Crude Culture Research Articles

One-pot synthesis of stable vitamin C analogue using trans-glycosylating enzyme from Aspergillus carbonarius BTCF 5

Biocatalysis using transglycosidases for one-pot glycosylation of small molecules is a greener alternative to chemical glycosylation. The transglycosylation potential of the crude culture filtrate of Aspergillus carbonarius BTCF 5 was evaluated based on the biotransformation of the target molecule, ascorbic acid (Vitamin-C), to its more stable glycosylated analogue -2-O-α-D-glucopyranosyl ascorbic acid (AA2G), using maltose as the glycosyl donor. Media engineering increased the transglycosylation yield by 64%, whereas the use of concentrated culture filtrate increased the transglycosylation yield by 70%. Protein purification studies identified the candidate transglycosylating enzyme to be a GH 31 family CAZyme (α-transglucosidase). A minimally purified enzyme could enhance the transglycosylation yield by 77% with a total AA2G yield of 91.5 mM (10.32 g/L) and improved the overall conversion rate of ascorbic acid and maltose in the reaction by 16% and 6.3% respectively from the base level. A two-step column chromatography helped in product purification with 46% recovery. The structural characterization of purified AA2G provided a detailed insight into the regioselectivity of the enzyme to glycosylate C2 of ascorbic acid and its ability to form AA2G as the predominant isomer, which is also the most preferred one for end applications.

Biostimulant effect of green soil microalgae Chlorella vulgaris suspensions on germination and growth of wheat (Triticum aestivum var. Achtar) and soil fertility

Microalgae-derived biostimulants are increasingly used as viable tools to improve growth, yield of crops, and reduce agricultural environmental footprint. However, microalgae isolated from aquatic environments are the main strains used in the production of biostimulants. This study aims to evaluate the biostimulant effect of a terrestrial Chlorella vulgaris strain isolated from arid soil on germination and growth of wheat (Triticum aestivum, var. Achtar). Different concentrations 0.2 %, 0.5 %, 25 % and 50 % (v/v) of four C. vulgaris treatments (aqueous extract, filtered culture, crude culture and sonified culture) were investigated by measuring several biometric parameters to assess wheat germination and growth performance. All C. vulgaris treatments significantly enhanced wheat germination and growth with concentration dependent effect. The crude culture and filtrate at 25 % were the most effective compared to untreated plants. They significantly improved wheat germination index by 16.86 %, germination rate coefficient by 7.87 %, germination energy by 20.40 % as well as growth parameters such as root length (62.82 % and 52.84 %), stem length (71.36 % and 59.19 %), and root (662.5 % and 391.67 %) and stem dry biomass (675 % and 370.83 %), respectively. Principal Component Analysis (PCA) revealed that crude culture and filtrate at the lowest doses of 0.5 and 25 % were closely correlated to enhanced wheat root and shoot length and dry weight. The plant nutrients, mineral elements, chlorophyll a, total proteins and carbohydrates contents were positively correlated with enhanced shoot length and dry weight. The soil physicochemical properties were significantly increased under all C. vulgaris treatments at higher dose (50 %) whereas plants N and P contents enhancement was correlated to the soil nutrients increase. Thus, the results highlighted the potential of soil microalgae as an eco-friendly biostimulant for promoting sustainable agriculture.

Biohydrogen production by a novel strain Petroclostridium sp. X23 isolated from the production water of oil reservoirs

Biohydrogen production from H2-producing has been considered a promising alternative for the replacement of fossil fuels. However, the mechanisms that affect the H2-producing capacities of wild-type strains remain largely elusive. To explore the capabilities of biohydrogen production from microorganisms in the deep subsurface oil-reservoir environment, we isolated an H2-producing bacteria identified as the genus Petroclostridium from a long-term methanogenic crude oil-degrading enrichment culture of the production water from oil reservoir. The impact of various temperature, pH, and glucose concentrations on hydrogen production by Petroclostridium sp. X23 was investigated. Under the conditions of 37 °C, initial pH 8.0, and glucose concentration of 4 g/L, the strain X23 achieved its maximum hydrogen yield at 1.82 ± 0.12 mol H2/mol glucose. When compared to the previously reported strain SK-Y3, X23 exhibited enhanced H2-producing ability, with lesser energy biomass production. Comparative genomic analysis revealed that the genome of X23 contains more genes associated with hydrogen generation than SK-Y3. These findings proposed Petroclostridium sp. X23 is a potential candidate for dark fermentative hydrogen production under alkaline and mesophilic conditions. It also increased our knowledge of genotypes related to high H2-producing capability, which may provide instructions for future selection of high-yielding strains and reasonable metabolic engineering of wild-type H2-producing strains.

