Nanocellulose-Based Ecofriendly Nanocomposite for Effective Wastewater Remediation: A Study on Its Process Optimization, Improved Swelling, Adsorption, and Thermal and Mechanical Behavior.

A nanocellulose (NC)-based cross-linked adsorbent has been employed herein for the removal of dye pollutants (e.g., methylene blue) from the textile industry. The synthesized hydrogel was optimized to achieve the best concentrations of the adsorbent constituents, i.e., 1.55% guar gum, 1.46% NC, and 0.84% borax for achieving the maximum swelling index (SI, 3741.42%) and higher adsorption capacity (qe, 24.05 mg g-1). 98.8% of dye qe was achieved at optimal conditions of pH 8 within 30 min at 30 °C. Adsorption isotherms and kinetics investigations showed good correlation with the Freundlich adsorption isotherm model (R2 > 0.9889; ΔG° = -4.71; ΔH° = -12.30; ΔS° = -0.025) as well as the pseudo-second-order kinetics model, indicating multilayered and intricate adsorption mechanisms for dye removal. The study of thermodynamic parameters confirmed the exothermic nature of the adsorption process. The adsorption-desorption study of the resulting hydrogel exhibited 64.58% dye removal efficiency even after 4 consecutive cycles of reuse. Further, scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction analysis revealed the surface morphology, functional moieties, thermal behavior, and crystallinity pattern of the hydrogel. Rheological analysis demonstrated pseudoplastic flow and improved mechanical behavior for the hydrogel. The current study found that the synthesized adsorbent with a higher SI and qe has a noticeable potential for the removal of dye pollutants from wastewater.

Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin.

Roseocin, the two-peptide lantibiotic from Streptomyces roseosporus, carries extensive intramolecular (methyl)lanthionine bridging in the peptides and exhibits synergistic antibacterial activity against clinically relevant Gram-positive pathogens. Both peptides have a conserved leader but a diverse core region. The biosynthesis of roseocin involves post-translational modification of the two precursor peptides by a single promiscuous lanthipeptide synthetase, RosM, to install an indispensable disulfide bond in the Rosα core along with four and six thioether rings in Rosα and Rosß cores, respectively. RosM homologs in the phylum actinobacteria were identified here to reveal twelve other members of the roseocin family which diverged into three types of biosynthetic gene clusters (BGCs). Further, the evolutionary rate among the BGC variants and analysis of variability within the core peptide versus leader peptide revealed a phylum-dependent lanthipeptide evolution. Analysis of horizontal gene transfer revealed its role in the generation of core peptide diversity. The naturally occurring diverse congeners of roseocin peptides identified from the mined novel BGCs were carefully aligned to identify the conserved sites and the substitutions in the core peptide region. These selected sites in the Rosα peptide were mutated for permitted substitutions, expressed heterologously in E. coli, and post-translationally modified by RosM in vivo. Despite a limited number of generated variants, two variants, RosαL8F and RosαL8W exhibited significantly improved inhibitory activity in a species-dependent manner compared to the wild-type roseocin. Our study proves that a natural repository of evolved variants of roseocin is present in nature and the key variations can be used to generate improved variants.

Thiopeptides encoding biosynthetic gene clusters mined from bacterial genomes.

Currently, we are at the threshold of the 'post-antibiotic era' due to the global emergence of antimicrobial resistance (AMR), and hence there is a dire need to discover new antibiotics. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse class of natural products (NPs), some of which are under clinical trials for their antimicrobial potential. Thiopeptides are structurally one of the most complex classes of RiPPs due to numerous post-translational modifications (PTMs), with [4+2] cycloaddition being the core PTM and are active against several gram-positive pathogens. Genome mining coupled with experimental work can harness the unexplored 'cryptic' gene clusters while minimizing the rate of the rediscovery of known metabolites and expand the molecular diversity of NPs with medicinal potential. Employing the genome mining approach using a series of freely available bioinformatics tools, we have identified eight novel putative thiopeptide encoding biosynthetic gene clusters (BGCs) from different bacterial genomes, most of which belong to the class Actinobacteria. Our results provide confidence in the newly identified BGCs, to proceed with wet-bench experiments and discover novel thiopeptide(s).

