Constructing a yeast to express the largest cellulosome complex on the cell surface.

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.

Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous ß-carotene and cellulase production.

Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the ß-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher ß-carotene (27.13 ± 0.66 mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous ß-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.

Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis.

BACKGROUND: Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis. RESULTS: According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon. CONCLUSIONS: In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.

Characterization of a novel exopolysaccharide produced by Lactobacillus gasseri FR4 and demonstration of its in vitro biological properties.

In this study, hetero-exopolysaccharide of Lactobacillus gasseri FR4 isolated from native chicken was produced and purified. The molecular weight of LgEPS was found to be 1.86×105Da. The gas chromatographic analysis revealed that the LgEPS was majorly composed of glucose (65.31%), mannose (16.51%), galactose (8.45%), rhamnose (6.55%) and a small fraction of fucose (3.18%). The functional groups of LgEPS were confirmed by FT-IR analysis. The 1D (1H and 13C) and 2D NMR (COSY and HSQC) analysis showed the presence of 1,6 linked-α-d-Glcp, 1,4 linked-α-d-Galp, 1,3,4 linked-α-d-Manp, 1,3 linked-α-l-Rhap, 1,4 linked-α-l-Fucp, 1,4 linked-ß-d-Glcp, and ß-d-Galp-1 residues. SEM and AFM micrographs revealed the fibrous and porous nature of LgEPS. Moreover, LgEPS exhibited in vitro antioxidant, antibacterial and antibiofilm activity against various food borne pathogens, which proved that this LgEPS might be used in food industries as an antioxidant agent, viscosifying agent and antimicrobial agent etc.

Characterization of Bile Salt Hydrolase from Lactobacillus gasseri FR4 and Demonstration of Its Substrate Specificity and Inhibitory Mechanism Using Molecular Docking Analysis.

Probiotic bacteria are beneficial to the health of poultry animals, thus are used as alternative candidates for antibiotics used as growth promoters (AGPs). However, they also reduce the body weight gain due to innate bile salt hydrolase (BSH) activity. Hence, the addition of a suitable BSH inhibitor along with the probiotic feed can decrease the BSH activity. In this study, a BSH gene (981 bp) encoding 326-amino acids was identified from the genome of Lactobacillus gasseri FR4 (LgBSH). The LgBSH-encoding gene was cloned and purified using an Escherichia coli BL21 (DE3) expression system, and its molecular weight (37 kDa) was confirmed by SDS-PAGE and a Western blot analysis. LgBSH exhibited greater hydrolysis toward glyco-conjugated bile salts compared to tauro-conjugated bile salts. LgBSH displayed optimal activity at 52°C at a pH of 5.5, and activity was further increased by several reducing agents (DTT), surfactants (Triton X-100 and Tween 80), and organic solvents (isopropanol, butanol, and acetone). Riboflavin and penicillin V, respectively, inhibited LgBSH activity by 98.31 and 97.84%. A homology model of LgBSH was predicted using EfBSH (4WL3) as a template. Molecular docking analysis revealed that the glycocholic acid had lowest binding energy of -8.46 kcal/mol; on the other hand, inhibitors, i.e., riboflavin and penicillin V, had relatively higher binding energies of -6.25 and -7.38 kcal/mol, respectively. Our results suggest that L. gasseri FR4 along with riboflavin might be a potential alternative to AGPs for poultry animals.

Physiochemical and biological characterization of novel exopolysaccharide produced by Bacillus tequilensis FR9 isolated from chicken.

Hetero-exopolysaccharide secreted by Bacillus tequilensis FR9 (BtqEPS), was produced and fractioned on DEAE-Sepharose and phenyl sepharose CL-6B column. HPLC analysis revealed the existence of five monosaccharides including glucose, galactose, mannose, arabinose and xylose. FT-IR spectroscopy confirmed the presence of carboxyl and hydroxyl groups. 1H NMR and 13C NMR spectra analysis showed the presence of α and ß-d-(+)-glucose residues and glycosidic linkages. The fibrous, porous nature and elemental composition (C, O, N, Cl, Na, P, S) of BtqEPS was inferred from SEM and EDX analysis. AFM proved that the micro-structure of BtqEPS is compact and rough. XRD analysis confirmed the amorphous nature of BtqEPS with 15.6% crystallinity index. TGA curve indicated the degradation temperature (Td) of 239.72°C. Furthermore, BtqEPS exhibited antioxidant effects by scavenging reactive oxygen species (ROS) and demonstrated strong reducing power. All the above findings on BtqEPS paves way to find novel insights of its potential applications in industries.

