High-level biosynthesis of enantiopure germacrene D in yeast.

Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (-) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast (Saccharomyces cerevisiae) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13-30 fold higher titers of (+) or (-)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80, LPP1 and rDNA loci generated CENses8(+D) and CENses8(-D) strains, which produced 41.36 µg/ml and 728.87 µg/ml of (+) and (-)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71 µg/ml and 815.81 µg/ml of (+) or (-)-germacrene D in CENses8(+D)* and CENses8(-D)* strains, which were 67-120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(-D)* produced 290.28 µg/ml and 2519.46 µg/ml (+) and (-)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. KEY POINTS: • Engineered S. cerevisiae produced enantiopure (+) and (-)-germacrene D at high titers • Engineered strain produced up to 120-fold higher germacrene D than the parental strain • Highest titers of enantiopure (+) and (-)-germacrene D achieved so far in shake-flask.

Exploration of molecular interactions between scoparone and associated compounds with Constitutive androstane receptor (CAR) leading to gallstone prevention: an in silico investigation.

Scoparone (6, 7 dimethylesculetin) is a biologically active compound derived from the herb Artemisia capillaris having anti-inflammatory, anti-lipemic, and anti-allergic roles. Activation of the constitutive androstane receptor (CAR) in primary hepatocytes of both wild-type and humanized CAR mice by scoparone, accelerates bilirubin and cholesterol clearance in vivo. This can prevent gallstones which is a dreaded gastrointestinal disease. To date, surgery is regarded as the gold standard for treating gallstones. The molecular interactions between scoparone and CAR leading to gallstone prevention are not yet explored. In this study, we have analyzed these interactions through an insilico approach. After extracting the CAR structures (mice and human) from the protein databank and 6, 7-dimethylesuletin from PubChem, energy minimization of both the receptors was done to make them stable followed by docking. Next, a simulation was performed to stabilize the docked complexes. Through docking, H-bonds and pi-pi interactions were found in the complexes, which imply a stable interaction, thus activating the CAR. A similarity search for scoparone was performed and the selected compounds were docked with the CAR receptors. Esculentin acetate and scopoletin acetate interacted with human CAR through pi-alkyl and H-bond respectively. While Fraxidin methyl ether, fraxinol methyl ether, and 6, 7 diethoxycoumarin interacted with mice CAR through H-bond and Pi-Pi T-shaped bonds. The selected complexes were simulated further. Our results are in accordance with the hypothesis in the literature. We have also analyzed the drug likeliness, absorption, non-carcinogenicity, and other properties of scoparone which can support further in vivo studies.Communicated by Ramaswamy H. Sarma.

Production of exopolysaccharides from Bacillus licheniformis, a thermophilic bacteria from hot spring of Jharkhand.

Thermophiles are the microorganisms which thrive under extreme conditions such as high temperature, making them significant for scientific interest. This study provides information based on isolation of thermophilic strain from Surajkund and Ramkund, hot spring of Jharkhand at 50, 60 and 70 °C. Two of the best isolates were used for the extraction of exopolysaccharides. Additionally, the lyophilized product obtained was further analyzed for protein and total sugar estimation. The FTIR analysis revealed the presence of different functional groups such as hydroxyl, C-H stretching, vibration of aliphatic CH2 and glycosidic linkage, thus proving the product obtained from bacteria was an exopolysaccharides The FESEM analysis of exopolysaccharides show varying surface morphology that is from porous to globular structure. Based on 16S rRNA sequences, the isolates from Surajkund (ON795919) and Ramkund (ON795916) were different strains of Bacillus licheniformis. This is the first report on exopolysaccharide secreting thermophilic strain from these hot springs.

Optimisation, characterization, and biological evaluation of novel exopolysaccharide from Bacillus licheniformis (BITSL006).

The study investigated production, characterisation, and biological properties of exopolysaccharide (EPS) from a thermophilic bacterium, Bacillus licheniformis using sucrose as a main carbon source at a temperature of 75 °C, resulting in a yield of 2.87 g/L. The surface topology of EPS was determined using FESEM indicating its porous nature. Subsequently, FTIR was employed to examine EPS and identified the presence of carboxyl and hydroxyl groups, which are believed to be associated with water-holding capacity (WHC). Comparing the FTIR spectrum of various exopolysaccharides, it was inferred that the exopolysaccharide derived from Ramkund closely resembles dextran. EDX and ICP-MS analysis revealed the presence of Sulphur and Selenium which might be involved in the anticancer properties of EPS. This is the first report on bacterial EPS from a hot spring (Ram kund) with antioxidant property, WHC, and high solubility. These properties offer beneficial resources for exploration in the pharmaceutical and agriculture industries.