Characterization of Extracellular Chitinase from Bacillus cereus SAHA 12.13 and Its Potency as a Biocontrol of Curvularia affinis

Bacillus cereus SAHA 12.13 can produce chitinase, an enzyme that digests chitin in the main compounds of cell walls, mycelia, and spores in pathogenic fungi that cause leaf spots on oil palm plants such as Curvularia affinis. This study aims to determine the properties of the chitinase enzyme B. cereus SAHA 12.13 that can inhibit the growth of C. affinis. Chitinase enzyme production and characterization were measured using the Spindler method. Antagonism test against pathogenic fungi using dual culture method by testing cell culture and enzyme crude extract. This result showed that the isolate produced a high level of specific chitinase activity at 37 °C for 45 h of incubation with 8.45 U mg-1 proteins with a growth rate (k) of 0.25 generation/h, and the generation time was 3.96 h/generation. The optimum chitinase activity was achieved at pH 7.0 and 45 °C and was stable for 3 h with a half-life (t1/2) of 770 min. The crude enzyme and cell culture of strain can inhibit the growth of C. affinis by 36.27±0.043% and 34.25±0.041%, respectively. These characteristics indicate that B. cereus strain SAHA12.13 can be used to inhibit C. affinis, which causes leaf blight of oil palm, under varying pH and temperature conditions.

Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System.

To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 106 cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.

Production of high-value oxidative enzymes by Cyathus bulleri on agricultural and agri-food wastes for application in the textile sector.

Ligninolytic and other oxidative enzymes have emerged as promising biocatalysts in several industries. Since their production at a low cost is necessary for any large-scale application, we demonstrate the use of rice bran (RB), an agricultural waste and agri-food wastes such as potato peelings (PP), banana peelings (BP), and green pea peelings (GPP) for their production. High activity of laccase (12 U/ml), manganese peroxidase (16.11 ± 1.43 U/ml), and aryl alcohol oxidase (1.25 U/ml) was obtained on the PP on the 12th day of growth and ~ 6 U/ml of lytic polysaccharide monooxygenase was obtained on the 14th day of growth demonstrating PP to be a good substrate for their production. RB served as the next best substrate for the production of these enzymes. While the GPP was effective for the production of laccase (9.2 U/ml), this and the BP were not good substrates for the production of other enzymes. Efficient (48-82%) decolorization of several azo-, triarylmethane- dyes, and real textile effluent, without the addition of any mediator, demonstrated the high oxidative ability of the crude culture filtrate produced on the PP (CF-PP), which was a significant improvement compared to the treatment given by the previously reported culture filtrate obtained on wheat bran (CF-WB). An extensive breakdown of Reactive Orange (RO) 16 was demonstrated using CF-PP resulting in the formation of a new product at m/z of 294.05 (6-acetamido-3,4-dioxo-3,4-dihydronapthalene-2-sulfonate), previously reported to be produced on ozonation/advanced oxidation of RO16. The predominant laccase and manganese peroxidase isoforms produced on the PP were also identified.

Integration of a perfusion reactor and continuous precipitation in an entirely membrane-based process for antibody capture.

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.

High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium

Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6–8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.

Genome-wide discovery of Pichia galeiformis-secreted proteins and their induction of green mold resistance in citrus fruit

Microbe-secreted proteins could alter plant defense systems, but yeast-secreted proteins and their impact on plant disease resistance remains uninvestigated. As our team had previously found that Pichia galeiformis (P. galeiformis) induced citrus green mold resistance, the purpose of this study was to identify its secreted proteins and their role in disease resistance. The genome of P. galeiformis was completely sequenced, and 4386 putative protein-coding genes were identified. The crude culture of P. galeiformis contained 1443 proteins. Among these proteins, 55 were identified as secreted proteins with an N-terminal signal peptide and lacking transmembrane structural domain. Overexpression of secreted proteins 2-gene0581, 22-gene0896, 23-gene0290, 12-gene3564, 9-gene0639, and 15-gene0649 significantly reduced citrus fruit disease incidence and lesion diameter. And the best effect was achieved by 15-gene0649 treatment. Notably, P. galeiformis 15-gene0649, which was named PgSCP, encoded a novel protein. The purified protein PgSCP demonstrated considerable ability to prevent green mold caused by Penicillium digitatum in citrus. This study presented here confirms that yeast could increase citrus green mold resistance through secreted proteins. Those secreted proteins offer a fresh insight for controlling postharvest disease in citrus fruit as well as a new commercialization and application method for antagonistic yeast.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

BackgroundFilamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.ResultsA novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.ConclusionsThis PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Metabolites Composition of Bacillus subtilis HussainT-AMU Determined by LC-MS and Their Effect on Fusarium Dry Rot of Potato Seed Tuber

Fusarium dry rot is considered to be the most critical soilborne and postharvest disease that damages potato tubers worldwide when they are stored for a long time. This study was performed to demonstrate the effect of crude extract, culture filtrate, and cell suspension obtained from the bacterium Bacillus subtilis HussainT-AMU, on the net house and field. From oil-contaminated soil, through the serial dilution method, biosurfactant bacteria were isolated on nutrient agar medium. To isolate and screen the prospective biosurfactant strain, various biosurfactant screening methods were used. Standard protocols were carried out for morphological, molecular, and chemical characterization. The disease incidences were significantly decreased under net house and field conditions after treatment with biosurfactant extract (20%, 70%), culture filtrate (23%, 49%), and bacterial cell suspension (46%, 27%), respectively. These results indicate a significant effect of B. subtilis HussainT-AMU against Fusarium oxysporum. The characterization of the partially purified extract by gas-chromatography indicated the existence of a high amount of methyl ester fatty acids. The quantitative analysis determined the presence of lipopeptide-surfactin, fengycin, and iturin in the biosurfactant extract. Scanning electron microscopy revealed that morphological variation was revealed in the structure of stomata size in leaves and in the mycelia of the pathogen that was investigated. This promising biocontrol agent can be a plausible option for further research and can also be used for the production of green biomolecules at an additional industrial level.

EasyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria.

The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.

Characterization of chaetoglobosin producing Chaetomium globosum for the management of Fusarium-Meloidogyne wilt complex in tomato.

Simultaneous management of FOL and RKN causing wilt complex in tomato by chaetoglobosin-producing Chaetomium globosum. Random survey was carried out to isolate Fusarium and Chaetomium. Twelve Fusarium isolates were characterized, and FOL4 (virulent) was molecularly identified. Wilt complex by FOL, RKN was assessed individually and in combination under greenhouse. RKN (1000 juveniles ml-1) inoculation followed by FOL4 (5×105 spores ml-1) accounted for 90% incidence. The chaetoglobosin-producing Chaetomium was isolated, characterized morphologically and molecularly. Among 55 isolates, nine showed >50% inhibition against FOL, and crude culture filtrate showed a significant reduction in RKN egg hatching (15.66%) and juvenile mortality (100%). Chaetomium Cg 40 was confirmed as C. globosum using SCAR marker (OK032373). Among 40 volatile compounds, hexadecanoic acid and 1,2-epoxy-5,9-cyclododecadiene exhibited antifungal and nematicidal properties in GC-MS. High-performance liquid chromatography revealed chaetoglobosin A (0.767μg μl-1), and the presence of bioactive molecules chaetoglobosin (528.25m/z), chaetomin (710m/z), chaetocin (692.8m/z), chaetoviridin (432.85m/z), and chaetomugilin (390m/z) was confirmed by LC/MS/MS. Cg 40 and Cg 6 were able to synthesize the pks1a, b gene responsible for chaetoglobosin, sporulation, and melanin biosynthesis was confirmed by PCR. The application of an aqueous formulation as seed treatment, seedling dip, and soil drenching (application) recorded lowest wilt incidence (11.11%) and gall index (1) with the maximum growth parameter (plant height 51.9cm), fruit yield (287.5g), and lycopene content (11.46mg/100g). Cg 40 and Cg 6, containing polyketides, secondary metabolites, antibiotics, chaetoglobosin, and plant growth-promoting ability, showed antifungal and nematicidal properties against the FOL-RKN wilt complex in tomato in vitro and pot culture experiments.

