Engineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

Gelation Kinetics and Mechanical Properties of Thiol-Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation.

Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.

Comparative Transcriptomics of Lowland Rice Varieties Uncovers Novel Candidate Genes for Adaptive Iron Excess Tolerance.

Iron (Fe) toxicity is a major challenge for plant cultivation in acidic waterlogged soil environments, where lowland rice is a major staple food crop. Only few studies have addressed the molecular characterization of excess Fe tolerance in rice, and these highlight different mechanisms for Fe tolerance. Out of 16 lowland rice varieties, we identified a pair of contrasting lines, Fe-tolerant Lachit and -susceptible Hacha. The two lines differed in their physiological and morphological responses to excess Fe, including leaf growth, leaf rolling, reactive oxygen species generation and Fe and metal contents. These responses were likely due to genetic origin as they were mirrored by differential gene expression patterns, obtained through RNA sequencing, and corresponding gene ontology term enrichment in tolerant vs. susceptible lines. Thirty-five genes of the metal homeostasis category, mainly root expressed, showed differential transcriptomic profiles suggestive of an induced tolerance mechanism. Twenty-two out of these 35 metal homeostasis genes were present in selection sweep genomic regions, in breeding signatures, and/or differentiated during rice domestication. These findings suggest that Fe excess tolerance is an important trait in the domestication of lowland rice, and the identified genes may further serve to design the targeted Fe tolerance breeding of rice crops.

Calcium-Promoted Interaction between the C2-Domain Protein EHB1 and Metal Transporter IRT1 Inhibits Arabidopsis Iron Acquisition.

Iron is a key transition element in the biosphere and is crucial for living organisms, although its cellular excess can be deleterious. Maintaining the balance of optimal iron availability in the model plant Arabidopsis (Arabidopsis thaliana) requires the precise operation of iron import through the principal iron transporter IRON-REGULATED TRANSPORTER1 (IRT1). Targeted inhibition of IRT1 can prevent oxidative stress, thus promoting plant survival. Here, we report the identification of an IRT1 inhibitor, namely the C2 domain-containing peripheral membrane protein ENHANCED BENDING1 (EHB1). EHB1 interacts with the cytoplasmically exposed variable region of IRT1, and we demonstrate that this interaction is greatly promoted by the presence of calcium. We found that EHB1 binds lipids characteristic of the plasma membrane, and the interaction between EHB1 and plant membranes is calcium-dependent. Molecular simulations showed that EHB1 membrane binding is a two-step process that precedes the interaction between EHB1 and IRT1. Genetic and physiological analyses indicated that EHB1 acts as a negative regulator of iron acquisition. The presence of EHB1 prevented the IRT1-mediated complementation of iron-deficient fet3fet4 yeast (Saccharomyces cerevisiae). Our data suggest that EHB1 acts as a direct inhibitor of IRT1-mediated iron import into the cell. These findings represent a major step in understanding plant iron acquisition, a process that underlies the primary production of bioavailable iron for land ecosystems.

Natural Variation in Physiological Responses of Tunisian Hedysarum carnosum Under Iron Deficiency.

Iron (Fe) is an essential element for plant growth and development. The cultivation of leguminous plants has generated strong interest because of their growth even on poor soils. Calcareous and saline soils with poor mineral availability are wide-spread in Tunisia. In an attempt to select better forage crops adapted to Tunisian soils, we characterized Fe deficiency responses of three different isolates of Hedysarum carnosum, an endemic Tunisian extremophile species growing in native stands in salt and calcareous soil conditions. H. carnosum is a non-model crop. The three isolates, named according to their habitats Karkar, Thelja, and Douiret, differed in the expression of Fe deficiency symptoms like morphology, leaf chlorosis with compromised leaf chlorophyll content and photosynthetic capacity and leaf metal contents. Across these parameters Thelja was found to be tolerant, while Karkar and Douiret were susceptible to Fe deficiency stress. The three physiological and molecular indicators of the iron deficiency response in roots, Fe reductase activity, growth medium acidification and induction of the IRON-REGULATED TRANSPORTER1 homolog, indicated that all lines responded to -Fe, however, varied in the strength of the different responses. We conclude that the individual lines have distinct adaptation capacities to react to iron deficiency, presumably involving mechanisms of whole-plant iron homeostasis and internal metal distribution. The Fe deficiency tolerance of Thelja might be linked with adaptation to its natural habitat on calcareous soil.

