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At least 181 records · Page 10

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↗

Rewetting of soil: Revisiting the origin of soil CO 2 emissions

Rewetting dry soils is associated with a burst of microbial activity and mineralization, which manifests itself as a pulse in soil CO 2 emissions, long-known as the Birch effect. In arid and semi-arid systems, soil CO 2 losses upon rewetting at the end of extended dry periods can contribute a significant fraction to the overall carbon (C) budget. Microbial biomass is one of the sources of mineralized C, as was demonstrated over 30 years ago (Kieft et al., 1987). The present paper offers a perspective on how the field has progressed since the 1987 paper was published in Soil Biology & Biochemistry, what it means in terms of current concerns about global climate change, and the needs and potential emphases of future research. Many studies since 1987 have addressed the origin of this CO 2 pulse, finding multiple possible C sources involving both biotic and abiotic processes. We propose that the magnitude of the rewetting event (Δψ) determines the relative contribution from the array of substrates that contribute to the soil CO 2 pulse upon rewetting. The magnitude of the CO 2 pulse is likely related to soil physical characteristics and to the size of the available C pool, which is partly controlled by plants. Further, the relative contributions of the mechanisms generating soil CO 2 pulses upon rewetting are likely to be modified by climate change. To understand and predict the magnitude of soil CO 2 pulses upon rewetting, we advocate continued cross-disciplinary research involving soil microbial ecology, soil physics, soil chemistry, as well as increased integration and recognition of the importance of plant-soil interactions in controlling the soil C pools contributing to soil CO 2 pulses.

54 ENVIRONMENTAL SCIENCES↗

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro , the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus . The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

59 BASIC BIOLOGICAL SCIENCES↗

Galba: genome annotation with miniprot and AUGUSTUS

The Earth Biogenome Project has rapidly increased the number of available eukaryotic genomes, but most released genomes continue to lack annotation of protein-coding genes. In addition, no transcriptome data is available for some genomes. Various gene annotation tools have been developed but each has its limitations. Here, we introduce GALBA, a fully automated pipeline that utilizes miniprot, a rapid protein-to-genome aligner, in combination with AUGUSTUS to predict genes with high accuracy. Accuracy results indicate that GALBA is particularly strong in the annotation of large vertebrate genomes. We also present use cases in insects, vertebrates, and a land plant. GALBA is fully open source and available as a docker image for easy execution with Singularity in high-performance computing environments. Our pipeline addresses the critical need for accurate gene annotation in newly sequenced genomes, and we believe that GALBA will greatly facilitate genome annotation for diverse organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Adapting enzymes to improve their functionality in plants: why and how

Synthetic biology creates new metabolic processes and improves existing ones using engineered or natural enzymes. These enzymes are often sourced from cells that differ from those in the target plant organ with respect to, e.g. redox potential, effector levels, or proteostasis machinery. Non-native enzymes may thus need to be adapted to work well in their new plant context (‘plantized’) even if their specificity and kinetics in vitro are adequate. Hence there are two distinct ways in which an enzyme destined for use in plants can require improvement: In catalytic properties such as substrate and product specificity, kcat, and KM; and in general compatibility with the milieu of cells that express the enzyme. Continuous directed evolution systems can deliver both types of improvement and are so far the most broadly effective way to deliver the second type. Accordingly, in this review we provide a short account of continuous evolution methods, emphasizing the yeast OrthoRep system because of its suitability for plant applications. We then cover the down-to-earth and increasingly urgent issues of which enzymes and enzyme properties can — or cannot — be improved in theory, and which in practice are the best to target for crop improvement, i.e. those that are realistically improvable and important enough to warrant deploying continuous directed evolution. We take horticultural crops as examples because of the opportunities they present and to sharpen the focus.

