Synthesis and Characterization of Zn(II) Complex of 4-chloro-2-(((2-phenoxyphenyl)imino)methyl)phenol and its Biological Efficacies: DNA Interaction, ADMET, DFT and Molecular Docking Study.

Condensing 2-phenoxyaniline with 5-chlorosalicyldehyde under reflux conditions, a 4-chloro-2-(((2-phenoxyphenyl)imino)methyl)phenol Schiff base has been Synthesized. A zinc complex was synthesized by combining the ligand in a 1:1 molar ratio with zinc sulphateheptahydrate. Mass spectroscopy, NMR, infrared, and elemental analysis were used to characterize the ligand and zinc complex. By measuring the molar conductance, the non-electrolytic character of the complex was confirmed. The zinc ion is coordinated in a pentadentate manner, according to an IR and NMR investigation. Viscosity measurements, absorption and fluorescence spectroscopy were utilized to examine the complex's interaction with CT (calf thymus) DNA. Furthermore, the ligand and complex's ADMET characteristics were ascertained through the use of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) study. Calculation of the different electronic parameters of the optimized structure through Density Functional Theory (DFT) indicated the stability of the Zn(II) complex. Molecular docking study reflected the future opportunity for the consideration of Zn(II) complex to fight against Alzheimer and Glaucoma diseases.

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Synthesis of Novel [{(2-Amino-5-Nitro-N-[(E)-Thiophen-2-yl-Methylidene]Aniline-κ3N1:N4:S)(Sulphato-κ2O1:O3)}Zinc(II)] Complex with Physico-Chemical and Biological Perspective Exploration: A Combined Experimental and Computational Studies.

A novel metal complex was synthesized using freshly prepared 2-Amino-5-nitro-N-[(E)-thiophen-2-yl-methylidene]aniline ligand with Zn (II) sulphate heptahydrate in a 1:1 molar ratio. The ligand and the complex were characterized using different spectroscopic techniques, and the complex was assigned a distorted square pyramidal geometry. Additionally, DNA binding assays and antibacterial activity were used to assess the biological perspectives for the synthesized complex, including the ligand and complex which was further confirmed by molecular docking. Fluorescence Spectroscopy, viscosity measurement, and adsorption measurement were used to investigate the interaction of the Zn (II) complex with CT-DNA. A comparative in vitro antibacterial activity study against Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus strains were studied with free ligand and Zn (II) metal complex. The stable geometry of the complex was additionally established through computational simulation utilizing density functional theory, which was followed by the calculation of several electronic properties. The ADMET characteristics of the complex and ligand were also assessed using ADMET analysis. The in-silico ADMET properties pointed to a significant drug-likeness feature in the synthesized compounds, based on the Lipinski criteria.

Nasal MRSA carriage is a risk factor for development of antibiotic resistance in diabetic foot ulcers and is significantly higher than diabetic and non-diabetic individuals without foot ulcer.

BACKGROUND: Diabetic foot ulcer (DFU) is a major complication of diabetes often impacted by polymicrobial infection in the wound site. Diabetic patients are immunocompromised in nature and hence vulnerable to infection once the skin barrier is breached. Microbiological culture-based methods show that Staphylococcus aureus (SA) is the most frequently isolated bacteria from the DFU wounds. SA and its most clinically important antibiotic resistant variant methicillin-resistant S. aureus (MRSA) are commonly found in the nasal vestibule and colonization of SA as well as MRSA in any wound site can aggravate the condition. We hypothesize that the presence of nasal MRSA carriage can serve as a potential risk factor contributing to the emergence of antibiotic resistance in diabetic foot ulcer wounds. METHODS: In the present study, we have compared the carriage of SA and MRSA in nasal cavity and foot skin among DFU patients (D+F+, n = 50), diabetic patients without any ulcer (D+F-, n = 50), and healthy controls (D-F-, n = 40) by using bacterial culture and PCR based methods. The D+F+, D+F- and D-F-individuals were further categorized based on the presence or absence of MRSA and clinical parameters were compared between MRSA+ ve and MRSA-ve individuals in each of the three groups mentioned above. RESULTS: Our results show that, (a) nasal MRSA carriage is significantly higher (p < 0.05) in D+F+ group than the D+F- and D-F- and significantly associated with wound MRSA carriage in D+ F+ individuals (O.R. = 4.09; 95% C.I. = 1.12-15.05) and (b) the HbA1C level is significantly higher (p < 0.02) in wound MRSA positive, compared to MRSA negative D+F+ patients. Interestingly more than half of the MRSA (64%) isolated from DFU wound were identified to be multidrug resistant. CONCLUSION: These findings strongly suggest that nasal MRSA carriage can act as a risk factor for development of antibiotic resistance in diabetic foot ulcers and it is therefore important to screen nasal and wound sites of these patients regularly. We have also developed a rapid multiplex PCR assay to detect MRSA from clinical isolates or microbial DNA isolated from clinical samples in the hospital settings.