Microstructural and heavy metal analysis of gallstones prevalent in Jharkhand and its implications in the treatment.

OBJECTIVES: In the present study the structural and compositional analysis of gallstones was carried out by various characterization techniques. Also, the utility of the study was evaluated on how the findings can enhance the treatment and dissolution therapies of gallstones? METHODS: Gallstones from the Jharkhand region were analyzed both structurally as well as chemically using Field emission scanning electron microscope (FESEM) and Energy-dispersive X-ray spectroscopy, inductively coupled optical emission spectrometry (ICP-OES), Carbon hydrogen and nitrogen analyzer (CHNS), Nuclear magnetic resonance (NMR), Thermal gravimetric analysis (TGA) and Differential Thermal Analysis (DTA) and other instruments. The content of heavy metal was represented statistically as a mean with standard deviation. RESULTS: FESEM analysis unveiled the crystal and globular structure of cholesterol and pigment gallstones respectively. Through ICP-OES analysis metals such as Fe, Mn, Cu, Pb, Cr, Zn etc. were detected in gallstones. FTIR and XRD analysis confirmed the presence of cholesterol and calcium carbonate and other compounds in mixed stones and calcium bilirubinate and bilirubin in pigment gallstones. NMR analysis revealed the presence of monohydrate cholesterol (Crystalline) in Cholesterol and mixed gallstones. Mixed gallstones were found to be predominant in this region in contrast to pigmented stones that were prevalent earlier. CONCLUSION: The present study uncovered the current composition and content of different heavy metals in gallstones of Jharkhand region which can be instrumental in enhancing the dissolution therapies for gallstone treatment. This can serve as a major tool for practitioners for gallstone treatment and change the way they are looked at.

Evaluation of gallstone classification and their diagnosis through serum parameters as emerging tools in treatment: a narrative review.

The treatment of gallstones is a matter of real concern as they may cause gallbladder cancer if not properly attended to. Evaluating the classification of gallstones can give major clues in their treatment as it will decide their etiology, chemical composition, and pathogenesis. Also, serum parameters have emerged as an efficient tool for diagnosing gallstones. They can be probed to evaluate different biochemicals and the changes in their levels in gallstone patients which can be correlated with early prediction of the formation of gallstones. In the present review, a thorough search of the available literature was done starting from the earliest approaches for the classification of gallstones up to the recent advancements. The alteration in the level of serum parameters was also studied in gallstone patients so that it can act as a potential diagnostic tool for early detection of gallstone formation. The earliest classification of gallstones was done in 1896 by Naunyn. He classified them into pure cholesterol stones, laminated cholesterol stones, ordinary gallbladder stones, mixed bilirubin stones, and rare forms. The most recent classification of gallstones was done by Peter et al in 2020 and they classified them as pure, mixed, composite cholesterol, carbonate stones, black and brown pigment stones. The altered of levels of serum parameters was analyzed by Reuben (1985) and in recent times by Peter et al (2020). The various serum parameters studied were as RDW-CV test (red blood cell distribution width), PCT (prolactin) test, MPV (mean platelet count), LYM (lymphocyte) test, and EOS (eosinophil and eosinophil count test). Also, we discussed some practical considerations for gallstones that can be taken into account for gallstone prevention and diagnosis. Further research is required to detect gallstone type in the gallbladder by using the alteration in the levels of serum parameters.

Bio-fabrication of thermozyme-based nano-biosensors: their components and present scenario.

