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At least 343 records · Page 19

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

59 BASIC BIOLOGICAL SCIENCES↗

Disentangling plant- and environment-mediated drivers of active rhizosphere bacterial community dynamics during short-term drought

Abstract Mitigating the effects of climate stress on crops is important for global food security. The microbiome associated with plant roots, the rhizobiome, can harbor beneficial microbes that alleviate stress, but the factors influencing their recruitment are unclear. We conducted a greenhouse experiment using field soil with a legacy of growing switchgrass and common bean to investigate the impact of short-term drought severity on the recruitment of active bacterial rhizobiome members. We applied 16S rRNA and 16S rRNA gene sequencing for both crops and metabolite profiling for switchgrass. We included planted and unplanted conditions to distinguish environment- versus plant-mediated rhizobiome drivers. Differences in community structure were observed between crops and between drought and watered and planted and unplanted treatments within crops. Despite crop-specific communities, drought rhizobiome dynamics were similar across the two crops. The presence of a plant more strongly explained the rhizobiome variation in bean (17%) than in switchgrass (3%), with a small effect of plant mediation during drought observed only for the bean rhizobiome. The switchgrass rhizobiome was stable despite changes in rhizosphere metabolite profiles between planted and unplanted treatments. We conclude that rhizobiome responses to short-term drought are crop-specific, with possible decoupling of plant exudation from rhizobiome responses.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic interactions shape emergent biofilm structures in a conceptual model of gut mucosal bacterial communities

Abstract The gut microbiome plays a major role in human health; however, little is known about the structural arrangement of microbes and factors governing their distribution. In this work, we present an in silico agent-based model (ABM) to conceptually simulate the dynamics of gut mucosal bacterial communities. We explored how various types of metabolic interactions, including competition, neutralism, commensalism, and mutualism, affect community structure, through nutrient consumption and metabolite exchange. Results showed that, across scenarios with different initial species abundances, cross-feeding promotes species coexistence. Morphologically, competition and neutralism resulted in segregation, while mutualism and commensalism fostered high intermixing. In addition, cooperative relations resulted in community properties with little sensitivity to the selective uptake of metabolites produced by the host. Moreover, metabolic interactions strongly influenced colonization success following the invasion of newcomer species. These results provide important insights into the utility of ABM in deciphering complex microbiome patterns.

Biotechnology & Applied Microbiology↗

Metabolic complexity drives divergence in microbial communities

Microbial communities are shaped by environmental metabolites, but the principles that govern whether different communities will converge or diverge in any given condition remain unknown, posing fundamental questions about the feasibility of microbiome engineering. Here, in this work, we studied the longitudinal assembly dynamics of a set of natural microbial communities grown in laboratory conditions of increasing metabolic complexity. We found that different microbial communities tend to become similar to each other when grown in metabolically simple conditions, but they diverge in composition as the metabolic complexity of the environment increases, a phenomenon we refer to as the divergence-complexity effect. A comparative analysis of these communities revealed that this divergence is driven by community diversity and by the assortment of specialist taxa capable of degrading complex metabolites. An ecological model of community dynamics indicates that the hierarchical structure of metabolism itself, where complex molecules are enzymatically degraded into progressively simpler ones that then participate in cross-feeding between community members, is necessary and sufficient to recapitulate our experimental observations. In addition to helping understand the role of the environment in community assembly, the divergence-complexity effect can provide insight into which environments support multiple community states, enabling the search for desired ecosystem functions towards microbiome engineering applications.

59 BASIC BIOLOGICAL SCIENCES↗

A microbial supply chain for production of the anti-cancer drug vinblastine

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

60 APPLIED LIFE SCIENCES↗

Mapping microhabitats of lignocellulose decomposition by a microbial consortium

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Manipulation of the Gut Microbiome Alters Acetaminophen Biodisposition in Mice

The gut microbiota is a vast and diverse microbial community that has co-evolved with its host to perform a variety of essential functions involved in the utilization of nutrients and the processing of xenobiotics. Shifts in the composition of gut microbiota can disturb the balance of organisms which can infuence the biodisposition of orally administered drugs. To determine how changes in the gut microbiome can alter drug disposition, the pharmacokinetics (PK), and biodistribution of acetaminophen were assessed in C57Bl/6 mice after treatment with the antibiotics ciprofoxacin, amoxicillin, or a cocktail of ampicillin/neomycin. Altered PK, and excretion profles of acetaminophen were observed in antibiotic exposed animals. Plasma C max was signifcantly decreased in antibiotic treated animals suggesting decreased bioavailability. Urinary metabolite profles revealed decreases in acetaminophen-sulfate metabolite levels in both the amoxicillin and ampicillin/neomycin treated animals. The ratio between urinary and fecal excretion was also altered in antibiotic treated animals. Analysis of gut microbe composition revealed that changes in microbe content in antibiotic treated animals was associated with changes in acetaminophen biodisposition. These results suggest that exposure to amoxicillin or ampicillin/neomycin can alter the biodisposition of acetaminophen and that these alterations could be due to changes in gut microbiome composition.

