Biology subjects

Masakapalli, S. K.

Publications and source records attributed to Masakapalli, S. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Confluent growth state dependent transcriptomic adaptation in A549 lung cancer cells

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

systems biology↗

bioRxiv2026

Alternaria solani infection reprograms potato leaf metabolism and highlights potential defence and metabolic markers

Potato (Solanum tuberosum L.), the worlds fourth most cultivated crop, suffers yield losses of up to 40-50% from early blight caused by the necrotrophic fungal pathogen Alternaria solani. In this study we performed gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics to characterize temporal alterations in metabolite composition, metabolic pathway regulation, and discriminatory biomarker metabolites in the susceptible Indian potato variety Kufri Jyoti, analyzing infected leaves, non-infected leaves, and lesion-associated necrotic tissues across four days post-inoculation (DPI).Metabolite annotation identified 58 compounds, including sugars, organic acids, amino acids, and secondary metabolites.. Multivariate analyses resolved distinct, largely non-overlapping metabolic clusters for control, infected leaves (1-4 DPI), and lesion tissue (Bs1-Bs3). A biphasic metabolic response was observed: early infection (1-2 DPI) was characterized by general suppression of primary metabolism, while late infection (3-4 DPI) showed pronounced upregulation of glycolysis, the TCA cycle, GS/GOGAT, and the shikimate pathway. Key discriminatory metabolites included asparagine, oxoproline, GABA, phenylalanine, and aromatic amino acids. Lesion tissues exhibited distinct metabolic fingerprints, with early disruption of amino acid recycling followed by a late rebound of defense-associated metabolites. Notably, defence-associated phenolics were detected exclusively within lesion tissue and were absent from whole-leaf profiles, demonstrating that spatially resolved lesion sampling captures defence chemistry that whole-leaf analysis alone would miss. The identified biomarker metabolites, particularly those linked to the shikimate and GS/GOGAT pathways, represent promising candidates for metabolite-assisted breeding and targeted crop protection strategies against early blight in potato. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/745268v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@100dd28org.highwire.dtl.DTLVardef@1b78326org.highwire.dtl.DTLVardef@103578corg.highwire.dtl.DTLVardef@68b214_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2026

Apple scar skin viroid disease induced physicochemical and metabolic alterations in Apples

Apple scar skin disease (ASSD), caused by Apple scar skin viroid (ASSVd), is characterized by peel scarring, cracking, dappling, and fruit deformation, resulting in reduced fruit quality and marketability. Despite its economic importance, the physicochemical and metabolic alterations underlying disease progression remain poorly understood. To address this knowledge gap, apple fruits representing four stages of ASSD (healthy, lightly infected, moderately infected, and highly infected) were comprehensively characterized. ASSVd infection was confirmed by RT-PCR, amplicon sequencing, and phylogenetic analysis. Fruit morphology and quality attributes, including firmness, total soluble solids (TSS), pH, titratable acidity (TA), and total phenolic content (TPC), were evaluated. ASSVd infection significantly reduced fruit weight and firmness and altered TSS and TA, indicating progressive deterioration of fruit quality. To investigate the underlying metabolic changes, peel and pulp tissues were analysed separately using gas chromatography-mass spectrometry (GC-MS), while major soluble sugars were quantified by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Integrated metabolomic analyses revealed distinct tissue-specific metabolic reprogramming during disease progression. Major soluble sugars declined significantly during early infection, followed by tissue-dependent recovery at later stages, whereas organic acids, amino acids, phenolics, lipids, polyols, and pentacyclic triterpenoids exhibited dynamic stage-dependent changes. Notably, lupeol accumulated progressively, whereas ursolic acid and oleanolic acid declined, indicating disease-associated alterations in host triterpenoid metabolism. Multivariate analyses demonstrated clear metabolic separation among disease stages. Lupeol, ursolic acid, and chlorogenic acid were identified as candidate discriminatory metabolites in the peel, whereas myo-inositol, chlorogenic acid, and aspartic acid were identified in the pulp. Collectively, these findings demonstrate that ASSD induces coordinated, tissue-specific physicochemical and metabolic reprogramming that compromises postharvest fruit quality and reshapes defence-associated metabolism. This study provides the first integrated metabolomic characterization of ASSD progression and identifies potential metabolic biomarkers for disease diagnosis and severity assessment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/741181v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@50763eorg.highwire.dtl.DTLVardef@82eaddorg.highwire.dtl.DTLVardef@1096b06org.highwire.dtl.DTLVardef@15161b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2026