Purification of Antilisterial Peptide (Subtilosin A) from Novel Bacillus tequilensis FR9 and Demonstrate Their Pathogen Invasion Protection Ability Using Human Carcinoma Cell Line.

This study focuses on isolation, screening, and characterization of novel probiotics from gastrointestinal tract of free-range chicken (Gallus gallus domesticus). Fifty seven colonies were isolated and three isolates (FR4, FR9, and FR12) were selected and identified as Lactobacillus gasseri FR4, Bacillus tequilensis FR9, and L. animalis FR12 by 16S rRNA sequencing. Three strains were able to survive in stimulated acidic and bile conditions and inhibit the growth of pathogens. Especially, FR9 exhibited maximum inhibition against Listeria monocytogenes and none of them exhibited hemolytic activity. Native-PAGE revealed the presence of low molecular weight (3.4-5.0 KDa) antimicrobial peptide. The peptide was further purified by Sephadex G-50 column and RP-HPLC using C18 column. N-terminal amino acid sequencing of antimicrobial peptide showed 100% consensus to antilisterial peptide Subtilosin A and SboA gene was amplified from FR9 genome. FR9 showed maximum aggregation activity, exopolysaccharide production (85.46 mg/L) and cholesterol assimilation (63.12 ± 0.05 µg/mL). Strong adhesion property (12.6%) and pathogen invasion protection ability was revealed by B. tequilensis FR9 towards HCT-116 human colon carcinoma cell line. This is the first study to demonstrate antilisterial Subtilosin A production of B. tequilensis. Our results indicate that B. tequilensis FR9 strain furnish the essential characteristics of a potential probiotics and might be incorporated into human and animal food supplements.

Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste.

Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40 °C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60 °C and pH 10. Ca(2+), Mn(2+), and Mg(2+) ions activated the protease, while Hg(2+), Cu(2+), Zn(2+), and Fe(2+) greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca(2+), which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications.

Determining the probiotic potential of cholesterol-reducing Lactobacillus and Weissella strains isolated from gherkins (fermented cucumber) and south Indian fermented koozh.

This study sought to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from traditionally fermented south Indian koozh and gherkin (cucumber). A total of 51 LAB strains were isolated, among which four were identified as Lactobacillus spp. and three as Weissella spp. The strains were screened for their probiotic potential. All isolated Lactobacillus and Weissella strains were capable of surviving under low pH and bile salt conditions. GI9 and FKI21 were able to survive at pH 2.0 and 0.50% bile salt for 3 h without losing their viability. All LAB strains exhibited inhibitory activity against tested pathogens and were able to deconjugate bile salt. Higher deconjugation was observed in the presence of sodium glycocholate (P < 0.05). Strain FKI21 showed maximum auto-aggregation (79%) and co-aggregation with Escherichia coli MTCC 1089 (68%). Exopolysaccharide production of LAB strains ranged from 68.39 to 127.12 mg/L (P < 0.05). Moreover, GI9 (58.08 µg/ml) and FKI21 (56.25 µg/ml) exhibited maximum cholesterol reduction with bile salts. 16S rRNA sequencing confirmed GI9 and FKI21 as Lactobacillus crispatus and Weissella koreensis, respectively. This is the first study to report isolation of W. koreensis FKI21 from fermented koozh and demonstrates its cholesterol-reducing potential.

Fermented fruits and vegetables of Asia: a potential source of probiotics.

As world population increases, lactic acid fermentation is expected to become an important role in preserving fresh vegetables, fruits, and other food items for feeding humanity in developing countries. However, several fermented fruits and vegetables products (Sauerkraut, Kimchi, Gundruk, Khalpi, Sinki, etc.) have a long history in human nutrition from ancient ages and are associated with the several social aspects of different communities. Among the food items, fruits and vegetables are easily perishable commodities due to their high water activity and nutritive values. These conditions are more critical in tropical and subtropical countries which favour the growth of spoilage causing microorganisms. Lactic acid fermentation increases shelf life of fruits and vegetables and also enhances several beneficial properties, including nutritive value and flavours, and reduces toxicity. Fermented fruits and vegetables can be used as a potential source of probiotics as they harbour several lactic acid bacteria such as Lactobacillus plantarum, L. pentosus, L. brevis, L. acidophilus, L. fermentum, Leuconostoc fallax, and L. mesenteroides. As a whole, the traditionally fermented fruits and vegetables not only serve as food supplements but also attribute towards health benefits. This review aims to describe some important Asian fermented fruits and vegetables and their significance as a potential source of probiotics.

