Mechanistic Insights into Cellular and Molecular Targets of Zinc Oxide Quantum Dots (ZnO QDs) in Fungal Pathogen, Candida albicans: One Drug Multi-Targeted Therapeutic Approach.

Rationally designed multitargeted drugs, known as network therapeutics/multimodal drugs, have emerged as versatile therapeutic solutions to combat drug-resistant microbes. Here, we report novel mechanistic insights into cellular and molecular targets of ZnO quantum dots (QDs) against Candida albicans, a representative of fungal pathogens. Stable, monodispersed 4-6 nm ZnO QDs were synthesized using a wet chemical route, which exhibited dose-dependent inhibition on the growth dynamics of Candida. Treatment with 200 µg/mL ZnO QDs revealed an aberrant morphology and a disrupted cellular ultrastructure in electron microscopy and led to a 23% reduction in ergosterol content and a 53% increase in intracellular reactive oxygen species. Significant increase in steady-state fluorescence polarization and fluorescence lifetime decay of membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in treated cells, respectively, implied reduction in membrane fluidity and enhanced microviscosity. The observed reduction in passive diffusion of fluorescent Rhodamine 6G across the membrane validated the intricate relationship between ergosterol, membrane fluidity, and microviscosity. An inverse relationship existing between ergosterol biosynthetic genes, ERG11 and ERG3 in treated cells, related well with displayed higher susceptibilities. Furthermore, treated cells exhibited impaired functionality and downregulation of ABC drug efflux pumps. Multiple cellular targets of ZnO QDs in Candida were validated by in silico molecular docking. Thus, targeting ERG11, ERG3, and ABC drug efflux pumps might emerge as a versatile, nano-ZnO-based strategy in fungal therapeutics to address the challenges of drug resistance.

Noninvasive Blood Pressure Monitoring via a Flexible and Wearable Piezoresistive Sensor.

In the present global context, continuous blood pressure (BP) monitoring is paramount in addressing the global mortality rates attributed to hypertension. Achieving precise insights into the human cardiovascular system necessitates accurate measurement of BP, and the accuracy depends on the faithful recording of oscillations or pulsations. This task ultimately depends on the caliber of the pressure sensor embedded in the BP device. In this context, we have fabricated a flexible resistive pressure sensor based on reduced graphene oxide (rGO) and a polydimethylsiloxane (PDMS) sponge that is highly flexible and sensitive. The designed device operates effectively with a minimal bias voltage of 500 mV, at which point it showed its maximum relative change in current, reaching approximately 25%. Additionally, the sensing device showed a notable change in resistance values, exhibiting almost 100% change in resistance when subjected to a pressure of 400 mmHg and high sensitivity of 0.27 mmHg-1. After promising outcomes were obtained during static pressure measurement, the sensor was used for BP monitoring in humans. The sensor accurately traced the oscillometric waveform (OMW) for distinct systolic blood pressure (SBP) and diastolic blood pressure (DBP) combinations to cover a range of medical situations, including hypotension, standard or normal, and hypertension. The values of SBP, DBP, and MAP were derived from the sensor's output using the MAA technique, and the errors in these values concerning the simulator and the traditional BP monitor follow the universal AAMI/ESH/ISO protocols. Bland-Altman (B&A) correlation and scatter plots were used to compare the sensor's results and further validate the proposed sensor. The sensor showed the mean and standard deviation error in the SBP, DBP, and MBP of -0.2 ± 5.9, -0.5 ± 7, and -0.9 ± 4.7 mmHg when compared with the noninvasive blood pressure (NIBP) simulator. The pulse rate (PR) was also calculated from the same OMW for the specified value of 80 beats per minute (bpm) given by the simulator and reported a mean PR value of ∼81 bpm, close to the reference value. The findings show that the flexible resistive sensing device can accurately measure BP and replace the existing sensors of BP devices.

Clinically Deployable Bioelectronic Sensing Platform for Ultrasensitive Detection of Transferrin in Serum Sample.