Decoding central metabolic rewiring induced by exogenous GABA shunt intermediates in Pea

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/741208v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1b38bfborg.highwire.dtl.DTLVardef@ab10b6org.highwire.dtl.DTLVardef@d65370org.highwire.dtl.DTLVardef@ffe08b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Legume-rhizobium symbiosis is constrained by carbon allocation and nutrient exchanges between the host plant and symbiotic bacteroids, emphasizing the necessity of biostimulant-based strategies to improve symbiotic efficiency. Studies suggest that plants provide carbon substrates (mainly TCA cycle intermediates) and nitrogen assimilation precursors to support bacteroid metabolism and nitrogen fixation. The GABA shunt represents a conserved bypass, linking the GS/GOGAT cycle, TCA cycle, and broader nitrogen metabolism. In the present study, the metabolic phenotypes upon rhizospheric application of exogenous {gamma}-aminobutyric acid (GABA) and succinate were investigated in pea. Application of exogenous GABA and succinate primarily altered the morpho-biochemical properties and root nodule establishment of the plant. While GABA supplementation reduced the root length and increased stomatal density, succinate treatment alone inhibited nodule organogenesis with disrupted symbiosome zoning. Metabolic rewirings associated with these phenotypic changes begin at the root nodule compartment, where exogenous GABA altered endogenous tricarboxylic acid (TCA) cycle pools, leading to higher turnover of TCA cycle intermediates, including succinate, fumarate, and malate, as well as elevated levels of sugars (sucrose, glucose, and fructose) and polyols (pinitol, mannitol, and myo-inositol). Concurrently, the plants accumulated substantially higher levels of asparagine (+3.9 log10-fold higher), accompanied by altered shoot protein content and enhanced elemental nitrogen content (+0.8-1%). Collectively, these findings demonstrate the potential of GABA as a biostimulant capable of altering the carbon-nitrogen dynamics and enhancing symbiotic functioning in pea root nodules, whereas succinate alters the process of nodule formation.

plant biology↗

bioRxiv2026

Identification of conserved age-associated aggregation-prone proteins for natural molecule targeting during aging in Caenorhabditis elegans

Aging is associated with proteome remodelling and progressive accumulation of insoluble proteins. Identifying age-enriched proteins that undergo aggregation and evaluating compounds capable of modulating their behaviour may provide insights into interventions that promote healthy aging. Here, we report a proteome-guided strategy to identify age-associated aggregation-prone proteins and evaluate phytochemicals targeting conserved proteins in Caenorhabditis elegans. We identified proteins whose abundance increased more than four-fold in aged worms compared with young worms, many of which also accumulated in the age-associated insoluble proteome, and subsequently identified their human orthologs for comparative analysis. Based on biological relevance, structural conservation, and availability of high-confidence structural models, glutamine-fructose-6-phosphate aminotransferase-2 (GFAT-2) was selected for molecular docking. Screening of fifteen phytochemicals against C. elegans GFAT-2 and its human ortholog GFPT1 identified quercetin as the strongest predicted binder, exhibiting conserved interactions with both proteins. However, treatment of worms with quercetin did not significantly alter global protein insolubility during aging. This may reflect its ability to modulate inappropriate protein-protein interactions without substantially affecting the overall aggregation burden. These findings underscore the need for experimental validation of favourable in silico docking predictions. More broadly, this study provides a proteome-guided framework for prioritizing age-associated aggregation-prone proteins as candidate therapeutic targets for preserving proteostasis during aging.

biochemistry↗

bioRxiv2026

Wavelength induced cultivar specific enrichment of essential amino acids and phenolics in Amaranthus tricolor