59 BASIC BIOLOGICAL SCIENCES↗

Acetyl-CoA Synthesis Through a Bicyclic Carbon-Fixing Pathway in Gas-Fermenting Bacteria

Gas-fermenting acetogens can upgrade one-carbon (C1) compounds (such as CO2 and CO) to the two-carbon (C2) metabolite acetyl coenzyme A (CoA) and convert sugar feedstocks to acetyl-CoA with minimal CO2 emissions. Fulfilling the biosynthetic potential of these microbes requires overcoming challenges in pathway engineering. Here we design a synthetic acetyl-CoA bi-cycle-in addition to the natural carbon-fixing pathways-for C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in sequence: carbon fixation, gluconeogenesis and non-oxidative glycolysis. The pathway was examined by in silico thermodynamic and kinetic analyses. The prototypic pathway was implemented in a syngas-fermenting organism, Clostridium ljungdahlii DSM 13528, by expressing a heterologous phosphoketolase that can work with other native enzymes in the host acetogen. The carbon conversion pathway is possible under various growth conditions and is independent of the Wood-Ljungdahl pathway for the valorization of H2 and CO2. This study reports the improvement of carbon conversion using a reductive acetyl-CoA bi-cycle and the potential impact of redox homoeostasis in the acetogenic host for industrial applications of gas fermentation.

Acetyl-CoA↗

Electrochemical and spectroelectrochemical characterization of bacteria and bacterial systems

Microbes, such as bacteria, can be described, at one level, as small, self-sustaining chemical factories. Based on the species, strain, and even the environment, bacteria can be useful, neutral or pathogenic to human life, so it is increasingly important that we be able to characterize them at the molecular level with chemical specificity and spatial and temporal resolution in order to understand their behavior. Bacterial metabolism involves a large number of internal and external electron transfer processes, so it is logical that electrochemical techniques have been employed to investigate these bacterial metabolites. In this mini-review, we focus on electrochemical and spectroelectrochemical methods that have been developed and used specifically to chemically characterize bacteria and their behavior. First, we discuss the latest mechanistic insights and current understanding of microbial electron transfer, including both direct and mediated electron transfer. Second, we summarize progress on approaches to spatiotemporal characterization of secreted factors, including both metabolites and signaling molecules, which can be used to discern how natural or external factors can alter metabolic states of bacterial cells and change either their individual or collective behavior. Finally, we address in situ methods of single-cell characterization, which can uncover how heterogeneity in cell behavior is reflected in the behavior and properties of collections of bacteria, e.g. bacterial communities. Recent advances in (spectro)electrochemical characterization of bacteria have yielded important new insights both at the ensemble and the single-entity levels, which are furthering our understanding of bacterial behavior. Furthermore, these insights, in turn, promise to benefit applications ranging from biosensors to the use of bacteria in bacteria-based bioenergy generation and storage.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Plant-based engineering for production of high-valued natural products

Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, in this paper, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

59 BASIC BIOLOGICAL SCIENCES↗

Cell-free synthetic biology for natural product biosynthesis and discovery

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Metabolic Modification of Sphingobium lignivorans SYK-6 for Lignin Valorization Through the Discovery of an Unusual Transcriptional Repressor of Lignin-Derived Dimer Catabolism

Sphingobium lignivorans SYK-6 catabolizes guaiacylglycerol-..beta..-guaiacyl ether (GGE, a ..beta..-O-4-type dimer) and 1,2-diguaiacylpropane-1,3-diol (DGPD, a ..beta..-1-type dimer) derived from lignin. Recently, SLG_35860 containing TetR- and MarR-type transcriptional regulator motifs was suggested to be involved in the regulation of GGE and DGPD catabolism. Here we investigated the role of SLG_35860 in the transcriptional regulation of GGE and DGPD catabolism genes. SLG_35860 designated ligS repressed 11 genes involved in GGE and DGPD catabolism. LigS binds directly to specific sequences in the promoter region of each gene. The MarR domain was shown to be involved in these bindings; however, GGE, DGPD, and their metabolites did not function as effectors of LigS. We discovered unidentified compound(s) in the black liquor of oxygen-soda anthraquinone pulping of Japanese cedar that SYK-6 cannot metabolize and that acted as effector(s). Therefore, LigS constantly represses the transcription of the GGE and DGPD catabolism genes to low levels. Based on these findings, we examined the productivity of a polymer building block, 2-pyrone-4,6-dicarboxylic acid (PDC), from GGE, DGPD, and a GGE metabolite using an engineered ligS mutant. The rates of PDC production from each compound by this strain were 1.5-6.0 times higher than those of a PDC-producing strain carrying ligS.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Maximizing long-term biohydrogen production with Clostridium thermocellum for high solids conversion of lignocellulosic biomass

Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems (i.e., cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation. To address these barriers to process intensification, we implemented novel reactor and process designs for high-solids lignocellulosic biomass fermentations using the C. thermocellum KJC19-9 strain, genetically engineered for co-utilization of cellulose and hemicellulose sugars (i.e., xylose). Via computational fluid dynamics (CFD) modeling and experimental validation, we achieved a >50% improvement in biohydrogen production with an improved anchor-type impeller morphology, coupled to a threefold reduction in agitation rate. To further reduce rheological constraints and accumulation of toxic metabolites, we then transitioned the process to sequencing fed-batch operation. The resulting process generated 24.87 L H 2 L −1 from 160 g L −1 of deacetylated and mechanically refined (DMR)-pretreated corn stover biomass over 16 days while solubilizing >95% of influent cellulose and hemicellulose, setting a new performance benchmark for continuous production of biohydrogen from lignocellulose.

