There is a compelling need across several industries to substitute non‐degradable, intentionally added microplastics with biodegradable alternatives. Nonetheless, stringent performance criteria in ...actives’ controlled release and manufacturing at scale of emerging materials hinder the replacement of polymers used for microplastics fabrication with circular ones. Here, the authors demonstrate that active microencapsulation in a structural protein such as silk fibroin can be achieved by modulating protein protonation and chain relaxation at the point of material assembly. Silk fibroin micelles’ size is tuned from several to hundreds of nanometers, enabling the manufacturing—by retrofitting spray drying and spray freeze drying techniques—of microcapsules with tunable morphology and structure, that is, hollow‐spongy, hollow‐smooth, hollow crumpled matrices, and hollow crumpled multi‐domain. Microcapsules degradation kinetics and sustained release of soluble and insoluble payloads typically used in cosmetic and agriculture applications are controlled by modulating fibroin's beta‐sheet content from 20% to near 40%. Ultraviolet‐visible studies indicate that burst release of a commonly used herbicide (i.e., saflufenacil) significantly decreases from 25% to 0.8% via silk fibroin microencapsulation. As a proof‐of‐concept for agrochemicals applications, a 6‐day greenhouse trial demonstrates that saflufenacil delivered on corn plants via silk microcapsules reduces crop injury when compared to the non‐encapsulated version.
A silk fibroin‐based technique is developed to fabricate biodegradable microcapsules. The payload release process and lifetime of microcapsules can be tuned by controlling their structures, morphologies, and compositions. An effective release control of herbicide on crops is demonstrated in a greenhouse.
The metabolism of microorganisms is regulated through two main mechanisms: changes of enzyme capacities as a consequence of gene expression modulation ("hierarchical control") and changes of enzyme ...activities through metabolite-enzyme interactions. An increasing body of evidence indicates that hierarchical control is insufficient to explain metabolic behaviors, but the system-wide impact of metabolic regulation remains largely uncharacterized. To clarify its role, we developed and validated a detailed kinetic model of Escherichia coli central metabolism that links growth to environment. Metabolic control analyses confirm that the control is widely distributed across the network and highlight strong interconnections between all the pathways. Exploration of the model solution space reveals that several robust properties emerge from metabolic regulation, from the molecular level (e.g. homeostasis of total metabolite pool) to the overall cellular physiology (e.g. coordination of carbon uptake, catabolism, energy and redox production, and growth), while allowing a large degree of flexibility at most individual metabolic steps. These properties have important physiological implications for E. coli and significantly expand the self-regulating capacities of its metabolism.
Abstract
Summary
Mass spectrometry (MS) is widely used for isotopic studies of metabolism and other (bio)chemical processes. Quantitative applications in systems and synthetic biology require to ...correct the raw MS data for the contribution of naturally occurring isotopes. Several tools are available to correct low-resolution MS data, and recent developments made substantial improvements by introducing resolution-dependent correction methods, hence opening the way to the correction of high-resolution MS (HRMS) data. Nevertheless, current HRMS correction methods partly fail to determine which isotopic species are resolved from the tracer isotopologues and should thus be corrected. We present an updated version of our isotope correction software (IsoCor) with a novel correction algorithm which ensures to accurately exploit any chemical species with any isotopic tracer, at any MS resolution. IsoCor v2 also includes a novel graphical user interface for intuitive use by end-users and a command-line interface to streamline integration into existing pipelines.
Availability and implementation
IsoCor v2 is implemented in Python 3 and was tested on Windows, Unix and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/MetaSys-LISBP/IsoCor/ and https://isocor.readthedocs.io/.
Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible ...process. However, dynamic
C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli.
Mass spectrometry (MS) is widely used for isotopic labeling studies of metabolism and other biological processes. Quantitative applications-e.g. metabolic flux analysis-require tools to correct the ...raw MS data for the contribution of all naturally abundant isotopes. IsoCor is a software that allows such correction to be applied to any chemical species. Hence it can be used to exploit any isotopic tracer, from well-known ((13)C, (15)N, (18)O, etc) to unusual ((57)Fe, (77)Se, etc) isotopes. It also provides new features-e.g. correction for the isotopic purity of the tracer-to improve the accuracy of quantitative isotopic studies, and implements an efficient algorithm to process large datasets. Its user-friendly interface makes isotope labeling experiments more accessible to a wider biological community.