Light wavelengths modulate plant growth, metabolism, and physiology. Amaranthus, a C4 underutilized climate resilient crop with promising nutritional properties remained unexplored in terms of metabolite enrichment under monochromatic light wavelengths of visible spectrum. In current study, two cultivars of Amaranthus tricolor (green and red) were exposed to seven light regimes of photosynthetically active radiation (PAR; 400-700 nm): deep blue, blue, green, amber, red, deep red, far red, and their metabolic responses were captured using Gas Chromatography-Mass Spectrometry. The metabolic analysis revealed wavelength-specific reprogramming in the levels of organic acids, sugars, amino acids, fatty acids as well as phenolics. In both the green and red Amaranthus, branched-chain amino acids and phenylalanine, which are nutritionally essential, were significantly elevated under far-red light. While the phenolics such as caffeic acid and ferulic acid were elevated under green and deep blue light respectively in green Amaranthus, amber light wavelengths enhanced these phenolics in red Amaranthus. The study highlighted cultivar-specific metabolic rewiring triggered by specific wavelengths. Altogether, these findings provides insights into metabolic adaptation and demonstrate the ability of light wavelength to specifically enrich the targeted metabolite of nutritional relevance in Amaranthus. It offers strategies to improve the nutritional value of crops in controlled agriculture systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/714947v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@8de67forg.highwire.dtl.DTLVardef@17a03corg.highwire.dtl.DTLVardef@118d18corg.highwire.dtl.DTLVardef@6568f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2026

Drought induced metabolomics of potato leaves highlight metabolic reprogramming and promising biomarkers for smart irrigation advisories

Smart irrigation management is essential for improving crop resilience under increasing drought frequency driven by climate change. Although satellite-based remote sensing provides valuable tools for monitoring crop water status at large spatial scales, its accuracy is often limited in mountainous and heterogeneous agricultural landscapes. In this study, we investigated drought-induced metabolic responses in potato (Solanum tuberosum L.) to identify biochemical biomarkers that could complement satellite-based irrigation advisories in the mid-Himalayan region of India. A field experiment was conducted using a gradient of soil moisture regimes corresponding to moderate (50% field capacity), critical (25% field capacity), and extreme drought stress (5-8% field capacity). Satellite-derived evapotranspiration-based irrigation advisories were validated against in situ soil moisture measurements, revealing discrepancies attributed to the inability of satellite estimates to capture actual water loss under drought stress conditions, highlighting the need for additional ground-truth biomarkers across heterogeneous field conditions. To capture plant-level physiological responses, untargeted metabolite profiling of potato leaves was performed using gas chromatography-mass spectrometry (GC-MS). Approximately fifty metabolites belonging to amino acids, organic acids, sugars, and sugar alcohols were detected. Multivariate statistical analyses revealed distinct metabolic signatures associated with progressive drought stress. Notably, accumulation of proline, serine, isoleucine, sucrose, fructose, glucose, and polyols such as mannitol and myo-inositol reflected key metabolic reprogramming associated with osmoprotection, redox homeostasis, and energy metabolism under drought conditions. Collectively, this ensemble of stress-responsive metabolites represents a robust panel of drought stress biomarkers. As a proof of concept, proline was validated as a qualitative biomarker of plant water status through a rapid and cost-effective colorimetric biochemical assay, demonstrating its practical applicability for field-level irrigation management. These findings demonstrate that metabolomics-derived biomarkers can provide sensitive plant-level indicators of drought stress that complement satellite-based monitoring systems. The integration of biochemical diagnostics with remote sensing platforms offers a promising approach for improving drought detection and developing low-cost, field-deployable tools for smart irrigation advisories in heterogeneous agricultural landscapes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/712810v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@5b4c6dorg.highwire.dtl.DTLVardef@1f6af44org.highwire.dtl.DTLVardef@6cd9f7org.highwire.dtl.DTLVardef@5a0f5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Kinetic 13CO2 mapping revealed distinct light-dark metabolic transition phenotypes in Brassica napus seedlings under visible and UV-B light