An amalgamation of microbiology, biocatalysis, recombinant molecular biology, and nanotechnology is crucial for groundbreaking innovation in developing nano-biomedicines and sensoristics. Enzyme-based nano-biosensor finds prospective applications in various sectors (environmental, pharmaceutical, food, biorefineries). These applications demand reliable catalytic efficiency and functionality of the enzyme under an extreme operational environment for a prolonged period. Over the last few years, bio-fabrication of nano-biosensors in conjunction with thermozymes from thermophilic microbes is being sought after as a viable design. Thermozymes are known for their robustness, are chemically resistant toward organic solvents, possess higher durability for constant use, catalytic ability, and stability at elevated temperatures. Additionally, several other attributes of thermozymes like substrate specificity, selectivity, and sensitivity make them desirable in developing a customized biosensor. In this review, crucial designing aspects of enzyme-based nano-biosensors like enzyme immobilization on an electrode surface, new materials derived from microbial sources (biopolymers based nanocomposites), improvisation measures for sensitivity, and selectivity have been addressed. It also covers microbial biosynthesis of nanomaterials used to develop sensoristic devices and its numerous applications such as wastewater treatment, biorefineries, and diagnostics. The knowledge will pave the way toward creating consistent eco-friendly, economically viable nanostructured-based technologies with broad applicability and exploitation for industrial use in the near future.

Vaccine for a neglected tropical disease Taenia solium cysticercosis: fight for eradication against all odds.

INTRODUCTION: Taenia solium infection is among the 17 most neglected tropical diseases identified by World Health Organization and to be eradicated by 2030. This parasite infects the central nervous system (Neurocysticercosis [NCC]) and intestine [Taeniasis]). NCC is the most frequent cause of acquired epilepsy in endemic regions and Taeniasis is responsible for the widespread malnutrition and abdominal discomfort among children. Epilepsy caused by T. solium is preventable and the total elimination of NCC can be achieved by good hygiene, mass therapy, and most importantly vaccination of pigs or humans. Vaccine for pig is available but not widely in use and for humans it's still elusive. AREA COVERED: Several vaccine candidates for porcine cysticercosis have been tried like TSOL18, SP3Vac, KETc7, TSOL45, etc. with good success in the limited field trial. This review highlights some seminal contributions for the anti-cestode vaccine, the associated challenges, current status, suggestive future directions, and the need of vaccine for human use. EXPERT OPINION: Though several vaccines are available, none is being widely used due to lack of awareness, economic constraints, accessibility, etc. Hence, there is a need for a newer, economic, and reliable vaccine for humans or pigs use to reduce the disease burden.

Application of virus-induced gene silencing in Andrographis paniculata, an economically important medicinal plant.

Kalmegh [Andrographis paniculata (Burm.f.) Wall. ex Nees] is one of the most studied medicinal plants for pharmaceutical properties and phytochemistry. However, functional genomics studies in kalmegh are so far limited due to the unavailability of a robust tool for gene silencing. Here, we tested the application of virus-induced gene silencing (VIGS) in kalmegh using the well-known Tobacco rattle virus (TRV)-based vectors and achieved targeted silencing of phytoene desaturase (ApPDS) which is essential in plants for carotenoid biosynthesis that protects chlorophyll from photooxidation. ApPDS silencing in kalmegh leaves developed a typical photobleaching phenotype. The silencing of ApPDS was confirmed by analysing ApPDS transcript level and determining chlorophyll content in the leaves of VIGS seedlings. The analysis revealed ~30% reduction in chlorophyll content, and 40 to 60% reduction in ApPDS transcript level in the leaves of VIGS seedlings. These findings clearly demonstrated the applicability of VIGS in kalmegh using TRV-based vectors. The VIGS protocol presented in this study might be useful for studying gene function related to medicinal and agricultural traits in kalmegh.

In vitro analysis of gallstone formation in the presence of bacteria.

AIM: In our previous study, we have isolated different genera of bacteria from gallstones and this intrigued us to study their role in gallstone formation. The isolates exhibited certain biliary activities like urease activity, slime production, and ß-glucuronidase production. We aimed to investigate the role of these factors in the formation of gallstone in in vitro conditions at a supersaturated concentration of cholesterol. METHODOLOGY: To mimic bile in in vitro state, Brilliant Green Bile Broth (BGBB) media having a composition similar to human bile was used. Four different experimental sets were prepared, each having nine flasks with varying concentrations of cholesterol and CaCO3 (calcium carbonate). Test sets I, II, III, and IV were inoculated with Salmonella, Enterococcus, Helicobacter, and Neisseria respectively, which were isolated from gallstone itself. Out of these four bacteria, only Helicobacter did not possess slime activity. A control set was also established which was devoid of bacteria. The control also had nine flasks with different concentrations of cholesterol and CaCO3. All the sets were incubated in the incubator shaker at 37 °C and 80 revolution per minutes (RPM) for 20 days. RESULT: It was observed that the sets having bacteria had a less nucleation time as compared to the control (F = 5.274; p < 0.001). Solidification of gallstone was observed only in the set with bacteria having slime activity (sets I, II, and IV). CONCLUSION: The slime activity of bacteria leads to solidification of gallstones, whereas the other activities accelerate the nucleation of gallstone formation enhancing the severity of the disease.