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Bioinformatics approach to understand nature's unified mechanism of stereo-divergent synthesis of isoprenoid skeletons.

In isoprenoid metabolism, cyclisation is the important gateway to chemical diversity. Terpene synthase is responsible for the cyclisation of a few universal substrates forming hundreds of often stereo-chemically complex mono- and poly-cyclic terpene hydrocarbons with a broad spectrum of functions in pharmaceuticals, flavours and fragrance industry. Although they are discovered and characterised mainly from plants and fungi, yet only a small share of bacterial terpenes has been investigated so far owing to their low level of expression in wild-type microorganisms. Extensive bacterial genome mining has revealed a treasure trove of terpene synthase genes and their regulated heterologous overexpression has pitched-in to describe the biochemical function of putative genes and sequester new terpene metabolites. This review deals with the modern genome mining techniques and molecular methods, providing more experimental tools for studying the structure and functions of terpenoid metabolites and strongly supports the idea that genome mining is a utile approach in deciphering the terpenoid diversity in bacteria.

Roseocin, a novel two-component lantibiotic from an actinomycete.

Lantibiotics are lanthionine ring containing natural products that belong to the class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Recent expansion in the availability of microbial genome data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic, roseocin, was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Posttranslationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in Escherichia coli, and were in vitro reconstituted to their bioactive form by exploiting commercial proteases like endoproteinase GluC, and proteinase K. The two peptides displayed synergistic antimicrobial activity against Gram-positive bacteria including the WHO high-priority pathogens, MRSA and VRE. Structural characterization confirmed the installation of four (methyl)lanthionine rings with an indispensable disulfide bond in the α-peptide, and six (methyl)lanthionine rings in the ß-peptide, by a single promiscuous lanthionine synthetase, RosM. Roseocin is the first two-component lantibiotic from a non-Firmicute, with extensive lanthionine bridging.

Electrochemical determination of M. tuberculosis antigen based on Poly(3,4-ethylenedioxythiophene) and functionalized carbon nanotubes hybrid platform.

An electrochemical DNA aptasensor for the detection of Mycobacterium tuberculosis (M. tb) antigen MPT64, was developed using Poly(3,4-ethylenedioxythiophene) (PEDOT) doped with carbon nanotubes (CNTs). The biotinylated aptamer was immobilized onto streptavidin attached to -COOH functionalized CNTs via streptavidin-biotin interaction. Various characterization studies as FT-IR, FE-SEM, EIS and DPV were done to validate each fabrication step of the aptasensor. Optimization studies related to aptamer concentration and response time were performed. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically by differential pulse voltammetry in the presence of [Fe(CN)6]3-/4- as a redox probe. The aptasensor exhibited limit of detection of 0.5±0.2fgmL-1 within 15min with stability of 27 days at 4°C and reusability of 7 times after repeated regeneration with 50mM NaOH. The potential application of the aptasensor was established by spike-in studies to obtain recovery in between (88-95)%.

Bioresolution of racemic phenyl glycidyl ether by a putative recombinant epoxide hydrolase from Streptomyces griseus NBRC 13350.

In order to produce enantiomerically pure epoxides for the synthesis of value-added chemicals, a novel putative epoxide hydrolase (EH) sgeh was cloned and overexpressed in pET28a/Escherichia coli BL21(DE3). The 1047 bp sgeh gene was mined from Streptomyces griseus NBRC 13350 genome sequence. The recombinant hexahistidyl-tagged SGEH was purified (16.6-fold) by immobilized metal-affinity chromatography, with 90% yield as a homodimer of 100 kDa. The recombinant E. coli whole cells overexpressing SGEH could kinetically resolve racemic phenyl glycidyl ether (PGE) into (R)-PGE with 98% ee, 40% yield, and enantiomeric ratio (E) of 20. This was achieved under the optimized reaction conditions i.e. cell/substrate ratio of 20:1 (w/w) at pH 7.5 and 20 °C in 10% (v/v) dimethylformamide (DMF) in a 10 h reaction. 99% enantiopure (R)-PGE was obtained when the reaction time was prolonged to 12 h with a yield of 34%. In conclusion, an economically viable and environment friendly green process for the production of enantiopure (R)-PGE was developed by using wet cells of E. coli expressing recombinant SGEH.