Prolonged Inflammation and Infectious Changes in the Corneal Epithelium Are Associated with Persistent Epithelial Defect (PED).

Purpose: Failure of rapid re-epithelialization within 10-14 days after corneal injury, even with standard supportive treatment, is referred to as persistent corneal epithelial (CE) defect (PED). Though an array of genes regulates reepithelization, their mechanisms are poorly understood. We sought to understand the network of genes driving the re-epithelialization in PED. Method: After obtaining informed consent, patients underwent an ophthalmic examination. Epithelial scrapes and tears samples of six PED patients and six individuals (control) undergoing photorefractive keratectomy (PRK) were collected. RNA isolation and quantification were performed using either the epithelial scrape taken from PED patients or from HCLE cells treated with control tears or tears of PED patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of a few important genes in CE homeostasis, inflammation, and cell-cell communication, viz., Kruppel-like factor 4 (KLF4), GPX4, IL6, TNFα, STING, IL8, desmoglein, and E-cadherin, among others. Their expressions were normalized with their respective housekeeping genes and fold changes were recorded. KLF4 localization and MMPs activity was carried out via immunofluorescence and zymography, respectively. Results: KLF4, a transcription factor important for CE homeostasis, was upregulated in tears-treated HCLE cells and downregulated in PED patients compared to the healthy PRK group. Cell-cell communication genes were also upregulated in tears-treated cells, whereas they were downregulated in the PED tissue group. Genes involved in proinflammation (IL6, 282-fold; TNFα, 43-fold; IL8, 4.2-fold) were highly upregulated in both conditions. MMP9 activity increased upon tears treatment. Conclusions: This study suggests that tears create an acute proinflammatory milieu driving the PED disease pathology, whereas the PED patients scrapes are an indicator of the chronic stage of the disease. Interferons, pro-inflammatory genes, and their pathways are involved in PED, which can be a potential target for inducing epithelialization of the cornea.

Comparative study of the efficiency of silicon carbide, boron nitride and carbon nanotube to deliver cancerous drug, azacitidine: A DFT study.

Herein we have made a comparative study of the efficiency of three different nanotubes viz. Carbon nanotube (CNT), boron nitride nanotube (BNNT) and silicon carbide nanotube (SiCNT) to deliver the cancerous drug, Azacitidine (AZD). The atomistic description of the encapsulation process of AZD in these nanotubes has been analyzed by evaluating parameters like adsorption energy, electrostatic potential map, reduced density gradient (RDG). Higher adsorption energy of AZD with BNNT (-0.66eV), SiCNT (-0.92eV) compared to CNT (-0.56eV) confirms stronger binding affinity of the drug for the former than the later. Charge density and electrostatic potential map suggest that charge separation involving BNNT and CNT is more prominent than SiCNT. Evaluation of different thermodynamic parameters like Gibbs free energy, enthalpy change revealed that the overall encapsulation process is spontaneous and exothermic in nature and much favorable with BNNT and SiCNT. Stabilizing interactions of the drug with BNNT and SiCNT has been confirmed from RDG analysis. ADMP molecular dynamics simulation supports that the encapsulation process of the drug within the NT at room temperature. These results open up unlimited opportunities for the applications of these NTs as a drug delivery system in the field of nanomedicine.

Correlation of fine needle aspiration cytology and cell block study of category III, IV, and V (TBSRTC-2017) thyroid lesions with special reference to cytokeratin-19 immunohistochemical staining.

Context: Ascertaining the role of cytokeratin-19 (CK19) and its staining pattern helps to differentiate papillary carcinoma from other thyroid lesions. Aims: To correlate fine needle aspiration cytology (FNAC) and cell block study of equivocal cases (Category III, IV, and V) with the role of CK19 staining in it. Settings and Design: A hospital-based cross-sectional observational study was designed and conducted at North Bengal Medical College and Hospital, Shusrutnagar, Darjeeling. Methods and Material: The FNAC performed and reported as per TBSRTC-2017.50 cases of Category III, IV, and V was selected for cell block study and CK19 staining followed by immunohistochemical scoring. Results: Out of 50 cases, 17 were follicular neoplasm, 21 papillary carcinoma, 6 lymphocytic thyroiditis, 1 Hürthle cell adenoma, 1 medullary carcinoma, 1 lymphoma, and 3 undifferentiated carcinomas. Among cases of papillary carcinoma, 10 showed 4+ positivity, 9 showed 3+ positivity, 2 showed focal 1+ and 2+ positivity. In the case of follicular neoplasm, 1 showed 3+ positivity, 3 cases had 1+ 2+ positivity, and 13 cases revealed negative staining. Conclusion: Role of CK19 in distinguishing papillary carcinoma of thyroid from other lesions in cytologically diagnosed Category III, IV, and V (TBSRTC-2017) cases can be demonstrated.