Varying levels of transferrin (Tf) have been associated with different disease conditions and are known to play a crucial role in various malignancies. Regular monitoring of the variations in Tf levels can be useful for managing related diseases, especially for the prognosis of certain cancers. We fabricated an immunosensor based on graphene oxide (GO) nanosheets to indirectly detect Tf levels in cancer patients. The GO nanosheets were deposited onto an indium tin oxide (ITO)-coated glass substrate and annealed at 120 °C to obtain reduced GO (rGO) films, followed by the immobilization of an antibody, anti-Tf. The materials and sensor probe used were systematically characterized by UV-Visible spectroscopy (UV-Vis), X-ray diffraction (XRD), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR). Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) were also used for the stepwise sensor probe characterizations and Tf detection in serum samples, respectively. The anti-Tf/rGO/ITO immunosensor DPV output demonstrated an excellent Tf detection capability in the linear range of 0.1 mg mL-1 to 12 mg mL-1 compared to the enzyme-linked immunosorbent assay (ELISA) detection range, with a limit of detection (LOD) of 0.010 ± 0.007 mg mL-1. Furthermore, the results of the fabricated immunosensor were compared with those of the ELISA and autobioanalyzer techniques, showing an outstanding match with < 5% error and demonstrating the immunosensor's clinical potential.

M1 to M2 induction in macrophages using a retinoic acid-releasing mesenchymal stem cell scaffold.

BACKGROUND: Modulation of macrophage polarization is required for effective tissue repair and regenerative therapies. Therapeutic modulation of macrophages from an inflammatory M1 to a fibrotic M2 phenotype could help in diseases, such as chronic wounds, which are stalled in a prolonged and heightened inflammatory stage within the wound healing process. OBJECTIVE: This study evaluates the efficiency of a pullulan/gelatin nanofiber scaffold loaded with retinoic acid (RA) and adipose-derived mesenchymal stem cells (ASCs) to modulate M1 to M2 anti-inflammatory transition. METHODS: Scaffolds were fabricated by electrospinning, and crosslinked using ethylene glycol diglycidyl ether (EGDE). Exposure of RA and/or ASCs to cultured macrophages have been shown to promote M1 to M2 transition. Pullulan was chosen as a scaffold material due to its ability to quench reactive oxygen species, key signaling molecules that play an important role in the progression of inflammation, as well as for its excellent mechanical properties. Gelatin was chosen as an additional scaffold component due to the presence of cell-binding motifs and its biocompatibility. Scaffold compositions examined were 75:25 and 50:50, pullulan:gelatin. The scaffolds were crosslinked in 1:70 and 1:50 EGDE:EtOH. The scaffold composition was determined via FTIR. For the present study, the 75:25 pullulan:gelatin crosslinked with 1:70 EGDE:EtOH, forming nanofibers 328 ± 47.9 nm (mean ± SD) in diameter, was chosen as the scaffold composition due to its lower degradation and release rate, which allows a sustained delivery of RA. RESULTS: The scaffold composition degraded to approximately 80% after 14 days, with approximately 38% of the drug released after 7 days. THP-1 monocytic cells were induced into a M1 macrophage phenotype through stimulation with lipopolysaccharide (LPS) and gamma interferon (IFN-γ). These M1 macrophages were the exposed to scaffolds loaded with RA and ASCs, to induce differentiation to an M2 phenotype. CONCLUSION: Gene expression quantitation by qPCR showed a reduction of M1 biomarkers, tumor necrosis factor alpha (TNFα) and interleukin 1ß (IL1ß), and an increase of M2 biomarker CCL22 after 2 days of exposure, suggesting successful M1 to M2 transition.

Complex Phase-Fluctuation Effects Correlated with Granularity in Superconducting NbN Nanofilms.

Superconducting nanofilms are tunable systems that can host a 3D-2D dimensional crossover leading to the Berezinskii-Kosterlitz-Thouless (BKT) superconducting transition approaching the 2D regime. Reducing the dimensionality further, from 2D to quasi-1D superconducting nanostructures with disorder, can generate quantum and thermal phase slips (PS) of the order parameter. Both BKT and PS are complex phase-fluctuation phenomena of difficult experiments. We characterized superconducting NbN nanofilms thinner than 15 nm, on different substrates, by temperature-dependent resistivity and current-voltage (I-V) characteristics. Our measurements evidence clear features related to the emergence of BKT transition and PS events. The contemporary observation in the same system of BKT transition and PS events, and their tunable evolution in temperature and thickness was explained as due to the nano-conducting paths forming in a granular NbN system. In one of the investigated samples, we were able to trace and characterize the continuous evolution in temperature from quantum to thermal PS. Our analysis established that the detected complex phase phenomena are strongly related to the interplay between the typical size of the nano-conductive paths and the superconducting coherence length.