Dissection of iron signaling and iron accumulation by overexpression of subgroup Ib bHLH039 protein.

Iron is an essential growth determinant for plants, and plants acquire this micronutrient in amounts they need in their environment. Plants can increase iron uptake in response to a regulatory transcription factor cascade. Arabidopsis thaliana serves as model plant to identify and characterize iron regulation genes. Here, we show that overexpression of subgroup Ib bHLH transcription factor bHLH039 (39Ox) caused constitutive iron acquisition responses, which resulted in enhanced iron contents in leaves and seeds. Transcriptome analysis demonstrated that 39Ox plants displayed simultaneously gene expression patterns characteristic of iron deficiency and iron stress signaling. Thereby, we could dissect iron deficiency response regulation. The transcription factor FIT, which is required to regulate iron uptake, was essential for the 39Ox phenotype. We provide evidence that subgroup Ib transcription factors are involved in FIT transcriptional regulation. Our findings pose interesting questions to the feedback control of iron homeostasis.

Aspherical, Nanostructured Microparticles for Targeted Gene Delivery to Alveolar Macrophages.

Introducing novel shapes to particulate carrier systems adds unique features to modern drug and gene delivery. Depending on the route of administration, particle geometry can influence deposition and fate within biological environments. In this work, a template-assisted engineering technique is applied, providing full control of size and shape in the preparation of aspherical, nanostructured microparticles. Based on the interconnection of nanoparticles, stabilized by a functional layer-by-layer (LbL) coating, the resulting cylindrical micrometer architecture is especially qualified for pulmonary delivery. Designed as gene delivery system, plasmid-DNA (pCMV-luciferase) and branched polyethylenimine are used to reach both structural integrity of the carrier system and delivery of genes into the cells of interest. Due to their size, particles are exclusively taken up by phagocytes, which also adds a targeting effect to the introduced system. The luciferase expression is demonstrated in macrophages showing increasing levels over a time period of at least 7 d. Furthermore, it is shown for the first time that the expression is depending on the LbL design. From in vivo experiments, corresponding luciferase expression is observed in mice alveolar macrophages. Combining site specific transport with the possibility of genetically engineering immunocompetent phagocytes, the presented system offers promising potential to improve applications for cell-based immunotherapy.

Interactions between DPPC as a component of lung surfactant and amorphous silica nanoparticles investigated by HILIC-ESI-MS.

This paper reports a rapid HILIC-ESI-MS assay to quantify dipalmitoylphosphatidylcholine (DPPC) as component of lung surfactant for nanosafety studies. The technique was used to investigate the concentration-dependent sorption of DPPC to two-sizes of amorphous SiO2 nanoparticles (SiO2-NPs) in a MeOH:H2O (50/50v/v) mixture and in cell culture medium. In MeOH:H2O (50/50v/v), the sorption of DPPC was positively correlated with the nanoparticles concentration. A substantial affinity of small amorphous SiO2-NPs (25nm) to DPPC standard solution compared to bigger SiO2-NPs (75nm) was not confirmed for biological specimens. After dispersion of SiO2-NPs in DPPC containing cell culture medium, the capacity of the SiO2-NPs to bind DPPC was reduced in comparison to a mixture of MeOH:H2O (50/50v/v) regardless from the nanoparticles size. Furthermore, HILIC-ESI-MS revealed that A549 cells internalized DPPC during growth in serum containing medium complemented with DPPC. This finding was in a good agreement with the potential of alveolar type II cells to recycle surfactant components. Binding of lipids present in the cell culture medium to amorphous SiO2-NPs was supported by means of HILIC-ESI-MS, TEM and ICP-MS independently.

p-Coumaric acid, a novel and effective biomarker for quantifying hypoxic stress by HILIC-ESI-MS.

