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.

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.

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.