Nano-Engineered Surface Comprising Metallic Dendrites for Biomolecular Analysis in Clinical Perspective.

Metallic dendrites, a class of three-dimensional nanostructured materials, have drawn a lot of interests in the recent years because of their interesting hierarchical structures and distinctive features. They are a hierarchical self-assembled array of primary, secondary, and terminal branches with a plethora of pointed ends, ridges, and edges. These features provide them with larger active surface areas. Due to their enormous active areas, the catalytic activity and conductivity of these nanostructures are higher as compared to other nanomaterials; therefore, they are increasingly used in the fabrication of sensors. This review begins with the properties and various synthetic approaches of nanodendrites. The primary goal of this review is to summarize various nanodendrites-engineered biosensors for monitoring of small molecules, macromolecules, metal ions, and cells in a wide variety of real matrices. Finally, to enlighten future research, the limitations and future potential of these newly discovered materials are discussed.

Hierarchical self-assembling and helical structure in focal conic domains in meniscus of ferroelectric liquid crystal.

We investigate experimentally the formation of focal conic domains of the ferroelectric phase of a liquid crystal, chiral smectic C (SmC^{*}), in the meniscus geometry. The meniscus geometry is formed in the gap between two glass plates which are placed on a common substrate. This gap is called here a physical cavity. Focal conic domains (FCDs) in the physical cavity with dimensions of micrometer scale are investigated under an optical polarizing microscope which enables us to extract the information on the helical structure formation in the constraint and gradient topological meniscus interface. The helical pitch in the FCD is observed to be shorter than in planar confined geometry. A crucial phenomenon of unwrapping and wrapping of helical structure from one FCD to another is also observed. In-plane application of an electric field on a FCD revealed the asymmetric helical unwinding process whereas an increase in temperature has shown symmetrical unwinding. The helical structure based observation is significant for understanding the ferroelectric phase in focal conic domains and their application in microlenses and optical components.

Graphene oxide based electrochemical immunosensor for rapid detection of groundnut bud necrosis orthotospovirus in agricultural crops.

Groundnut bud necrosis orthotospovirus (GBNV) is one of the causative plant viruses responsible for the outbreak of many viral epidemics in food crops across India and other south-Asian countries. Its management is a major challenge due to fast vector transmission, and the non-availability of appropriate agrochemical treatment. The timely detection of GBNV becomes indispensable for the effective management of viral infection and the periodic monitoring of plant health. We report the fabrication of graphene oxide (GO) based electrochemical immunosensor for the rapid and sensitive detection of GBNV. The immunoelectrode is prepared by depositing GO onto indium-tin oxide (ITO) coated glass substrates and functionalized by anti-GBNV antibodies using N-ethyl-N'-(3- dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) conjugation chemistry. The response measurements of the immunoelectrodes revealed a sensitivity of 221 ± 1 µA µg-1 mL-1(n = 3) and limit of detection (LOD) of 5.7 ± 0.7 ng mL-1(n = 3) for the standard concentrations of GBNV antigen. Further, the GBNV detection was carried out in infected leaf extracts of three different host plants i.e., Tomato, Cowpea, and N. benthamiana, and the results have been compared with the conventionally used direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) technique. The comparable results obtained for the detection of GBNV in infected plants using electrochemical immunosensing and DAC-ELISA techniques advocated the immense potential of GO based immunosensor as a point-of-care sensing device that is poised to overcome the limitations of the traditional methods of virus detection in field conditions and may transform the diagnostics in agriculture.

Sesamum indicum Oleosin L improves oil packaging in Nicotiana benthamiana leaves.

Plant oil production has been increasing continuously in the past decade. There has been significant investment in the production of high biomass plants with elevated oil content. We recently showed that the expression of Arabidopsis thaliana WRI1 and DGAT1 genes increase oil content by up to 15% in leaf dry weight tissue. However, triacylglycerols in leaf tissue are subject to degradation during senescence. In order to better package the oil, we expressed a series of lipid droplet proteins isolated from bacterial and plant sources in Nicotiana benthamiana leaf tissue. We observed further increases in leaf oil content of up to 2.3-fold when we co-expressed Sesamum indicum Oleosin L with AtWRI1 and AtDGAT1. Biochemical assays and lipid droplet visualization with confocal microscopy confirmed the increase in oil content and revealed a significant change in the size and abundance of lipid droplets.