Biological control of fall webworm larva (Hyphantria cunea Drury) and growth promotion of poplar seedlings (Populus × canadensis Moench) with Bacillus licheniformis PR2

The production of Populus × canadensis Moench seedling, as an important restoration resource has been constrained by the invasion of Hyphantria cunea Drury, a polyphagous pest that causes leaf feeding damage on nursery seedling production. This study investigated the larvicidal potential of cuticle degrading chitinase and protease enzymes secreted by the Bacillus licheniformis PR2 against H. cunea larvae. In addition, we investigated the growth promotion of P. canadensis seedling through the auxin belonging to plant growth hormones. Treatment of H. cunea larvae with either the bacterial crude enzymes or bacterial culture broth from B. licheniformis PR2 under laboratory conditions resulted in larvae mortality due to the severe degradation of the cuticle characterized by the loss of setae, swelling of sockets, and rupturing of the epicuticle and procuticle, and also caused a repellent effect from leaves, when the larvae suffered minor lacerations only in the epicuticle except the procuticle. Application of B. licheniformis PR2 culture broth on P. canadensis seedlings in the greenhouse experiment caused a mortality rate of 34.4 % and a repellency rate of 75.0 % against H. cunea larvae and reduced the leaf damage rate by 3.7-folds to only 25.9 % compared to 96.3 % in the control group. Accordingly, treatment with B. licheniformis PR2 improved seedling growth by reducing foliar damage caused by H. cunea larvae. In addition, B. licheniformis PR2 produced indole-3-acetic acid (IAA), which improves root hair growth and lateral root development to increase the surface area for nutrient uptake. Thus, inoculation of B. licheniformis PR2 improved the leaf chlorophyll content (photosynthetic rate) of the P. canadensis seedlings by 1.9-folds to 43.26 units compared to 23.26 units in the control group. Consequently, the application of B. licheniformis PR2 also improved the shoot, root, and leaf dry weights by 3.4-, 1.5- and 1.6-folds compared to the control group, respectively. Therefore, this study demonstrates the dual larvicidal and plant growth-promoting potential of B. licheniformis PR2, as an environmentally friendly alternative to chemical pesticides in the control of H. cunea larvae and production of healthy P. canadensis seedlings in the forest nursery.

An antibacterial compound pyrimidomycin produced by Streptomyces sp. PSAA01 isolated from soil of Eastern Himalayan foothill

Selective isolation of soil Actinobacteria was undertaken to isolate a new class of antibiotics and bioactive molecules. A Streptomyces sp. PSAA01 (= MTCC 13,157), isolated from soil of Eastern Himalaya foothill was cultivated on a large scale for the production of the antimicrobial SM02. It has been found that the maximum amount of SM02 produced while PSAA01 was grown in ISP-2 medium (pH 7.0) for 7 days at 30 °C in shaking (180 rpm) condition. A significant zone of inhibition against Staphylococcus aureus MTCC 96 has been found with the crude cell-free culture media (50 µL) of 7 days grown PSAA01. After the purification and chemical structural characterization, we found that SM02 is a new antimicrobial having 746 dalton molecular weight. The compound SM02 contains pyrimidine moiety in it and is produced by a species of Streptomyces and thus we have named this antibiotic pyrimidomycin. The antimicrobial spectrum of pyrimidomycin has been found to be restricted in Gram-positive organisms with a MIC of 12 µg/mL. SM02 was found active against Mycobacterium sp. and also multi-drug resistant Gram-positive bacteria with similar potency and found to disrupt the bacterial cell wall. Pyrimidomycin also showed significant impairment in the biofilm formation by S. aureus. Furthermore, pyrimidomycin showed synergy with the most used antibiotic like ampicillin, vancomycin and chloramphenicol. Pyrimidomycin did not have cytotoxicity towards human cell lines indicating its limited activity within bacteria.

Extracellular Secretion and Simple Purification of Bacterial Collagen from Escherichia coli

Because of structural similarities with type-I animal collagen, recombinant bacterial collagen-like proteins have been progressively used as a source of collagen for biomaterial applications. However, the intracellular expression combined with current costly and time-consuming chromatography methods for purification makes the large-scale production of recombinant bacterial collagen challenging. Here, we report the use of an adapted secretion pathway, used natively byEscherichia colito secrete curli fibers, for extracellular secretion of the bacterial collagen. We confirmed that a considerable fraction of expressed collagen (∼70%) is being secreted freely into the extracellular medium, with an initial purity of ∼50% in the crude culture supernatant. To simplify the purification of extracellular collagen, we avoided cell lysis and used cross-flow filtration or acid precipitation to concentrate the voluminous supernatant and separate the collagen from impurities. We confirmed that the secreted collagen forms triple helical structures, using Sirius Red staining and circular dichroism. We also detected collagen biomarkers via Raman spectroscopy, further supporting that the recombinant collagen forms a stable triple helical conformation. We further studied the effect of the isolation methods on the morphology and secondary structure, concluding that the final collagen structure is process-dependent. Overall, we show that the curli secretion system can be adapted for extracellular secretion of the bacterial collagen, eliminating the need for cell lysis, which simplifies the collagen isolation process and enables a simple cost-effective method with potential for scale-up.