Repurposing of anti-lung cancer drugs as multi-target inhibitors of SARS-CoV-2 proteins: An insight from molecular docking and MD-simulation study.

Herein we have selected seventeen anti-lung cancer drugs to screen against Mpro, PLpro and spike glycoproteins of SARS-CoV-2to ascertain the potential therapeutic agent against COVID-19. ADMET profiling were employed to evaluate their pharmacokinetic properties. Molecular docking studies revealed that Capmatinib (CAP) showed highest binding affinity against the selected proteins of SARS-CoV-2. Molecular Dynamics (MD) simulation and the analysis of RMSD, RMSF, and binding energy confirmed the abrupt conformational changes of the proteins due to the presence of this drug. These findings provide an opportunity for doing advanced experimental research to evaluate the potential drug to combat COVID-19.

Potential Anti-leishmanial Activity of a Semi-purified Fraction Isolated from the Leaves of Parthenium hysterophorus.

PURPOSE: In the present perspective, emergence of resistant strains of Leishmania donovani and severe side effects resulting from the use of conventional anti-leishmanial therapies present an urgent need for developing novel agents against this parasite. We have explored the effectiveness of secondary plant metabolites as alternative choices in the treatment for visceral leishmaniasis (vl). METHODS: The plant Parthenium hysterophorus L. (Asteraceae) was collected from the West Bengal State University Campus, Barasat, West Bengal, India. The leaves of this plant were extracted by different solvents, such as ethyl acetate, water, petroleum ether and hexane. Gas chromatography-mass spectrometry (GC-MS) analysis was also carried out for the identification of compounds in the hexane soluble fraction (PHFd) with substantial anti-leishmanial activities. The antipromastigote activity and cytotoxicity of this fraction were evaluated by the tetrazolium MTT assay. Other biochemical and physiological parameters were studied by microscopic observation and flow cytometric analyses. RESULTS: PHFd showed considerable activity against L. donovani promastigotes (IC50: 20 µg/ml). The PHFd also inhibited in vitro growth of L. major LV39 promastigotes dose dependently with an IC50 of 40 µg/ml. The GC-MS studies of this particular fraction revealed the presence of four major compounds with different retention times (RT) of 26.08, 33.11, 36.41, and 41.20 min. In this study, we also established that PHFd could induce DNA damage and subsequent apoptosis of L. donovani promastigotes with a concomitant increase in generations of reactive oxygen species (ROS) in a time-dependent manner. This fraction was also found to be effective in nitric oxide-mediated inhibition of intracellular amastigotes (IC50:12.5 µg/ml) without any noticeable cytotoxicity towards murine splenocytes in vitro. CONCLUSION: This study provides the basis for additional phytochemical and pharmacological studies on the antiprotozoal applications of P. hysterophorus.

Inhibitory effect of anti-HIV compounds extracted from Indian medicinal plants to retard the replication and transcription process of SARS-CoV-2: an insight from molecular docking and MD-simulation studies.

Outbreak of Coronavirus (SARS-CoV-2) has thrown a big challenge to the globe by snatching millions of human lives from the world. In this study, inhibitory efficiency of ten anti-HIV compounds from different Indian medicinal plant parts have been virtually screened against Mpro, PLpro and RdRp proteins of SARS-CoV-2. The molecular docking study reflected that among these compounds, Proptine (PTP) has the highest binding affinity for the three cases. Introduction of PTP molecules within the binding pocket of these proteins showed a large structural and conformational changes on the structure of proteins which is revealed from molecular dynamics (MD) simulation studies. RMSD, RMSF and analysis of thermodynamic parameters also revealed that PTP makes a huge impact on the structures of the respective proteins which will pave an opportunity for doing advanced experimental research to evaluate the potential drug to combat COVID-19. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13721-021-00309-3.

Inhibitory efficacy of RNA virus drugs against SARS-CoV-2 proteins: An extensive study.