Targeted Delivery for Neurodegenerative Disorders Using Gene Therapy Vectors: Gene Next Therapeutic Goals.

The technique of gene therapy, ever since its advent nearly fifty years ago, has been utilized by scientists as a potential treatment option for various disorders. This review discusses some of the major neurodegenerative diseases (NDDs) like Alzheimer's disease (AD), Parkinson's Disease (PD), Motor neuron diseases (MND), Spinal Muscular Atrophy (SMA), Huntington's Disease (HD), Multiple Sclerosis (MS), etc. and their underlying genetic mechanisms along with the role that gene therapy can play in combating them. The pathogenesis and the molecular mechanisms specifying the altered gene expression of each of these NDDs have also been discussed in elaboration. The use of gene therapy vectors can prove to be an effective tool in the field of curative modern medicine for the generations to come. Therefore, consistent efforts and progressive research towards its implementation can provide us with powerful treatment options for disease conditions that have so far been considered as incurable.

Correlation between alignment geometries and memory effect in a surface-stabilized ferroelectric liquid crystal.

Memory effect in weakly aligned surface stabilized ferroelectric liquid crystal (SSFLC) material has been investigated by electro-optical and dielectric spectroscopy in three configurations of alignment: antiparallel, 90^{∘} twisted, and unaligned planar samples. It has been observed that two types of molecular dynamics exist in antiparallel rubbed cell in which memory effect is observed for longer duration than in other samples. One dielectric relaxation process is near the surface of the electrode and a second is in the bulk of the SSFLC. Both the molecular dynamics contribute in the switching process and affect the memory phenomenon in surface stabilized geometries. However, a single dielectric process is observed in twisted geometry in which the sample is showing shorter memory effect than in antiparallel and this is compared with unaligned samples also having cell thickness less than the pitch value of FLC. In an unaligned sample, a single dielectric process is observed and the smaple does not show memory effect at all. The investigation is significant to understand the anomalies occurring in memory observations in various geometries.

ZnO-rGO nanocomposite based bioelectrode for sensitive and ultrafast detection of dopamine in human serum.

We present a tyrosinase-conjugated zinc oxide-reduced graphene oxide (Tyr/ZnO-rGO) nanocomposite system as a biosensing test-bed for rapid and sensitive detection of dopamine (DA). The bioelectrodes (Tyr/ZnO-rGO/ITO) were designed by covalently immobilizing tyrosinase enzyme on spin-coated films of ZnO-rGO nanocomposite prepared via self-assembly approach. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed fast electron transfer kinetics of ZnO-rGO/ITO electrode. The response studies of the Tyr/ZnO-rGO/ITO bioelectrode revealed ultrafast (0.34 ± 0.09 s) detection of DA in a wide linear dynamic range of 0.1-1500 pM. The significant performance of the biosensor in terms of low detection limit (8.75 ± 0.64 pM) and high sensitivity (39.56 ± 0.41 µA nM-1) values is attributed to the fast and unhindered electron transfer mechanism of ZnO-rGO matrix having low electrochemical band gap. The nanoplatform exhibited high selectivity toward DA in human sera, and remained stable up to 3 months at 4 °C, representing its suitability for clinical applications.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Can mesenchymal stem cell therapy be the interim management of COVID-19?

COVID-19 pandemic has accounted for ~ 4.3 million confirmed cases and ~ 292,000 deaths (till 12th May, 2020) across the globe since its outbreak. Several anti-viral drugs such as RNA dependent RNA polymerase inhibitors (remdesivir, favipiravir, ribavirin), protease inhibitors (lopinavir, ritonavir) and drugs targeting endocytic pathway (hydroxychloroquine) are being evaluated for COVID-19 but standard therapeutics yet not available. Severe health deterioration in critically ill patients is characterized by pulmonary edema, severe respiratory distress, cytokine storm and septic shock. To combat cytokine storm, immune-therapy targeting IL-1, IL-2, IL-6 and TNFα are being evaluated and one of the promising immune-modulator is the mesenchymal stem cells (MSCs) that can surmount the severity of COVID-19 infections. Recent studies have shown that MSC-therapy significantly dampens the cytokine storm in critically ill COVID-19 patients. This communication endows with the insight of stem cell therapy and summarizes the recent studies on COVID-19 patients.