Dynamic stable-isotope tracing using 13CO2 has gained significant attention in systems biology due to its potential to visualise carbon assimilation patterns. However, the plant metabolic phenotypes under visible and UV-B light, explaining the light-to-dark transition between photoperiods, remain unexplored. In this study, we investigated the dynamics of photosynthetic carbon assimilation and resource partitioning during the day-night transition under visible and UV-B light in Brassica napus seedlings. Kinetic 13CO2 tracing via the analysis of mass isotopomer distributions of metabolite fragments using GC-MS revealed reprogramming of the source-sink carbon dynamics. While visible light enabled the dynamic redistribution of newly fixed carbon during the light and dark photoperiod, de novo biosynthesis of shikimic acid, TCA cycle intermediates and metabolites of the glutamate-GABA metabolism was strongly favoured in dark metabolism. In contrast, a delayed and reduced photosynthetic carbon assimilation response was observed in the UV-B phenotype during the light period. Moreover, towards the late light period, de novo biosynthesis of sucrose, shikimic acid, phenylalanine, citric, succinic and malic acid was favoured, along with a greater reliance on pre-existing carbon pools for other metabolites. However, ketoglutarate, succinic acid, malic acid and GABA showed limited de novo synthesis in the dark period. Across both light regimes, amino acid pools largely remained in constant sync with the pre-existing pools during the light-dark transition. Overall, our findings demonstrate that light quality and photoperiod-driven metabolic transitions distinctly shape plant metabolic phenotypes.

plant biology↗

bioRxiv2025

Genome-scale metabolic model guided metabolic flux analysis in the endophyte Alternaria burnsii NCIM1409

Camptothecin (CPT), a potent anticancer alkaloid, is traditionally derived from plants like Camptotheca acuminata and Nothapodytes nimmoniana, but sustainable production remains challenging. This study explores the metabolic network of the fungal endophyte Alternaria burnsii NCIM1409, known for camptothecin production, using genome sequencing, genome scale metabolic modeling, and 13C-based pathway mapping. A genome-scale metabolic model (AltGEM iDD1552) was reconstructed, comprising 2188 reactions, 2148 metabolites, and 1552 genes, along with manual curation to include camptothecin biosynthesis pathways. Flux balance analysis identified key enzymatic targets, including secologanin synthase and tryptophan decarboxylase, for enhancing CPT production. Further, metabolic analysis of A. burnsii subjected to 20%[U-13C6] glucose or 99% [1-13C] glucose revealed active glycolysis, the pentose phosphate pathway, and the TCA cycle. This integrative approach provides insights into A. burnsiis metabolic capabilities and highlights strategies for optimizing camptothecin biosynthesis, offering a foundation for sustainable production methods.

systems biology↗

bioRxiv2024

Isotopic tracing in Mycobacterium tuberculosis-infected alveolar epithelial cells indicates metabolic specificity to their virulence and drug-resistance status

Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis (TB), infects different host cells; however, studies on its metabolic impact on the type 2 alveolar epithelial cells are limited. In this study, comparative metabolic insights of A549 cells infected with laboratory (H37RV, H37Ra) and clinical isolates (drug-resistant: S6, S11 and sensitive: S4, S5) were derived from read out of the 13C-based proteinogenic amino acid kinetics. Mtb H37Rv (virulent) exhibited higher growth kinetics within A549 cells compared to H37Ra (avirulent), whereas drug-resistant clinical isolate S6 showed the highest intracellular growth. The viability of H37Rv-infected A549 cells was significantly low. Interestingly, the drug-resistant clinical Mtb isolates maintained better cell viability over time. A [13C6] glucose tracer and mass isotopomer distributions revealed that A549 cells infected with virulent Mtb strains exhibited higher dependency on the central carbon metabolism like glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle for de novo amino acid biosynthesis. Drug-resistant Mtb isolates infected A549 cells showed robust 13C incorporation in the proteinogenic Threonine, Methionine and Lysine, derived from the tricarboxylic acid cycle. A549 cells infected with Mtb, irrespective of their drug resistance status, showed 13C incorporation in the essential amino acids (lysine, methionine, threonine, valine), underlining the dependencies of the pathogen on the de novo host metabolic precursors. These findings elucidate the importance of deciphering the strain-specific host-pathogen metabolic interactions to develop targeted therapies against drug-resistant Mtb infections.

biochemistry↗

bioRxiv2024

Time course metabolomics and 13CO2 mapping establish BBX31/miP1b mediated metabolic readjustments conferring UV-B tolerance in Arabidopsis