Biochemistry & Molecular Biology↗

Critical Enzyme Reactions in Aromatic Catabolism for Microbial Lignin Conversion

The application of microbes to valorize aromatic compounds derived from the abundant plant biopolymer lignin is a rapidly developing area of research that may ultimately enable viable conversion of this recalcitrant and heterogeneous resource to valuable bio-based chemical products. Starting from the three canonical lignin building blocks, which differ in the extent of aromatic ring methoxylation, several common classes of enzymatic reaction occur in the upper pathways of aromatic catabolism to prepare aromatic compounds for assimilation into central carbon metabolism, including aromatic O-demethylation, hydroxylation and decarboxylation. These critical enzymatic steps can often be rate-limiting for efficient biological funnelling of aromatic compounds. Here we review the known enzymatic mechanisms for these reactions that are relevant for aerobic aromatic catabolism of lignin-related monomers, highlighting opportunities at the intersection of biochemistry, enzyme engineering and metabolic engineering for applications in the expanding field of microbial lignin valorization.

aromatic catabolism↗

Let's Exploit Available Knowledge on Vegetation Fluorescence

The potential to measure vegetation fluorescence from space (1) and to derive from it direct information on the gross primary productivity (GPP) of terrestrial ecosystems is probably the most thrilling development in remote sensing and global ecology of recent years, as it moves Earth observation techniques from the detection of canopy biophysics (e.g., fraction of absorbed radiation) and biochemistry (chlorophyll and nitrogen content) to the realm of ecosystem function. The existence of a functional relationship between fluorescence and photosynthesis has been elucidated over the last decade by several laboratories, notably as part of the preliminary studies of the European Space Agency Fluorescence Explorer (FLEX) Earth Explorer Mission. The empirical observation presented by Guanter et al. (2) of a linear relationship between fluorescence radiance and GPP, however, provides the first experimental confirmation of the feasibility of the approach— already thoroughly tested at leaf level—at the desired scale, despite the confounding effects associated with the satellite detection of such a faint signal. A word of clarification is needed here. The use of fluorescence as a probe of leaf photochemistry has been a staple of plant ecophysiology for decades, rooted in a sound understanding of photosynthetic energy dissipation. However, most past studies had to rely for the interpretation of results on active (pulse-saturated) techniques, making them unsuitable for remote-sensing applications. Over recent years, however, novel process based models have been developed for the interpretation of steady-state, solar-induced fluorescence at the leaf to canopy scale (3). We are therefore in a position to move beyond the mere empirical observation of an association between GPP and fluorescence radiance. In particular, Guanter et al. (2) base their analysis on the assumption of a constant ratio between photosynthetic and fluorescence light use efficiencies (equation 3 in ref. 2).We know, however, that the ratio is not constant, but changes widely in response to light, CO2, stomatal limitations, and extreme stress (4, 5). What’s more, we can make sense of such changes, thus extracting valuable information from the very scatter that is apparent in their data. However, this process will require the availability of more tailored instruments, such as the one planned for the FLEX mission. As already stressed by Guanter et al. (2), the spatial resolution of the Global Ozone Monitoring Experiment-2 sensor (40 × 80 km) makes it difficult to compare meaningfully the fluorescence signal with ground measurements, when only 60–70% of the footprint consists of the desired land-cover type (table S1 in ref. 2), suggesting that this could be largely responsible for the low signals observed in European grasslands. Moreover, the overpass time of the MetOp-A satellite (9:30 AM) implies that fluorescence is generally measured under light-limiting conditions, when fluorescence is only marginally affected by stomatal closure even under stress conditions. This result could explain the seasonal mismatch with daily GPP observed in natural ecosystems in the absence of irrigation (figure 4 in ref. 2). We hope, therefore, that this welcome contribution to this fast-advancing field will help demonstrate the potential of the new technique, and pave the way for more refined studies under both a technological and scientific point of view.

Space↗

The cytochrome bc 1 complex as an antipathogenic target

The cytochrome bc 1 complex is a key component of the mitochondrial respiratory chains of many eukaryotic microorganisms that are pathogenic for plants or humans, such as fungi responsible for crop diseases and Plasmodium falciparum, which causes human malaria. Cytochrome bc 1 is an enzyme that contains two (ubi)quinone/quinol-binding sites, which can be exploited for the development of fungicidal and chemotherapeutic agents. In this article, we review recent progress in determination of the structure and mechanism of action of cytochrome bc 1 , and the associated development of antimicrobial agents (and associated resistance mechanisms) targeting its activity.

59 BASIC BIOLOGICAL SCIENCES↗

Macroalgal germplasm banking for conservation, food security, and industry

Ex situ seed banking was first conceptualized and implemented in the early 20th century to maintain and protect crop lines. Today, ex situ seed banking is important for the preservation of heirloom strains, biodiversity conservation and ecosystem restoration, and diverse research applications. However, these efforts primarily target microalgae and terrestrial plants. Although some collections include macroalgae (i.e., seaweeds), they are relatively few and have yet to be connected via any international, coordinated initiative. In this piece, we provide a brief introduction to macroalgal germplasm banking and its application to conservation, industry, and mariculture. We argue that concerted effort should be made globally in germline preservation of marine algal species via germplasm banking with an overview of the technical advances for feasibility and ensured success.