Advent of Proteomic Tools for Diagnostic Biomarker Analysis in Alzheimer's Disease.

Locating remedies for Alzheimer's disease (AD) has been majorly restricted by the inefficiency to establish a definitive detection model for early-stage diagnosis of pathological events. This current lapse in AD diagnosis also limits the therapeutic efficiency of the drugs, which might have been effective if given at the earlier stages of the disease. The indicated situation directs towards the burgeoned need for an effective biomarker technique that will help in early detection of AD and would be imminently useful to facilitate improved diagnosis and stimulate therapeutic trials. Till date, the major biomarkers, specifically associated with AD detection, may help in determining the early-stage AD diagnosis and identifying alterations in the cellular proteome, offering deeper insight into disease etiology. Currently existing multidisciplinary clinical diagnosis of AD is a very tedious, expensive procedure and requires highly trained and skilled professionals who are rarely available outside the specialty clinics. Mutations in amyloid precursor protein (APP) or Presenilin 1 and 2 (PSEN1 and PSEN2) are some biomarkers acting as critical checkpoints for AD diagnosis. However, the presence of some associated biomarkers in cerebrospinal fluid (CSF) such as total-Tau (tTau), phosphorylated- Tau (pTau) 181 and Amyloid-ß (Aß) 1-42 using structural or functional imaging techniques is considered for confirmatory diagnosis of AD. Furthermore, the molecular diagnosis of AD incorporates various sophisticated techniques including immuno-sensing, machine learning, nano conjugation-based detections, etc. In the current review description, we have summarized the various diagnostic approaches and their relevance in mitigating the long-standing urgency of targeted diagnostic tools for detection of AD.

Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

A Synergistic Genetic Engineering Strategy Induced Triacylglycerol Accumulation in Potato (Solanum tuberosum) Leaf.

Potato is the 4th largest staple food in the world currently. As a high biomass crop, potato harbors excellent potential to produce energy-rich compounds such as triacylglycerol as a valuable co-product. We have previously reported that transgenic potato tubers overexpressing WRINKLED1, DIACYLGLYCEROL ACYLTRANSFERASE 1, and OLEOSIN genes produced considerable levels of triacylglycerol. In this study, the same genetic engineering strategy was employed on potato leaves. The overexpression of Arabidopsis thaliana WRINKED1 under the transcriptional control of a senescence-inducible promoter together with Arabidopsis thaliana DIACYLGLYCEROL ACYLTRANSFERASE 1 and Sesamum indicum OLEOSIN driven by the Cauliflower Mosaic Virus 35S promoter and small subunit of Rubisco promoter respectively, resulted in an approximately 30- fold enhancement of triacylglycerols in the senescent transgenic potato leaves compared to the wild type. The increase of triacylglycerol in the transgenic potato leaves was accompanied by perturbations of carbohydrate accumulation, apparent in a reduction in starch content and increased total soluble sugars, as well as changes of polar membrane lipids at different developmental stages. Microscopic and biochemical analysis further indicated that triacylglycerols and lipid droplets could not be produced in chloroplasts, despite the increase and enlargement of plastoglobuli at the senescent stage. Possibly enhanced accumulation of fatty acid phytyl esters in the plastoglobuli were reflected in transgenic potato leaves relative to wild type. It is likely that the plastoglobuli may have hijacked some of the carbon as the result of WRINKED1 expression, which could be a potential factor restricting the effective accumulation of triacylglycerols in potato leaves. Increased lipid production was also observed in potato tubers, which may have affected the tuberization to a certain extent. The expression of transgenes in potato leaf not only altered the carbon partitioning in the photosynthetic source tissue, but also the underground sink organs which highly relies on the leaves in development and energy deposition.

Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family.

Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

Producing Cyclopropane Fatty Acid in Plant Leafy Biomass via Expression of Bacterial and Plant Cyclopropane Fatty Acid Synthases.

Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.