Herein we have made a comprehensive analysis of inhibitory efficacy of 16 RNA virus drugs against RdRp, Mpro and PLpro proteins of SARS-CoV-2. Analysis of docked conformation revealed that Baloxavir marboxil (BMX) corresponds to the highest binding energy. Analysis of residue confirmed that BMX strongly interact with these three proteins involving H-bonding, ionic as well as hydrophobic interactions. Molecular dynamics simulation and analysis of parameters like RMSD, RMSF, binding energy confirmed noticeable conformational alternation with these proteins with makeable effect on RdRp. The potentially inhibitory action of BMX against these three proteins suggests the inhibition of overall transcription process of SARS-CoV-2. These observation along with the recently observed inhibitory action of BMX on influenza with clinically proven no side effects emphasizes to uncover the role of BMX by in-vitro and in-vivo analysis.

Inhibitory efficiency of potential drugs against SARS-CoV-2 by blocking human angiotensin converting enzyme-2: Virtual screening and molecular dynamics study.

Till date millions of people are infected by SARS-CoV-2 throughout the world, while no potential therapeutics or vaccines are available to combat this deadly virus. Blocking of human angiotensin-converting enzyme 2 (ACE-2) receptor, the binding site of SARS-CoV-2 spike protein, an effective strategy to discover a drug for COVID-19. Herein we have selected 24 anti-bacterial and anti-viral drugs and made a comprehensive analysis by screened them virtually against ACE-2 receptor to find the best blocker by molecular docking and molecular dynamics studies. Analysis of results revealed that, Cefpiramide (CPM) showed the highest binding affinity of -9.1 kcal/mol. Furthermore, MD study for 10 ns and evaluation of parameters like RMSD, RMSF, radius of gyration, solvent accessible surface area analysis confirmed that CPM effectively binds and blocks ACE-2 receptor efficiently.

Screening of potential drug from Azadirachta Indica (Neem) extracts for SARS-CoV-2: An insight from molecular docking and MD-simulation studies.

Azadirachta Indica (Neem) extracts have been known for their anti-bacterial and other effects since ancient times. The present work examines the inhibitory activity of Neem extracts on Papain like protease (PLpro) of the novel coronavirus SARS-CoV-2. The activity is analysed by molecular docking study along with molecular dynamics simulation. All the studied Neem compounds showed decent level of inhibitory activity against PLpro of SARS-CoV-2. Among them, desacetylgedunin (DCG) found in Neem seed showed the highest binding affinity towards PLpro. Furthermore, MD-simulation studies supported by standard analysis (e.g. root mean square deviation and fluctuation (RMSD, RMSF), radius of gyration, solvent accessible surface area (SASA)) showed large impact on the structure of PLpro by DCG. We believe that the significant effect of DCG on PLpro may help in therapeutic efforts against SARS-CoV-2.

Performance of three delignifying pretreatments on hardwoods: hydrolysis yields, comprehensive mass balances, and lignin properties.

BACKGROUND: In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, ß-aryl ether content, and response to depolymerization by thioacidolysis. RESULTS: All three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and ß-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high ß-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13C NMR-determined ß-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality. CONCLUSIONS: Overall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of ß-aryl ether bonds, is shown to be detrimental to subsequent depolymerization.

Selective in vitro inhibition of Leishmania donovani by a semi-purified fraction of wild mushroom Grifola frondosa.

The current study was designed to assess the anti-leishmanial effect of a semi-purified fraction of wild mushroom Grifola frondosa against Leishmania donovani, in vitro. A total of five extracts from three wild mushrooms [Grifola frondosa (family, Meripilaceae) Laetiporus sulphurous (family, Polyporaceae) and Meripilus giganteus (family, Meripilaceae) were explored for novel anti-leishmanial leads against promastigotes. The ethanol extract of G. frondosa was selected as the most efficient against L. donovani promastigotes (IC50: 93.9 µg/mL). A semi-purified fraction was obtained from an active ethanol extract of G. frondosa and found to inhibit the survival of promastigotes of L. donovani (MHOM/IN/83/AG83) significantly (IC50: 20.37 µg/mL) and it also had some effect against L. major LV39 (MRHO/Sv/59/P strain) and L. tropica WR683 (MHOM/SU/58/OD) strains at higher concentrations (IC50: 46.08 µg/mL and 53.79 µg/mL respectively). The semi-purified fraction also interfered in lipid biosynthesis, altered parasite morphology and induced apoptosis in L. donovani promastigotes. The semi-purified fraction was also effective against intracellular amastigotes in infected macrophages and enhanced the release of nitric oxide and pro-inflammatory cytokines, in vitro. Interestingly, the 50% inhibitory concentration of the semi-purified fraction against the intracellular amastigotes (IC50: 2.48 µg/mL) was much lower in comparison to promastigotes (IC50: 20.37 µg/mL). The semi-purified fraction was found to inhibit the intracellular amastigotes slightly more efficiently in comparison to conventional anti-leishmanial drugs; sodium antimony gluconate, amphotericin B, miltefosine and paromomycin and noticeably non-toxic towards host splenocytes. The findings of the present study established that G. frondosa might be a natural resource for development of a new anti-leishmanial lead.

Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts.

BACKGROUND: Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts. RESULTS: In this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars. CONCLUSIONS: This study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.

Lignin Valorization: Two Hybrid Biochemical Routes for the Conversion of Polymeric Lignin into Value-added Chemicals.

Naturally, many aerobic organisms degrade lignin-derived aromatics through conserved intermediates including protocatechuate and catechol. Employing this microbial approach offers a potential solution for valorizing lignin into valuable chemicals for a potential lignocellulosic biorefinery and enabling bioeconomy. In this study, two hybrid biochemical routes combining lignin chemical depolymerization, plant metabolic engineering, and synthetic pathway reconstruction were demonstrated for valorizing lignin into value-added products. In the biochemical route 1, alkali lignin was chemically depolymerized into vanillin and syringate as major products, which were further bio-converted into cis, cis-muconic acid (ccMA) and pyrogallol, respectively, using engineered Escherichia coli strains. In the second biochemical route, the shikimate pathway of Tobacco plant was engineered to accumulate protocatechuate (PCA) as a soluble intermediate compound. The PCA extracted from the engineered Tobacco was further converted into ccMA using the engineered E. coli strain. This study reports a direct process for converting lignin into ccMA and pyrogallol as value-added chemicals, and more importantly demonstrates benign methods for valorization of polymeric lignin that is inherently heterogeneous and recalcitrant. Our approach also validates the promising combination of plant engineering with microbial chassis development for the production of value added and speciality chemicals.

Efficient dehydration and recovery of ionic liquid after lignocellulosic processing using pervaporation.

BACKGROUND: Biomass pretreatment using certain ionic liquids (ILs) is very efficient, generally producing a substrate that is amenable to saccharification with fermentable sugar yields approaching theoretical limits. Although promising, several challenges must be addressed before an IL pretreatment technology can become commercially viable. One of the most significant challenges is the affordable and scalable recovery and recycle of the IL itself. Pervaporation (PV) is a highly selective and scalable membrane separation process for quantitatively recovering volatile solutes or solvents directly from non-volatile solvents that could prove more versatile for IL dehydration. RESULTS: We evaluated a commercially available PV system for IL dehydration and recycling as part of an integrated IL pretreatment process using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) that has been proven to be very effective as a biomass pretreatment solvent. Separation factors as high as 1500 were observed. We demonstrate that >99.9 wt% [C2C1Im][OAc] can be recovered from aqueous solution (≤20 wt% IL) and recycled five times. A preliminary technoeconomic analysis validated the promising role of PV in improving overall biorefinery process economics, especially in the case where other IL recovery technologies might lead to significant losses. CONCLUSIONS: These findings establish the foundation for further development of PV as an effective method of recovering and recycling ILs using a commercially viable process technology.

SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum.

Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maize that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units.

Impact of lignin polymer backbone esters on ionic liquid pretreatment of poplar.

BACKGROUND: Biomass pretreatment remains an essential step in lignocellulosic biofuel production, largely to facilitate the efficient removal of lignin and increase enzyme accessibility to the polysaccharides. In recent years, there have been significant efforts in planta to reduce lignin content or modify its composition to overcome the inherent recalcitrance that it imposes on lignocellulosic biomass during processing. Here, transgenic poplar lines in which monolignol ferulate conjugates were synthesized during cell wall development to introduce, during lignification, readily cleavable ester linkages into the lignin polymer backbone (i.e., "zip lignin"), along with wild-type (WT) controls, were pretreated with different ionic liquids (ILs). RESULTS: The strategic introduction of ester bonds into the lignin backbone resulted in increased pretreatment efficiency and released more carbohydrates with lower energy input. After pretreatment with any of three different ILs, and after limited saccharification, the transgenic poplars, especially those with relatively higher amounts of incorporated monolignol ferulate conjugates, yielded up to 23% higher sugar levels compared to WT plants. CONCLUSION: Our findings clearly demonstrate that the introduction of ester linkages into the lignin polymer backbone decreases biomass recalcitrance in poplar has the potential to reduce the energy and/or amount of IL required for effective pretreatment, and could enable the development of an economically viable and sustainable biorefinery process.