An ethnobotanical study of indigenous knowledge on medicinal plants used by the village peoples of Thoppampatti, Dindigul district, Tamilnadu, India.

ETHNOPHARMACOLOGICAL RELEVANCE: This study provides significant ethnopharmacological information, both qualitative and quantitative on medicinal plants in the Thoppampatti village, Dindigul district, Tamilnadu, India. There is urgency in recording such data to conserve the traditional medicinal plants. This is the first ethnobotanical study which records the traditional important medicinal plants of Thoppampatti village. To collect, analyze and evaluate the ethnopharmacologic knowledge in Thoppampatti village in order to protect it. This study reports the results of an ethnopharmacological survey on the uses of medicinal plants by inhabitants of the Thoppampatti. MATERIALS AND METHODS: The field study was carried out in a period of about one year (April 2012-May 2013) in Thoppampatti village. The information was obtained through open and semi-structured interviews with 48 (27 males, 21 females) knowledgeable local people and traditional healers (THs). The collected data were analyzed qualitatively and quantitatively. In addition, use value (UV), relative importance (RI) and Jaccard index (JI) was determined. RESULTS: A total of 139 species of plants, mostly trees and herbs, belonging to 54 families were identified in this study. This is used to treat 142 diseases and ailments. These ailments were categorized into 18 major categories. Leaves were the most frequently used parts, while decoction and juice are the most common method of preparation to treat various diseases. Based on IUCN Red data the identified medicinal plants include 11 least concerned species, 3 vulnerable species and one endangered species. The most important species according to their use value were Cynodon dactylon (0.79), Azadirachta indica (0.73), Ocimum tenuiflorum (0.71), Moringa oleifera (0.68), Coriandrum sativum (0.62), Abelmoschus esculentus (0.61), Acalypha indica (0.59) and Hibiscus rosa-sinensis (0.59). CONCLUSION: As a result of the present study, medicinal plants play an important role in the health care of Thoppampatti village inhabitants; they rely on medicinal plants to treat various ailments. In addition, the medicinal plants with high RI values might give some useful leads for further pharmacological investigations. Deforestation and overexploitation of a particular plant species leads to extinction so sustainable utilization of medicinal plants is recommended in the study area.

Isolation of potential probiotic Lactobacillus oris HMI68 from mother's milk with cholesterol-reducing property.

The objective of this study was to evaluate the probiotic properties of Lactobacillus strains isolated from mother's milk and their effects on cholesterol assimilation. In this study 120 isolates from mother's milk were phenotypically and genotypically characterized. Among these, only 6 predominant strains were identified as Lactobacillus spp. The following parameters were selected as important test variables in model stomach passage survival trials: acid and bile tolerance, antimicrobial activity, antibiotic susceptibility and cholesterol reduction. Results showed that the considerable variation existed among six strains. Moreover, the strain HMI68 is the most acid-tolerant and the HMI28 and HMI74 is the most acid-sensitive of all strains tested. HMI118 did not grow at 0.5% and 1% bile concentration after 5 h but the HMI68 and HMI43 showed some tolerance to such bile concentration. The differences found in the growth rate were not significant (P > 0.05). HMI68 showed resistance to most of the antibiotics as well as antagonistic activity against the tested pathogens. The amount of cholesterol reduction is increased when the media supplemented with bile salts. HMI68 assimilate 61.05 ± 0.05 µg/ml cholesterol with the presence of 0.3% bile salt this could be significantly decreased by 25.41 ± 1.09 µg/ml without bile salt. HMI68 was identified to be Lactobacillus oris HMI68 and 16S rRNA sequence was deposited in the National Center for Biotechnological Information (GenBank). For the first time the cholesterol-reducing property of L. oris isolated from mother's milk were investigated in this study. Therefore the effective L. oris HMI68 strain was regarded as a candidate probiotic.

A study on traditional medicinal plants of Uthapuram, Madurai District, Tamilnadu, South India.

OBJECTIVE: To record the medicinal plants of Uthapuram Village, Madurai district, Tamilnadu, South India for the first time and the usage of these medicinal plants to remediate the diseases among the peoples. METHODS: Explorative field trips were made to the village for about twelve months from April 2012 to May 2013 to survey the medicinal plants and collect the information from the villagers. RESULTS: From this study 52 species of valuable medicinal plants belonging to 36 families were recorded and their ethnomedicinal values were collected from the village peoples. CONCLUSION: This study focuses the importance, utilization and conservation of the medicinal plants among the people.