThiS, ThiF, and other proteins that can produce, relay, or use persulfide or thiocarboxylate sulfur. These recurring genomic associations raise the possibility that, in some microorganisms, suicide Thi4s may interact with sulfur-relay systems, i.e., they can possibly operate in a nonsuicide mode. This proof-of-concept study explores this possibility via complementation assays using Escherichia coli as a heterologous platform. A representative bacterial Thi4 that clustered with thiS and thiF complemented an E. coli ΔthiG (thiazole auxotroph) single mutant better than a ΔthiG ΔthiF ΔthiS triple mutant. Although (in)direct sulfur transfer could not be assessed in the scope of our investigation, the initial results suggest a dependence on host sulfur relay components, consistent with predicted interactions with the host sulfide transfer chain. Collectively, this new perspective provides a useful guide for future biochemical studies on alternative modes of action for “suicide Thi4s” and accessory proteins.

Bacteria↗

Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG’s role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

59 BASIC BIOLOGICAL SCIENCES↗

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

NASA Discipline Cell Biology↗

Patchy burn severity explains heterogeneous soil viral and prokaryotic responses to fire in a mixed conifer forest

ABSTRACT Effects of fire on soil viruses and virus–host dynamics are largely unexplored, despite known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we assessed how viral and prokaryotic communities responded to a prescribed burn in a mixed conifer forest. We sequenced 91 viral-size fraction metagenomes (viromes) and 115 16S rRNA gene amplicon libraries from 120 samples: four samples at five timepoints (two before fire and three after fire) at six sites (four treatment, two control). We hypothesized that compositional differences would be most significant between burned and unburned soils, but instead, plot location best distinguished viral communities, more than treatment (burned or not), depth (0–3 or 3–6 cm), or timepoint. For both viruses and prokaryotes, some burned communities resembled unburned controls, while others were significantly different, revealing heterogeneous responses to fire. These patterns were explained by burn severity, here defined by soil chemistry. Viral but not prokaryotic richness decreased significantly with burn severity, and low viromic DNA yields indicated substantial loss of viral biomass at higher severity. The relative abundances of Firmicutes, Actinobacteriota, and the viruses predicted to infect them increased significantly with burn severity, suggesting survival and viral infection of these fire-responsive and potentially spore-forming taxa. The degree of burn severity experienced by each patch of soil, rather than burn status alone, differed over mere meters in the same fire. Therefore, our analyses highlight the importance of high-resolution, paired biogeochemical data to explain soil community responses to fire. IMPORTANCE The impact of fire on the soil microbiome, particularly on understudied soil viral communities, warrants investigation, given known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we collected 120 soil samples before and after a prescribed burn in a mixed conifer forest to assess the impacts of this disturbance on soil viral and prokaryotic communities. We show that simple categorical comparisons of burned and unburned areas were insufficient to reveal the underlying community response patterns. The patchy nature of the fire (indicated by soil chemistry data) led to significant changes in viral and prokaryotic community composition in areas of high burn severity, while communities that experienced lower burn severity were indistinguishable from those in unburned controls. Our results highlight the importance of considering highly resolved burn severity and biogeochemical measurements, even in nearby soils after the same fire, in order to understand soil microbial responses to prescribed burns.

Microbiology↗

RCSB Protein Data Bank: improved annotation, search and visualization of membrane protein structures archived in the PDB

Abstract Motivation Membrane proteins are encoded by approximately one fifth of human genes but account for more than half of all US FDA approved drug targets. Thanks to new technological advances, the number of membrane proteins archived in the PDB is growing rapidly. However, automatic identification of membrane proteins or inference of membrane location is not a trivial task. Results We present recent improvements to the RCSB Protein Data Bank web portal (RCSB PDB, rcsb.org) that provide a wealth of new membrane protein annotations integrated from four external resources: OPM, PDBTM, MemProtMD and mpstruc. We have substantially enhanced the presentation of data on membrane proteins. The number of membrane proteins with annotations available on rcsb.org was increased by ∼80%. Users can search for these annotations, explore corresponding tree hierarchies, display membrane segments at the 1D amino acid sequence level, and visualize the predicted location of the membrane layer in 3D. Availability and implementation Annotations, search, tree data and visualization are available at our rcsb.org web portal. Membrane visualization is supported by the open-source Mol* viewer (molstar.org and github.com/molstar/molstar). Supplementary information Supplementary data are available at Bioinformatics online.

59 BASIC BIOLOGICAL SCIENCES↗

MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters

Abstract With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

59 BASIC BIOLOGICAL SCIENCES↗

Catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans

ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

59 BASIC BIOLOGICAL SCIENCES↗

Multi-site Milling Strategy Reveals Significant Variation in Biomass Composition of Switchgrass ( Panicum virgatum ) Grown at Ten Locations

Cell wall composition influences biomass use as a forage and as a feedstock for biofuel and chemical conversion. To examine the influence of environment on composition of switchgrass (Panicum virgatum L.), we utilized a multi-environment experiment consisting of clones of switchgrass genotypes grown at up to ten locations in the continental US. We tested the influence of different milling treatments on biomass composition trait predictions via near-infrared reflectance spectroscopy (NIRS). We found that most compositional trait predictions (29/34) were significantly different (P < 0.05) when a single lot of biomass was subjected to disparate milling treatments, i.e., knife milling vs. knife milling with an additional cyclone milling. Further, depending on the plant material tested, three to eight compositional trait predictions vary (P < 0.05) when identical biomass was knife milled at different sites followed by cyclone milling at a single site, including for traits such as Klason lignin, nitrogen, and carbon. In some cases, variation due to milling site exceeded environmentally induced compositional variation of a single switchgrass genotype grown at different sites. From these observations, we recommend a protocol with two sequential millings that decouples growth environment from a particular mill. Utilizing this approach, we found that 46/46 biomass composition traits from the warm season herbaceous forage and switchgrass bioethanol NIRS equations vary significantly (P < 0.001) in clones of a switchgrass genotype (WBC) grown at ten sites, with the growth site representing the largest average source of variation (41%). This multi-site milling approach can be used to examine environmental and gene-by-environment influences on composition with the goal of optimizing cell wall composition in different environments for biomass utilization.

09 BIOMASS FUELS↗