An Overview on the Enhancement of Enantioselectivity and Stability of Microbial Epoxide Hydrolases.

Epoxide hydrolases (EHs; 3.3.2.x) catalyze the enantioselective ring opening of racemic epoxides to the corresponding enantiopure vicinal diols and remaining equivalent unreacted epoxides. These epoxides and diols are used for the synthesis of chiral drug intermediates. With an upsurge in the methods for identification of novel microbial EHs, a lot of EHs have been discovered and utilized for kinetic resolution of racemic epoxides. However, there is still a constraint on the account of limited EHs being successfully applied on the preparative scale for industrial biotransformations. This limitation has to be overcome before application of identified functional EHs on large scale. Many strategies such as optimizing reaction media, immobilizing EHs and laboratory-scale directed evolution of EHs have been adopted for enhancing the industrial potential of EHs. In this review, these approaches have been highlighted which can serve as a pathway for the enrichment of already identified EHs for their application on an industrial scale in future studies.

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product.

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

Trigger factor assisted folding of the recombinant epoxide hydrolases identified from C. pelagibacter and S. nassauensis.

Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85µmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases.

Novel LanT associated lantibiotic clusters identified by genome database mining.

BACKGROUND: Frequent use of antibiotics has led to the emergence of antibiotic resistance in bacteria. Lantibiotic compounds are ribosomally synthesized antimicrobial peptides against which bacteria are not able to produce resistance, hence making them a good alternative to antibiotics. Nisin is the oldest and the most widely used lantibiotic, in food preservation, without having developed any significant resistance against it. Having their antimicrobial potential and a limited number, there is a need to identify novel lantibiotics. METHODOLOGY/FINDINGS: Identification of novel lantibiotic biosynthetic clusters from an ever increasing database of bacterial genomes, can provide a major lead in this direction. In order to achieve this, a strategy was adopted to identify novel lantibiotic biosynthetic clusters by screening the sequenced genomes for LanT homolog, which is a conserved lantibiotic transporter specific to type IB clusters. This strategy resulted in identification of 54 bacterial strains containing the LanT homologs, which are not the known lantibiotic producers. Of these, 24 strains were subjected to a detailed bioinformatic analysis to identify genes encoding for precursor peptides, modification enzyme, immunity and quorum sensing proteins. Eight clusters having two LanM determinants, similar to haloduracin and lichenicidin were identified, along with 13 clusters having a single LanM determinant as in mersacidin biosynthetic cluster. Besides these, orphan LanT homologs were also identified which might be associated with novel bacteriocins, encoded somewhere else in the genome. Three identified gene clusters had a C39 domain containing LanT transporter, associated with the LanBC proteins and double glycine type precursor peptides, the only known example of such a cluster is that of salivaricin. CONCLUSION: This study led to the identification of 8 novel putative two-component lantibiotic clusters along with 13 having a single LanM and 3 with LanBC genes. Putative lantibiotic clusters identified here hold the potential for the discovery of novel lantibiotic(s).

Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34.

A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ∼2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.

A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening.

Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM.

Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman.

Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production. A targeted disruption of the native mshC gene in M. tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH. However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained. These clones produced normal levels of MSH. These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M. tuberculosis Erdman under laboratory conditions.

ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low molecular weight thiol in mycobacteria. The mycothiol biosynthetic pathway has been postulated to involve ATP-dependent ligation of L-cysteine (Cys) with 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside; GlcN-Ins) catalyzed by MshC to produce Cys-GlcN-Ins. The ligase activity was purified approximately 2400-fold from Mycobacterium smegmatis and two proteins of slightly different M(r) approximately 47000 were identified with MshC activity. The N-terminal sequence of the smaller protein revealed that it was coded by a gene in the databases for M. smegmatis and M. tuberculosis previously designated as cysS2. The larger protein was coded by the same gene in M. smegmatis but included an eight amino acid N-terminal extension involving a different start codon. The ligase was found to have K(m) values of 40 +/- 3 and 72 +/- 9 microM for Cys and GlcN-Ins, respectively. The cysS2 gene was thought to encode a second cysteinyl-tRNA synthetase in addition to cysS but the present results indicate that cysS2 is actually the mshC gene encoding ATP-dependent Cys:GlcN-Ins ligase.