Virus-Induced Gene Silencing in Sweet Basil (Ocimum basilicum).

Virus-induced gene silencing (VIGS) is a powerful reverse genetic tool for rapid functional analysis of plant genes. Over the last decade, VIGS has been widely used for conducting rapid gene knockdown experiment in plants and played a crucial role in advancing applied and basic research in plant science. VIGS was studied extensively in model plants Arabidopsis and tobacco. Moreover, several non-model plants such as Papaver (Hileman et al., Plant J 44:334-341, 2005), Aquilegia (Gould and Kramer, Plant Methods 3:6, 2007), Catharanthus (Liscombe and O'Connor, Phytochemistry 72:1969-1977, 2011), Withania (Singh et al., Plant Biol J 13:1287-1299, 2015), and Ocimum (Misra et al., New Phytol 214:706-720, 2017) were also successfully explored. We have recently developed a robust protocol for VIGS in sweet basil (Ocimum basilicum). Sweet basil, a popular medicinal/aromatic herb, is being studied for the diversity of specialized metabolites produced in it.

Cellulolytic, amylolytic and xylanolytic potential of thermophilic isolates of Surajkund hot spring.

A total of 41 isolates were obtained from various samples (soil, mud, and water) of Surajkund hot spring, Jharkhand, at three different isolation temperatures of 50°C, 60°C, and 70°C. However, our interest was in the thermophilic strains that were isolated at 60°C and 70°C. Four isolates at 70°C (BITSNS038, BITSNS039, BITSNS040, BITSNS041) are the producers of thermozymes, namely amylase, xylanase, and cellulase, respectively. The highlights of the present study also showed that three out of four isolates demonstrated all three enzymatic activities, i.e. amylolytic, xylanolytic and cellulolytic on agar plate assay conditions at 70°C. One of the isolates, BITSNS038, was further chosen for phenotypic characterization as well as 16S rRNA gene sequencing and was affiliated to Geobacillus icigianus. The presence of Geobacillus icigianus was reported first time from hot spring, Surajkund, which showed amylolytic index of 1.58, xylanolytic index of 1.5 and cellulolytic index of 2.3 based on plate assay, and amylase activity of 0.81 U/mL, xylanase activity of 0.72 U/mL and very less cellulase activity of 0.15 U/mL after 24 h of growth in submerged conditions. One isolate at 60°C BITSNS024 was found to exhibit maximum amylase activity with an enzymatic index value of 3.5 and was identified as Anoxybacillus gonensis.

Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis.

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and ß-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

BACKGROUND: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. RESULTS: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs. CONCLUSIONS: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

Analysis of gallstone composition and structure in Jharkhand region.

AIM: The aim of this study was to analyze gallstones structurally and chemically as this may help to direct the measures for its treatment. METHODOLOGY: On the basis of morphology, 459 gallstones were categorized into pigmented, cholesterol, and mixed gallstones and analyzed for their chemical structure and composition. Elementary analysis was done with the help of inductively coupled plasma, optical emission spectrophotometry. Fourier transform infrared spectroscopy was used for compound analysis. The effects of thermal stability were analyzed by thermal gravimetric analysis (TGA). Statistical analysis was done to correlate gallstone composition with their shape and number in gallbladder. RESULTS: Pigmented gallstones were predominant and generally occurred in multiple forms, cholesterol as solitaire and black pigmented as slug (χ (2) = 3.56; p < 0.001). Scanning electron microscopy showed the crystals of cholesterol in cholesterol gallstones while pigmented gallstones were more compact in structure. Both types of structure were seen in mixed stones while black pigmented stones were amorphous in nature. This difference in structures might be due to difference in chemical compositions. Cholesterol and mixed stones contained basically cholesterol, brown pigmented constituted bilirubin as a major component, and black stones differed from brown pigmented stones by the presence of Ca palmitate. Bilirubin and palmitate were thermally more stable than cholesterol; hence pigmented gallstones were thermally more stable than cholesterol gallstones, as seen in TGA study. CONCLUSION: Pigmented gallstones were most common in this study of gallstones from the Jharkhand region.