In this study, we report p-coumaric acid as novel and effective response marker for indirectly measuring the levels of hypoxia in normal primary bronchial epithelial cells. We developed a simple and rapid technique based on hydrophilic interaction chromatography-electrospray ionization-mass spectrometry (HILIC-ESI-MS). During 168h of hypoxia without induction of reactive oxygen species (ROS), an almost linear increase of p-coumaric acid levels was observed. We interpret the increasing p-coumaric acid concentrations during hypoxia as a result of cell damage, triggered by reduced co-enzyme Q10 levels, because the oxidative cascade was not able to supply sufficient energy. The HILIC-ESI-MS assay within p-coumaric acid exhibited a linear dynamic range from 60 to 610 ng/µL with correlation coefficient of 0.9998. The precision of the assay was ≤15% RSD and method accuracies between 97 and 108%.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.

Iron and FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent regulation of proteins and genes in Arabidopsis thaliana roots.

Iron is an essential micronutrient for plants, and iron deficiency requires a variety of physiological adaptations. FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is essential for the regulation of iron uptake in Arabidopsis thaliana roots. FIT is transcriptionally as well as posttranscriptionally regulated in response to iron supply. To investigate to which extent posttranscriptional regulation upon iron deficiency applies to proteins and to determine the dependency on FIT, we performed a parallel proteomic and transcriptomic study with wild-type, a fit knock-out mutant, and a FIT overexpressing Arabidopsis line. Among 92 proteins differentially regulated by iron and/or FIT, we identified 30 proteins, which displayed differential regulation at the transcriptional level. Eleven protein spots were regulated in at least one of the data points even contrary to the respective genes dependent on FIT. We found ten proteins in at least two forms. The analysis of functional classification showed enriched GO terms among the posttranscriptionally regulated genes and of proteins, that were downregulated or absent in the fit knock-out mutant. Taken together, we provide evidence for iron and FIT-dependent posttranscriptional regulation in iron homeostasis in A. thaliana.

SORTING NEXIN1 is required for modulating the trafficking and stability of the Arabidopsis IRON-REGULATED TRANSPORTER1.

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.

Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis.

The metal chelator nicotianamine promotes the bioavailability of Fe and reduces cellular Fe toxicity. For breeding Fe-efficient crops, we need to explore the fundamental impact of nicotianamine on plant development and physiology. The quadruple nas4x-2 mutant of Arabidopsis thaliana cannot synthesize any nicotianamine, shows strong leaf chlorosis, and is sterile. To date, these phenotypes have not been fully explained. Here, we show that sink organs of this mutant were Fe deficient, while aged leaves were Fe sufficient. Upper organs were also Zn deficient. We demonstrate that transport of Fe to aged leaves relied on citrate, which partially complemented the loss of nicotianamine. In the absence of nicotianamine, Fe accumulated in the phloem. Our results show that rather than enabling the long-distance movement of Fe in the phloem (as is the case for Zn), nicotianamine facilitates the transport of Fe from the phloem to sink organs. We delimit nicotianamine function in plant reproductive biology and demonstrate that nicotianamine acts in pollen development in anthers and pollen tube passage in the carpels. Since Fe and Zn both enhance pollen germination, a lack of either metal may contribute to the reproductive defect. Our study sheds light on the physiological functions of nicotianamine.

Interaction between the bHLH transcription factor FIT and ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE3-LIKE1 reveals molecular linkage between the regulation of iron acquisition and ethylene signaling in Arabidopsis.