B-box proteins (BBXs) are transcription factors that act as signal transducers in light signaling pathways. The microprotein BBX31/miP1b is known to play a positive role in promoting photomorphogenesis and stress tolerance under UV-B. However, the BBX31-mediated metabolic reprogramming to confer UV-B tolerance in plants is not well characterised. Here, we integrate metabolomics with kinetic 13CO2 tracer-based metabolic mapping, morpho-physiological and biochemical analysis to determine the metabolic rewiring in the UV tolerant genotypes. Our results suggest that BBX31 modulates the levels of photosynthetic compounds, reduces TCA cycle intermediates and enhances GS/GOGAT metabolic intermediates and secondary metabolic pathways. 13CO2 tracing studies established BBX31 modulates phenylpropanoid and GS/GOGAT pathways to divert flux towards the accumulation of UV-B protective metabolites phenylalanine, oxoproline, glutamine, and others. Although metabolomics indicated a higher accumulation of branch chain amino acids (BCAAs) under UV-B, they had negligible 13C incorporation, indicating their biosynthesis from pre-existing intermediates or via protein degradation. Further, we demonstrate that the exogenous application of phenylalanine, identified as one of the marker metabolites, confers tolerance to plants under UV-B. This study sheds light on BBX31-mediated metabolic rewiring under UV-B, which can assist targeted enrichment of metabolites and metabolic engineering to promote UV-B tolerance in plants. HighlightBBX31/miP1b modulates the levels of photosynthetic compounds, reduces TCA cycle intermediates and enhances GS/GOGAT metabolic intermediates to confer UV-B tolerance.

plant biology↗

bioRxiv2023

Mass spectrometry and NMR spectroscopy profiles of red and pink Rhododendron flower petals establish them as rich sources of bioactive secondary metabolites

Rhododendron petals are considered high-value owing to their commercial utility, national/state flower status in certain countries, and bioactive potential from recent studies. Profiling and quantitative analysis of the bioactive metabolites would evaluate if they can be natural sources. This study is focused on comprehensive profiling of secondary metabolites in the petals of Red and Pink Rhododendron flowers (R. arboreum and R. campanulatum) using Mass Spectrometry (GC-MS, LC-MS/MS) and Proton-Nuclear Magnetic Resonance (1H-NMR) Spectroscopy. The profiling highlighted the presence of secondary metabolites belonging to phenolic acids and flavonoids. Specifically, the flowers are rich in promising bioactive molecules such as quinic acid, chlorogenic acid (3-O-caffeoyl quinic acid), protocatechuic acid, coumaroyl quinic acids, catechin, epigallocatechin, and shikimic acid. The profiles are correlated with the metabolic pathways which reflected the activity of shikimic acid, phenolic acid and flavonoid biosynthetic pathways. These metabolites are well reported for their bioactive potential as anti-oxidative, anti-viral, anti-cancerous, anti-diabetic, anti-inflammatory, etc. While the quantitative and multivariate analysis showed variations in the levels of phenolic acids and flavonoids, it is established that red and pink Rhododendron flower petals are a rich source of bioactive phytochemicals of interest to the phytochemical Industry.

plant biology↗

bioRxiv2023

Metabolic, Biochemical, Mineral and Fatty acid profiles of edible Brassicaceae microgreens establish them as promising functional food