59 BASIC BIOLOGICAL SCIENCES↗

Antifungal and Phytotoxic Activities of Isolated Compounds from Helietta parvifolia Stems

The identification of natural and environmentally friendly pesticides is a key area of interest for the agrochemical industry, with many potentially active compounds being sourced from numerous plant species. In this study, we report the bioassay-guided isolation and identification of phytotoxic and antifungal compounds from the ethyl acetate extract of Helietta parvifolia stems. We identified eight compounds, consisting of two coumarins and six alkaloids. Among these, a new alkaloid, 2-hydroxy-3,6,7-trimethoxyquinoline-4-carbaldehyde (6), was elucidated, along with seven known compounds. The phytotoxicity of purified compounds was evaluated, and chalepin (4) was active against Agrostis stolonifera at 1 mM with 50% inhibition of seed germination and it reduced Lemna pausicotata (duckweed) growth by 50% (IC50) at 168 μM. Additionally, we evaluated the antifungal activity against the fungal plant pathogen Colletotrichum fragariae using a thin-layer chromatography bioautography assay, which revealed that three isolated furoquinoline alkaloids (flindersiamine (3), kokusagenine (7), and maculine (8)) among the isolated compounds had the strongest inhibitory effects on the growth of C. fragariae at all tested concentrations. Our results indicate that these active natural compounds, i.e., (3), (4), (7), and (8), could be scaffolds for the production of more active pesticides with better physicochemical properties.

59 BASIC BIOLOGICAL SCIENCES↗

Diel dynamics of multi-omics in elkhorn fern provide new insights into weak CAM photosynthesis

Crassulacean acid metabolism (CAM) has high water-use efficiency (WUE) and is widely recognized to have evolved from C 3 photosynthesis. Different plant lineages have convergently evolved CAM, but the molecular mechanism that underlies C 3 -to-CAM evolution remains to be clarified. Platycerium bifurcatum (elkhorn fern) provides an opportunity to study the molecular changes underlying the transition from C 3 to CAM photosynthesis because both modes of photosynthesis occur in this species, with sporotrophophyll leaves (SLs) and cover leaves (CLs) performing C 3 and weak CAM photosynthesis, respectively. Here, we report that the physiological and biochemical attributes of CAM in weak CAM-performing CLs differed from those in strong CAM species. We investigated the diel dynamics of the metabolome, proteome, and transcriptome in these dimorphic leaves within the same genetic background and under identical environmental conditions. We found that multi-omic diel dynamics in P. bifurcatum exhibit both tissue and diel effects. Our analysis revealed temporal rewiring of biochemistry relevant to the energy-producing pathway (TCA cycle), CAM pathway, and stomatal movement in CLs compared with SLs. We also confirmed that PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) exhibits convergence in gene expression among highly divergent CAM lineages. Gene regulatory network analysis identified candidate transcription factors regulating the CAM pathway and stomatal movement. Taken together, our results provide new insights into weak CAM photosynthesis and new avenues for CAM bioengineering.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanism and potential applications of bio-ligninolytic systems in a CELSS

A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms. Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.

Review↗

Exploring the Interplay between Polyphenols and Lysyl Oxidase Enzymes for Maintaining Extracellular Matrix Homeostasis

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

59 BASIC BIOLOGICAL SCIENCES↗

Functional Meta-Analysis of the Proteomic Responses of Arabidopsis Seedlings to the Spaceflight Environment Reveals Multi-Dimensional Sources of Variability across Spaceflight Experiments