08 HYDROGEN↗

Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. Here, the reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO 2 -derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Glyceraldehyde-3-phosphate dehydrogenase homologs as bifunctional gatekeepers of metabolic segregation in Pseudomonas putida

Metabolically versatile Pseudomonas species can assimilate various glycolytic and gluconeogenic substrates. Simultaneous assimilation is known to segregate carbons from each substrate type into different metabolic pathways. However, the mechanisms of this metabolic segregation remain unresolved. Here, we investigate Pseudomonas putida KT2440 during processing of the sugar glucose through glycolysis versus the phenolic acid ferulate through gluconeogenesis. Metabolome profiling reveals up to twofold less tricarboxylic acid cycle metabolites but up to 10-fold higher metabolites of upper glycolysis, pentose-phosphate, and Entner–Doudoroff pathways in glucose-grown cells compared to ferulate-grown cells. After 13C-substrate switching, kinetic isotopic profiling captures rapid assimilation of new substrate carbons into initial catabolic pathways, but incorporation into downstream pathways is absent or incomplete. Proteomics identifies a 22-fold higher abundance of one homolog of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GapA) in cells fed on glucose relative to ferulate, while abundance of another homolog (GapB) remains unchanged. Growth phenotypes and quantitative metabolomics for single and double knockout mutants of these GAPDH homologs indicate only GapA involvement in glycolytic flux, which can be compensated by the Entner–Doudoroff pathway, and distinct preference of GapB with minimal role of GapA for gluconeogenic flux. Accordingly, growth of triple knockout mutant with deletion of gapA, gapB, and edd is possible only when glycolytic and gluconeogenic substrates are provided together to meet metabolic demands in a segregated fashion, but metabolic tradeoffs lead to slow growth. A mathematical, experimentally constrained, model of the GAPDH node shows that tuning of GapA and GapB concentrations enables transition between flux regimes for nutritional adaptability.

Zhou, Nanqing [Northwestern University, Evanston]↗

Gut enterotype-dependent modulation of gut microbiota and their metabolism in response to xanthohumol supplementation in healthy adults

Xanthohumol (XN), a polyphenol found in the hop plant (Humulus lupulus), has antioxidant, anti-inflammatory, prebiotic, and anti-hyperlipidemic activity. Preclinical evidence suggests the gut microbiome is essential in mediating these bioactivities; however, relatively little is known about XN’s impact on human gut microbiota in vivo. We conducted a randomized, triple-blinded, placebo-controlled clinical trial (ClinicalTrials.gov NCT03735420) to determine safety and tolerability of XN in healthy adults. Thirty healthy participants were randomized to 24 mg/day XN or placebo for 8 weeks. As secondary outcomes, quantification of bacterial metabolites and 16S rRNA gene sequencing were utilized to explore the relationships between XN supplementation, gut microbiota, and biomarkers of gut health. Although XN did not significantly change gut microbiota composition, it did re-shape individual taxa in an enterotype-dependent manner. High levels of inter-individual variation in metabolic profiles and bioavailability of XN metabolites were observed. Moreover, reductions in microbiota-derived bile acid metabolism were observed, which were enterotype-dependent. These results suggest interactions between XN and gut microbiota in healthy adults are highly inter-individualized and potentially indicate that XN elicits effects on gut health in an enterotype-dependent manner.

59 BASIC BIOLOGICAL SCIENCES↗

A device for volatile organic compound (VOC) analysis from skin using heated dynamic headspace sampling

Abstract Human skin is an important source of volatile organic compounds (VOCs) offering noninvasive methods to gain clinical metabolite information. This work was focused on the development of a skin sampling device based on a dynamic headspace sampling method with the addition of temperature to increase VOC metabolite recovery. The device preconcentrates skin VOC emissions onto a sorbent substrate, which can either be preserved for offline analysis or attached to a real time sensor downstream. In this work, skin VOC samples were analyzed offline using thermal desorption-gas chromatography-mass spectrometry. A list of 10 common skin VOCs was pre-selected to optimize parameters of sampling time, sampling temperature, and sorbent selection. Overall, this study highlights an effective skin VOC sampling technology with a heating dimension (40 °C, rather than 30 °C or no heating) with a sampling time of 15 min (rather than 5 or 30 mins) and onto Tenax TA sorbent (rather than PDMS), which collectively increases the recovery of compounds with lower vapor pressure and decreases the observed variability in skin VOC measurements. Finally, a list of 79 skin VOC compounds were detected and identified within a cohort of 20 young, healthy volunteers.

Biochemistry & Molecular Biology↗