Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock. carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps

From waste to health-supporting molecules: biosynthesis of natural products from lignin-, plastic- and seaweed-based monomers using metabolically engineered Streptomyces lividans

Background Transforming waste and nonfood materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry to optimize the use of resources while reducing environmental footprint. However, despite these advancements, the production of high-value natural products often continues to depend on the use of frst-generation substrates, underscoring the intricate processes and specifc requirements of their bio‑ syntheses. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce a wide array of natural products, which is attributed to its genetic versatility and potent secondary metabolic activ‑ ity. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifcally from waste and nonfood substrates. Results We metabolically engineered S. lividans to heterologously produce the ribosomally synthesized and post‑ translationally modifed peptide bottromycin, as well as the polyketide pamamycin. The modifed strains success‑ fully produced these compounds using waste and nonfood model substrates such as protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive tran‑ scriptomic and metabolomic analyses ofered insights into how these substrates infuenced the cellular metabolism of S. lividans. In terms of production efciency, S. lividans showed remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, which led to enhanced and highly selective bottromycin production. Additionally, the strain generated a unique spectrum of pamamycins when cul‑ tured in mannitol-rich seaweed extract with no additional nutrients. Conclusion Our study showcases the successful production of high-value natural products based on the use of var‑ ied waste and nonfood raw materials, circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience when grown on these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide pro‑ duction. Future research could be focused on enhancing S. lividans substrate utilization pathways to process the intri‑ cate mixtures commonly found in waste and nonfood sources more efciently

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes

Background Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5–10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. Results Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases

Dynamic metabolic rewiring enables efficient acetyl-CoA assimilation in Paracoccus denitrificans

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl-CoA, which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies have been described in bacteria so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism’s central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling fast growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism

Hotspots of land use change in Europe

Die Zweitveröffentlichung der Publikation wurde durch Studierende des Projektseminars "Open Access Publizieren an der HU" im Sommersemester 2017 betreut. Nachgenutzt gemäß den CC-Bestimmungen des Lizenzgebers bzw. einer im Dokument selbst enthaltenen CC-Lizenz.Assessing changes in the extent and management intensity of land use is crucial to understanding land-system dynamics and their environmental and social outcomes. Yet, changes in the spatial patterns of land management intensity, and thus how they might relate to changes in the extent of land uses, remains unclear for many world regions.Wecompiled and analyzed high-resolution, spatiallyexplicit land-use change indicators capturing changes in both the extent and management intensity of cropland, grazing land, forests, and urban areas for all of Europe for the period 1990–2006. Based on these indicators, we identified hotspots of change and explored the spatial concordance of area versus intensity changes.Wefound a clear East–West divide with regard to agriculture, with stronger cropland declines and lower management intensity in the East compared to the West. Yet, these patterns were not uniform and diverging patterns of intensification in areas highly suitable for farming, and disintensification and cropland contraction in more marginal areas emerged. Despite the moderate overall rates of change, many regions in Europe fell into at least one land-use change hotspot during 1990–2006, often related to a spatial reorganization of land use (i.e., co-occurring area decline and intensification or co-occurring area increase and disintensification). Our analyses highlighted the diverse spatial patterns and heterogeneity of land-use changes in Europe, and the importance of jointly considering changes in the extent and management intensity of land use, as well as feedbacks among land-use sectors. Given this spatial differentiation of land-use change, and thus its environmental impacts, spatially-explicit assessments of land-use dynamics are important for context-specific, regionalized land-use policy making.Peer Reviewe

Independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae)

Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.Austrian Science Fund (FWF) PZ00P3-161472National Science Foundation (NSF) 1811965 1645256Triad FoundationGerman Research Foundation (DFG) DFG-PE 2059/3-1Agencia Estatal de Investigacion CGL2017-86626-C2-2-PLOEWE Program Insect Biotechnology and BioresourcesJunta de Andalucía A-RNM505-UGR1

Emergence of fractal geometries in the evolution of a metabolic enzyme

Fractals are patterns that are self-similar across multiple length-scales. Macroscopic fractals are common in nature; however, so far, molecular assembly into fractals is restricted to synthetic systems. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution

Модернизация автоматизированной системы блока стабилизации давления магистрального насосного агрегата на нефтеперекачивающей станции

Объектом исследования является блок стабилизации давления магистрального насосного агрегата. Цель работы – модернизация автоматизированной системы блока стабилизации давления магистрального трубопровода нефтеперекачивающей станции с использованием ПЛК, на основе выбранной SCADA-системы. В данном проекте была разработана система контроля и управления технологическим процессом на базе промышленных контроллеров Modicon M340 BMXP34 2020, с применением SCADA-системы.The object of investigation is the pressure stabilization unit of the main pump unit. The purpose of the work is the modernization of the automated system for stabilizing the pressure of the main pipeline of the oil pumping station using PLCs, based on the SCADA system chosen. In this project, a system for monitoring and controlling the process was developed on the basis of industrial controllers Modicon M340 BMXP34 2020, using a SCADA system

Ten facts about land systems for sustainability

Land use is central to addressing sustainability issues, including biodiversity conservation, climate change, food security, poverty alleviation, and sustainable energy. In this paper, we synthesize knowledge accumulated in land system science, the integrated study of terrestrial social-ecological systems, into 10 hard truths that have strong, general, empirical support. These facts help to explain the challenges of achieving sustainability in land use and thus also point toward solutions. The 10 facts are as follows: 1) Meanings and values of land are socially constructed and contested; 2) land systems exhibit complex behaviors with abrupt, hard-to-predict changes; 3) irreversible changes and path dependence are common features of land systems; 4) some land uses have a small footprint but very large impacts; 5) drivers and impacts of land-use change are globally interconnected and spill over to distant locations; 6) humanity lives on a used planet where all land provides benefits to societies; 7) land-use change usually entails trade-offs between different benefits—"win–wins" are thus rare; 8) land tenure and land-use claims are often unclear, overlapping, and contested; 9) the benefits and burdens from land are unequally distributed; and 10) land users have multiple, sometimes conflicting, ideas of what social and environmental justice entails. The facts have implications for governance, but do not provide fixed answers. Instead they constitute a set of core principles which can guide scientists, policy makers, and practitioners toward meeting sustainability challenges in land use