IsoCor is distributed under OpenSource license at http://metasys.insa-toulouse.fr/software/isocor/
Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia pastoris (Komagataella phaffii), demonstrating the potential of this cell factory ...to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g., significantly lower product yield and/or growth rate. To gain an understanding on the metabolic constraints underlying these observations, the fluxome (metabolic flux phenotype) of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput .sup.13C-metabolic flux analysis platform. Such platform enabled the operation of an optimised workflow to obtain comprehensive maps of the carbon flux distribution in the central carbon metabolism in a parallel-automated manner, thereby accelerating the time-consuming strain characterization step in the design-build-test-learn cycle for metabolic engineering of P. pastoris. We generated detailed maps of the carbon fluxes in the central carbon metabolism of the 3-HP producing strain series, revealing the metabolic consequences of different metabolic engineering strategies aimed at improving NADPH regeneration, enhancing conversion of pyruvate into cytosolic acetyl-CoA, or eliminating by-product (arabitol) formation. Results indicate that the expression of the POS5 NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions, whereas an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA synthesis pathway was overexpressed. Results also show that the tight control of the glycolytic flux hampers cell growth due to limited acetyl-CoA biosynthesis. When the cytosolic acetyl-CoA synthesis pathway was overexpressed, the cell growth increased, but the product yield decreased due to higher growth-associated ATP costs. Finally, the six most relevant strains were also cultured at pH 3.5 to assess the effect of a lower pH on their fluxome. Notably, similar metabolic fluxes were observed at pH 3.5 compared to the reference condition at pH 5. This study shows that existing fluoxomics workflows for high-throughput analyses of metabolic phenotypes can be adapted to investigate P. pastoris, providing valuable information on the impact of genetic manipulations on the metabolic phenotype of this yeast. Specifically, our results highlight the metabolic robustness of P. pastoris's central carbon metabolism when genetic modifications are made to increase the availability of NADPH and cytosolic acetyl-CoA. Such knowledge can guide further metabolic engineering of these strains. Moreover, insights into the metabolic adaptation of P. pastoris to an acidic pH have also been obtained, showing the capability of the fluoxomics workflow to assess the metabolic impact of environmental changes.
Overflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glucose even when oxygen is abundant. Two theories have been proposed to explain acetate ...overflow in
- global control of the central metabolism and local control of the acetate pathway - but neither accounts for all observations. Here, we develop a kinetic model of
metabolism that quantitatively accounts for observed behaviours and successfully predicts the response of
to new perturbations. We reconcile these theories and clarify the origin, control, and regulation of the acetate flux. We also find that, in turns, acetate regulates glucose metabolism by coordinating the expression of glycolytic and TCA genes. Acetate should not be considered a wasteful end-product since it is also a co-substrate and a global regulator of glucose metabolism in
. This has broad implications for our understanding of overflow metabolism.
13C-metabolic flux analysis (13C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current 13C-MFA approaches model label ...propagation starting from the extracellular 13C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current 13C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of Saccharomyces cerevisiae mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.
: In Acute Myeloid Leukemia (AML), a complete response to chemotherapy is usually obtained after conventional chemotherapy but overall patient survival is poor due to highly frequent relapses. As ...opposed to chronic myeloid leukemia, B lymphoma or multiple myeloma, AML is one of the rare malignant hemopathies the therapy of which has not significantly improved during the past 30 years despite intense research efforts. One promising approach is to determine metabolic dependencies in AML cells. Moreover, two key metabolic enzymes, isocitrate dehydrogenases (IDH1/2), are mutated in more than 15% of AML patient, reinforcing the interest in studying metabolic reprogramming, in particular in this subgroup of patients.
: Using a multi-omics approach combining proteomics, lipidomics, and isotopic profiling of U-
C glucose and U-
C glutamine cultures with more classical biochemical analyses, we studied the impact of the IDH1 R132H mutation in AML cells on lipid biosynthesis.
: Global proteomic and lipidomic approaches showed a dysregulation of lipid metabolism, especially an increase of phosphatidylinositol, sphingolipids (especially few species of ceramide, sphingosine, and sphinganine), free cholesterol and monounsaturated fatty acids in IDH1 mutant cells. Isotopic profiling of fatty acids revealed that higher lipid anabolism in IDH1 mutant cells corroborated with an increase in lipogenesis fluxes.
: This integrative approach was efficient to gain insight into metabolism and dynamics of lipid species in leukemic cells. Therefore, we have determined that lipid anabolism is strongly reprogrammed in IDH1 mutant AML cells with a crucial dysregulation of fatty acid metabolism and fluxes, both being mediated by 2-HG (2-Hydroxyglutarate) production.
The coupling of the reversible addition fragmentation chain transfer (RAFT) polymerization technique with the copper-catalyzed Huisgen 1,3-dipolar cycloaddition (“click chemistry”) as a simple and ...effective way to generate polystyrene (PS) macrocycles is presented. The novel strategy entails the synthesis of linear PS backbones followed by endgroup modification to facilitate click chemistry for the formation of ring shaped polymers. An azido group modified 4-cyanopentanoic acid dithiobenzoate is employed as the chain transfer agent in the RAFT mediated polymerization of styrene to form PS with Mn from 2000gmol−1 to 6000gmol−1 and PDI<1.2. To facilitate the cyclization of the polystyrene chains by click coupling, the thiocarbonylthio endgroup is removed and concomitantly replaced by an alkyne bearing function. This is carried out via the radical decomposition of excess azobis(4-cyano valeric acid) (ACVA) modified with an alkyne endgroup in the presence of the thiocarbonylthio-capped PS. The successful click endgroup modifications of several polystyrenes along with the results from the cyclization of a PS with Mn=4300gmol−1 are discussed in detail. This improved method avoids the presence of thiocarbonylthio functions in the macrocycle, thus considerably increasing the chemical stability of these polymers.
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