Understanding the regulation of key genes involved in plant iron acquisition is of crucial importance for breeding of micronutrient-enriched crops. The basic helix-loop-helix protein FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), a central regulator of Fe acquisition in roots, is regulated by environmental cues and internal requirements for iron at the transcriptional and posttranscriptional levels. The plant stress hormone ethylene promotes iron acquisition, but the molecular basis for this remained unknown. Here, we demonstrate a direct molecular link between ethylene signaling and FIT. We identified ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1) in a screen for direct FIT interaction partners and validated their physical interaction in planta. We demonstrate that the ein3 eil1 transcriptome was affected to a greater extent upon iron deficiency than normal iron compared with the wild type. Ethylene signaling by way of EIN3/EIL1 was required for full-level FIT accumulation. FIT levels were reduced upon application of aminoethoxyvinylglycine and in the ein3 eil1 background. MG132 could restore FIT levels. We propose that upon ethylene signaling, FIT is less susceptible to proteasomal degradation, presumably due to a physical interaction between FIT and EIN3/EIL1. Increased FIT abundance then leads to the high level of expression of genes required for Fe acquisition. This way, ethylene is one of the signals that triggers Fe deficiency responses at the transcriptional and posttranscriptional levels.

Multilayer coating of gold nanoparticles with drug-polymer coadsorbates.

The aim of our present study was the development of a drug multilayer-based carrier system for delivery of water-insoluble drugs. As drug, we applied the anticancer drug 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, mTHPP, which is a model photosensitizer for photodynamic therapy. Gold nanoparticles (AuNP) with a diameter of 14.5 ± 0.9 nm were prepared and used as template for the layer-by-layer approach. The drug and the negatively charged polyelectrolyte (PE) poly(styrene sulfonate) sodium salt (PSS) were complexed with a new developed method using freeze-drying. The complexation efficiency was determined to be ∼11-12 monomers PSS per mTHPP molecule by CHNS analysis and UV/vis measurement. Molecular docking simulations revealed π-π interactions and H-bonding to be the responsible mechanisms. A drug multilayer system based on the layer-by-layer (LbL) technique utilized the water-soluble complex as anionic layer material and poly(allylamine hydrochloride) (PAH) as cationic layer. The modified AuNP were characterized by different physicochemical techniques such as UV/vis, ζ-potential, ICP-OES, and TEM. To the best of our knowledge, we could demonstrate for the first time the adsorption of three drug layers to a nanoparticulate system. Furthermore, the adaptation of the LbL-technique resulted in drastically increased drug deposition efficiency (factor of 100). Furthermore, we developed a new and comfortable way to solubilize water-insoluble drugs in water.

The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses.

Nicotianamine chelates and transports micronutrient metal ions in plants. It has been speculated that nicotianamine is involved in seed loading with micronutrients. A tomato (Solanum lycopersicum) mutant (chloronerva) and a tobacco (Nicotiana tabacum) transgenic line have been utilized to analyze the effects of nicotianamine loss. These mutants showed early leaf chlorosis and had sterile flowers. Arabidopsis (Arabidopsis thaliana) has four NICOTIANAMINE SYNTHASE (NAS) genes. We constructed two quadruple nas mutants: one had full loss of NAS function, was sterile, and showed a chloronerva-like phenotype (nas4x-2); another mutant, with intermediate phenotype (nas4x-1), developed chlorotic leaves, which became severe upon transition from the vegetative to the reproductive phase and upon iron (Fe) deficiency. Residual nicotianamine levels were sufficient to sustain the life cycle. Therefore, the nas4x-1 mutant enabled us to study late nicotianamine functions. This mutant had no detectable nicotianamine in rosette leaves of the reproductive stage but low nicotianamine levels in vegetative rosette leaves and seeds. Fe accumulated in the rosette leaves, while less Fe was present in flowers and seeds. Leaves, roots, and flowers showed symptoms of Fe deficiency, whereas leaves also showed signs of sufficient Fe supply, as revealed by molecular-physiological analysis. The mutant was not able to fully mobilize Fe to sustain Fe supply of flowers and seeds in the normal way. Thus, nicotianamine is needed for correct supply of seeds with Fe. These results are fundamental for plant manipulation approaches to modify Fe homeostasis regulation through alterations of NAS genes.