The human quest for sustainable habitation of extraterrestrial environments necessitates a robust understanding of life’s adaptability to the unique conditions of spaceflight. This study provides a comprehensive proteomic dissection of the Arabidopsis plant’s responses to the spaceflight environment through a meta-analysis of proteomics data from four separate spaceflight experiments conducted on the International Space Station (ISS) in different hardware configurations. Raw proteomics LC/MS spectra were analyzed for differential expression in MaxQuant and Perseus software. The analysis of dissimilarities among the datasets reveals the multidimensional nature of plant proteomic responses to spaceflight, impacted by variables such as spaceflight hardware, seedling age, lighting conditions, and proteomic quantification techniques. By contrasting datasets that varied in light exposure, we elucidated proteins involved in photomorphogenesis and skotomorphogenesis in plant spaceflight responses. Additionally, with data from an onboard 1 g control experiment, we isolated proteins that specifically respond to the microgravity environment and those that respond to other spaceflight conditions. This study identified proteins and associated metabolic pathways that are consistently impacted across the datasets. Notably, these shared proteins were associated with critical metabolic functions, including carbon metabolism, glycolysis, gluconeogenesis, and amino acid biosynthesis, underscoring their potential significance in Arabidopsis’ spaceflight adaptation mechanisms and informing strategies for successful space farming.

59 BASIC BIOLOGICAL SCIENCES↗

Vegetation species composition and canopy architecture information expressed in leaf water absorption measured in the 1000 nm and 2200 spectral region by an imaging spectrometer

Plant species composition and plant architectural attributes are critical parameters required for the measuring, monitoring, and modeling of terrestrial ecosystems. Remote sensing is commonly cited as an important tool for deriving vegetation properties at an appropriate scale for ecosystem studies, ranging from local to regional and even synoptic scales. Classical approaches rely on vegetation indices such as the normalized difference vegetation index (NDVI) to estimate biophysical parameters such as leaf area index or intercepted photosynthetically active radiation (IPAR). Another approach is to apply a variety of classification schemes to map vegetation and thus extrapolate fine-scale information about specific sites to larger areas of similar composition. Imaging spectrometry provides additional information that is not obtainable through broad-band sensors and that may provide improved inputs both to direct biophysical estimates as well as classification schemes. Some of this capability has been demonstrated through improved discrimination of vegetation, estimates of canopy biochemistry, and liquid water estimates from vegetation. We investigate further the potential of leaf water absorption estimated from Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) data as a means for discriminating vegetation types and deriving canopy architectural information. We expand our analysis to incorporate liquid water estimates from two spectral regions, the 1000-nm region and the 2200-nm region. The study was conducted in the vicinity of Jasper Ridge, California, which is located on the San Francisco peninsula to the west of the Stanford University campus. AVIRIS data were acquired over Jasper Ridge, CA, on June 2, 1992, at 19:31 UTC. Spectra from three sites in this image were analyzed. These data are from an area of healthy grass, oak woodland, and redwood forest, respectively. For these analyses, the AVIRIS-measured upwelling radiance spectra for the entire Jasper Ridge scene were transformed to apparent surface reflectance using a radiative transfer code-based inversion algorithm.

Green, Robert O.↗

Information-incorporated gene network construction with FDR control

Abstract Motivation Large-scale gene expression studies allow gene network construction to uncover associations among genes. To study direct associations among genes, partial correlation-based networks are preferred over marginal correlations. However, FDR control for partial correlation-based network construction is not well-studied. In addition, currently available partial correlation-based methods cannot take existing biological knowledge to help network construction while controlling FDR. Results In this paper, we propose a method called Partial Correlation Graph with Information Incorporation (PCGII). PCGII estimates partial correlations between each pair of genes by regularized node-wise regression that can incorporate prior knowledge while controlling the effects of all other genes. It handles high-dimensional data where the number of genes can be much larger than the sample size and controls FDR at the same time. We compare PCGII with several existing approaches through extensive simulation studies and demonstrate that PCGII has better FDR control and higher power. We apply PCGII to a plant gene expression dataset where it recovers confirmed regulatory relationships and a hub node, as well as several direct associations that shed light on potential functional relationships in the system. We also introduce a method to supplement observed data with a pseudogene to apply PCGII when no prior information is available, which also allows checking FDR control and power for real data analysis. Availability and implementation R package is freely available for download at https://cran.r-project.org/package=PCGII.

59 BASIC BIOLOGICAL SCIENCES↗

OxyR regulates the oxidative stress response in Zymomonas mobilis during oxic growth and anoxic biofuel fermentation

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. RNA-Sequencing was perfromed on wild-type Z. mobilis (ZM4) and ∆oxyR in both oxic and anoxic conditions in rich media (ZRMG). This study reveals the indirect regulon of OxyR in Z. mobilis, which is important for both oxic growth and anoxic biofuel fermentation.

bioenergy↗