Morphological and molecular reinvestigation of acanthoecid species I. – Enibas urnula (Thomsen, 1973) comb. nov. (=Stephanoeca urnula Thomsen, 1973)

In the course of the last decades, several acanthoecid choanoflagellates have been assigned to the tectiform reproducing genus Stephanoeca based on lorica morphological comparability. The recent description of a Stephanoeca-like nudiform genus, Enibas Schiwitza, Arndt and Nitsche, 2019, has emphasized the possibility that some species assigned to Stephanoeca (based solely on lorica morphological features) might in the end have to be relegated to other genera. Stephanoeca urnula Thomsen, 1973, shows a striking morphological resemblance to E. thessalia Schiwitza and Nitsche, 2020. Also, the inability to document from microscopy any bundles of supernumerary costal strips in the collar region of this species, makes it an obvious Stephanoeca outcast candidate. We have revisited the S. urnula type locality and established this species in crude culture. A detailed SEM morphological analysis of cultured specimens, and a molecular analysis of the SSU and LSU rRNA, has unveiled its undisputed affiliation to the nudiform genus Enibas. Here, we redescribe S. urnula Thomsen, 1973 as E. urnula (Thomsen, 1973) comb. nov. Based on microscopical examinations of species of Stephanoeca and Enibas we have been able to pinpoint anterior lorica chamber construction details, that in the future renders possible a morphological distinction between the two genera.

Structural, phylogenetic and expression analysis of genes producing lignin degrading enzymes in the basidiomycete Cyathus bulleri and their effect on the release of reducing sugars from agro-residues

One of the major impediments in the use of agricultural by-products, including rice husk and wheat straw, as a raw material for biofuel production is the availability of a suitable biological method for delignification. The current physical, chemical and physico-chemical methods are harsh, expensive and are accompanied by the release of toxic end-products such as furan derivatives, weak carboxylic acids and phenolic compounds. The aim of this study was to explore the lignin degrading enzymes of Cyathus bulleri, an avid lignin degrader, for their use in delignification of agricultural by-products. A total of 13 laccase (Lcc) encoding and 6 manganese peroxidase or MnP (MnP) encoding genes were identified in the draft genome of C. bulleri. The Lcc genes could be sub-divided in to 3 classes based on their structural organization whereas no common structures were identified in the MnPs. Similarity at the genomic level for all laccases (except Lcc12) indicated their evolution as a result of gene duplication and these appeared to be very closely related based on the phylogenetic relationship. All the copper binding domains and the heme signature sequences were conserved in the laccases and the MnPs respectively. A transmembrane helix was predicted at the C-terminus of Lcc12 suggesting its localization on the mycelial surface. A temporal analysis of the transcription of Lcc and MnP genes was carried out on wheat bran (WB) and the data indicated Lcc1 to be transcribed the most on Day 4 while Lcc12 was transcribed during later stages of growth. The crude culture filtrate obtained from WB-grown fungus released reducing sugars from rice husk (∼18.5 mg/g of rice husk) and wheat straw (∼9.5 mg/g of WS) indicating effective delignification as well as saccharification of these materials.

Extracellular resveratrol producing endophytic fungus, Quambalaria cyanescens

A fungal endophyte of Vitis vinifera, Quambalaria cyanescens is shown to produce high amount of resveratrol, a well-known anti-cancer agent in culture media. The ethyl acetate extract of the crude culture filtrate yielded 240 mg of which 40 mg of purified Resveratrol was recovered. The endophyte is first of its kind to be reported from Vitis vinifera, and was identified by taxonomic and molecular phylogenetic methods. The fungal resveratrol exhibited a potent cytotoxic activity against A549, with 82% reduction in the cell count. It also exhibited a potent anti-oxidant activity with 83% radical scavenging activity. Further, the fungal resveratrol from Q. cyanescens also exhibited a potent activity against methicillin resistant Staphylococcus aureus. Thus, this endophytic Quambalaria cyanescens holds a promise for development into a commercial strain for fermentative production of resveratrol.