Drug resistance through ribosome splitting and rRNA disordering in mycobacteria.

HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX-mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic-bound inactive 50S subunits.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture.

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate.

A novel Lip+ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip+Amy+host. Lipase hydrolyzed methyloleate into methanol that sustained PAOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip+Amy+ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy+ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip+ host to validate disparity between lipase background and PAOX1 induction in presence of methyloleate.

Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity.

Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett-Burman (PB). Of these, yeast extract, MgSO4 and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO4 (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC-MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.

Disrupting putative N-glycosylation site N17 in lipase Lip11 of Yarrowia lipolytica yielded a catalytically efficient and thermostable variant accompanying conformational changes.

Lip11 gene from oleaginous yeast Yarrowia lipolytica MSR80 was recombinantly expressed in Pichia pastoris X33. Native secretion signal present in its sequence resulted in 92 % expression in comparison to α-secretion factor which resulted to 900 U/L in the extracellular broth. Catalytic triad in Lip11, like most lipases, was formed by serine, histidine, and aspartate residues. While point mutation disrupting putative glycosylation site (N389) present towards the C-terminus ruinously effected its stability and catalytic activity, disruption of the first putative glycosylation site (N17) located towards the N-terminus presented interesting insights. Mutation resulted in a variant N1 exhibiting higher thermal and acid stability; a t1/2 of 198 min was obtained at 50 °C and it retained almost 80 % activity following incubation at pH 3. Catalytic efficiency was improved by 2.7 fold and a 10 °C rise in temperature optima was accompanied by higher relative activity in acidic range. Thermal stability corresponded to convoying structural modifications in the tertiary structure, findings of fluorescence spectroscopy suggested. Thermal fluorescence studies revealed a Tm of 65 °C for both Lip11 and N1 and λmax of Lip11 exhibited a blue shift upon refolding while no shift in the λmax of N1 was observed. A resilient tertiary structure which could fold back to its native confirmation upon thermal denaturation and increase in surface-exposed hydrophobic residues as revealed by ANS binding assay summed up to thermal stability of N1. Furthermore, circular dichroism data disclosed an alternate ratio of alpha-helices and beta-sheets; respective values changed from 36 % and 8%-27% and 19 %. Following mutation, substrate specificity remained unaffected and similar to native protein, N1 showed activation in presence of organic solvents and most divalent cations.

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications.

Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.

DS86760016, a Leucyl-tRNA Synthetase Inhibitor with Activity against Pseudomonas aeruginosa.

DS86760016 is a new leucyl-tRNA-synthetase inhibitor at the preclinical development stage. DS86760016 showed potent activity against extended-spectrum multidrug-resistant Pseudomonas aeruginosa isolated from clinical samples and in vitro biofilms. In a murine catheter-associated urinary tract infection model, DS86760016 treatment resulted in significant eradication of P. aeruginosa from the kidney, bladder, and catheter without developing drug resistance. Our data suggest that DS86760016 has the potential to act as a new drug for the treatment of Pseudomonas infections.

Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.