Hidden hunger due to micronutrient deficiencies affecting one in three people is a global concern. Identifying functional foods which provide vital health beneficial components in addition to the nutrients is of immense health relevance. Microgreens are edible seedlings enriched with concentrated minerals and phytochemicals whose dietary potential as functional foods needs evaluation. In this study, comprehensive biochemical, mineral, metabolic, and fatty acid profiles of four Brassicaceae microgreens - mustard (Brassica juncea), pak choi (Brassica rapa subsp. chinensis), radish pink (Raphanus sativus), and radish white (Raphanus ruphanistrum) was investigated. The biochemical and mineral profiling confirmed their promising nutritional and antioxidant nature and as excellent sources of minerals. Mineral profiling using inductively coupled plasma mass spectrometry (ICP-MS) exhibited promising levels of Fe, Mn, Mg, K, and Ca in microgreens. Gas chromatography-mass spectrometry (GC-MS) based metabolite profiling highlighted a range of phytochemicals-sugars, amino acids, organic acids, amines, fatty acids, phenol, and other molecules. Fatty acid profiling established promising levels of Oleic acid (C18:1; Monounsaturated fatty acids-MUFA) and linoleic acids (C18:2; omega-6 Poly unsaturated fatty acid-PUFA), which are health beneficial. It is estimated that fresh microgreens (100 g) can meet about 20 % to 50 % recommended dietary allowance (RDA) of macro- and micro-minerals along with providing useful fatty acids and antioxidants. Overall, the study highlighted Brassicaceae microgreens as an excellent nutrient source that can act as functional foods with promising potential to overcome "hidden hunger". Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/541100v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@d3b9c9org.highwire.dtl.DTLVardef@4a7604org.highwire.dtl.DTLVardef@8a164corg.highwire.dtl.DTLVardef@1be187b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBrassicaceae microgreens are rich in molecules with relevance to nutrition and health C_LIO_LIThe biochemical analysis supported the antioxidant nature of microgreens C_LIO_LIComprehensive metabolite profiles of edible microgreens of Brassica juncea (Mustard), Brassica rapa subsp. chinensis (Pak Choi), Raphanus sativus (Radish Pink), and Raphanus ruphanistrum (Radish white) using GC-MS are reported C_LIO_LIIonomics analysis using the Brassicaceae microgreens exhibited promising levels of microminerals Fe, Mn, Mg, K, and Ca C_LIO_LIFatty acid profiles show promising levels of Linoleic acid and Oleic acid, which have health relevance C_LI

plant biology↗

bioRxiv2022

Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables

Improved technologies are needed for sustainable conversion of cellulosic waste to valuable products. Here we demonstrate the successful integration of a synthetic microbial consortium (SynCONS) based consolidated bioprocessing with pyrolysis to produce commodity chemicals from cellulose. Promising microbial partners were rationally identified from 7626 organisms via comparative metabolic mapping which led to establishing two promising SynCONS with abilities to convert cellulose to ethanol and lactate in bioreactors. The partners in the two SynCONS were a) the mesophilic fungus Trichoderma reesei grown sequentially with the thermophilic bacterium Parageobacillus thermoglucosidasius NCIMB 11955 (TrPt) and b) a thermophilic bacterium Thermobifida fusca grown together with Parageobacillus thermoglucosidasius NCIMB 11955 (TfPt). TrPt sequential bioprocessing resulted in 39% (g/g) cellulose consumption with product yields up to 9.3% g/g (ethanol + lactate). The TfPt co-cultures demonstrated a cellulose consumption of 30% (g/g) and combined yields of ethanol and lactic acid up to 23.7% g/g of consumed cellulose. The total product yields were further enhanced (51% g/g cellulose) when commercially available cellulases were used in place of T. fusca. Furthermore, when the metabolically engineered ethanol-producing strain of P. thermoglucosidasius TM242 (TfPt242) was substituted in the thermophilic TfPt co-culture consortium, ethanol yields were substantially higher (32.7% g/g of consumed cellulose). Finally, subjecting the residual cellulose and microbial biomass to pyrolysis resulted in carbon material with physicochemical properties similar to commercially available activated carbon as analysed using Scanning Electron Microscopy, X-Ray Diffraction and Raman spectroscopy. Overall, the integration of this synthetic microbial consortia-based bioprocessing strategy with pyrolysis demonstrated a promising strategy for conversion of waste cellulose to chemicals, biofuels, and industrial carbon potentially suitable for several industrial applications.

bioengineering↗

bioRxiv2021

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis

Studies on specialised metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibits spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimised the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved by through a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment b) nitrogen gas drying and c) their derivatisation using MSTFA for GC-MS analysis. The optimised protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), hydroxycinnamic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. Notably, vanillin (253 m/z) was detected only in the UV-B exposed tissues. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems.

plant biology↗

bioRxiv2021

Mutant strains of Escherichia coli lacking global regulators, arcA and fis, demonstrate better growth fitness by pathway reprogramming under acetate metabolism

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A systems-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the substrates available in a range of nutrient niches such as the mammalian gut and high-fat diet. Therefore, investigating the study on acetate metabolism is highly significant. It is well known that the global regulators arcA and fis regulate acetate uptake genes in E. coli under glucose condition. In this study, we deciphered the growth and flux distribution of E.coli transcription regulatory knockout mutants {Delta}arcA, {Delta}fis and double deletion mutant, {Delta}arcAfis under acetate using 13C-Metabolic Flux Analysis which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~1.2-1.6 fold) with a proportionate increase in acetate uptake rates compared to the wild-type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes, which conferred an advantage of faster growth with better carbon usage in all the mutants. Under acetate metabolism, the mutants exhibited higher fluxes in the TCA cycle (~18-90%) and lower gluconeogenesis flux (~15-35%) with the proportional increase in growth rate. This study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate to acetyl-CoA and therefore are potential candidates that can serve as a precursor for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products. ImportanceUnravelling the role of global regulatory genes on microbial metabolism of substrates available in various growth niche is important. Studies have shown that the global transcriptional regulators arcA and fis, under glucose availability, suppress the acetate uptake genes indicating a link between nutrient source and gene regulatory control. This work is focused on deciphering the influence of these regulators on acetate metabolism in E.coli. Growth studies using knockout strains ({Delta}arcA, {Delta}fis and {Delta}arcAfis) and 13C Metabolic flux analysis defined precise metabolic phenotypes under acetate metabolism. Interestingly, the mutants showed metabolic readjustment to facilitate optimal biomass requirements and a better balance between energy and precursor synthesis, resulting in better growth, which lacked in the wild-type strain. The outcomes of this study will be leveraged in understanding the regulatory control under various nutrient shifts.

microbiology↗

bioRxiv2021

Oleate metabolism using kinetic 13C dilution strategy deciphered the potential role of global transcription regulator arcA in Escherichia coli

Microbial metabolism of long-chain fatty acids (LCFA; > C12) is of relevance owing to their presence in various nutrient niches. Microbes have evolved to metabolize LCFA by expressing relevant genes coordinated by various transcriptional regulators. Among the global transcriptional regulators, the metabolic control conferred by arcA (aerobic respiration control) under a LCFA medium is lacking. This work is targeted to unravel the metabolic features of E.coli MG1655 and its knockout strain {Delta}arcA under oleate (C18:1) as a sole carbon source, providing novel insights into the flexibility of the global regulators in maintaining the cellular physiology. Owing to the availability and cost of stable isotope LCFA tracers, we adopted a novel kinetic 13C dilution strategy. This allowed us to quantify the 13C dilution rates in the amino acids that retro-biosynthetically shed light on the central metabolic pathways in actively growing cells. Our data comprehensively mapped oleate oxidization in E.coli via the pathways of {beta}-oxidation, TCA cycle, anaplerotic and gluconeogenesis. Interestingly, arcA knockout showed expeditious growth (~60%) along with an increased oleate utilization rate (~55%) relative to the wild-type. {Delta}arcA also exhibited higher 13C dilution rates (> 20%) in proteinogenic amino acids than the wild-type. Overall, the study established the de-repression effect conferred by {Delta}arcA in E.coli, which resulted in a phenotype with reprogrammed metabolism favouring higher oleate assimilation. The outcomes suggest rational metabolic engineering of regulators as a strategy to develop smart cells for enhanced biotransformation of LCFA. This study also opens an avenue for adopting a kinetic 13C dilution strategy to decipher the cellular metabolism of a plethora of substrates, including other LCFA in microbes.

systems biology↗

bioRxiv2020

Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using glucose

13C based Isotopic tracers of the media components can be used to kinetically track their contribution in the cell systems. A tracer ([U-13C6] glucose) was used to monitor its contribution into the central carbon metabolic pathways of human lung carcinoma (A549) cells by Gas chromatography-mass spectrometry (GC-MS) based mass isotopomer analysis. Calculated average 13C of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), protein acid hydrolysates (serine: 4.51{+/-}0.21 %, glycine: 2.44{+/-}0.31 %, alanine: 24.56{+/-}0.59 %, glutamate: 8.81{+/-}0.85 %, proline: 6.96{+/-}0.53 % and aspartate: 10.72{+/-}0.95 %) and the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), showed significant contribution of 13C glucose. We observed the Warburg effect with higher levels of 13C lactate in the culture filtrate. 13C glycerol levels in culture supernatant showed significant increase with time and amino acids of glucogenic origin also contributed cellular protein biomass. The workflow adopted in this study for 13C analysis could be useful for the metabolic phenotyping of other mammalian cell systems under normal and perturbed (cancer and infection) conditions.

biochemistry↗