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Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.

β-Mannans are a heterogeneous group of polysaccharides with a common main chain of β-1,4-linked mannopyranoside residues. The cleavage of β-mannan chains is catalyzed by glycoside hydrolases called β-mannanases. In the CAZy database, β-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides., Competing Interests: The authors have declared that no competing interests exist.

Objective: O-linked N-acetylglucosaminylation (O-GlcNAcylation), controlled by O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), is an important post-translational modification of eukaryotic proteins and plays an essential role in regulating gut inflammation. Gut microbiota encode various enzymes involved in O-GlcNAcylation. However, the characteristics, abundance and function of these enzymes are unknown., Design: We first investigated the structure and taxonomic distribution of bacterial OGAs and OGTs. Then, we performed metagenomic analysis to explore the OGA genes abundance in health samples and different diseases. Finally, we employed in vitro and in vivo experiments to determine the effects and mechanisms of bacterial OGAs to hydrolyse O-GlcNAcylated proteins in host cells and suppress inflammatory response in the gut., Results: We found OGAs, instead of OGTs, are enriched in Bacteroidetes and Firmicutes , the major bacterial divisions in the human gut. Most bacterial OGAs are secreted enzymes with the same conserved catalytic domain as human OGAs. A pooled analysis on 1999 metagenomic samples encompassed six diseases revealed that bacterial OGA genes were conserved in healthy human gut with high abundance, and reduced exclusively in ulcerative colitis. In vitro studies showed that bacterial OGAs could hydrolyse O-GlcNAcylated proteins in host cells, including O-GlcNAcylated NF-κB-p65 subunit, which is important for activating NF-κB signalling. In vivo studies demonstrated that gut bacteria-derived OGAs could protect mice from chemically induced colonic inflammation through hydrolysing O-GlcNAcylated proteins., Conclusion: Our results reveal a previously unrecognised enzymatic activity by which gut microbiota influence intestinal physiology and highlight bacterial OGAs as a promising therapeutic strategy in colonic inflammation., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high inter-individual variation in specific and nonspecific, or trained, immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown., Results: Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling after 2 weeks and 3 months. Metagenomics based on de novo genome assembly reveals 43 immunomodulatory taxa. The nonspecific, trained immune response is detected by altered production of cytokines IL-6, IL-1β, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlates with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, is associated positively with Ruminococcus and Eggerthella lenta. The identified immunomodulatory taxa also have the strongest effects on circulating metabolites, with Roseburia affecting phenylalanine metabolism. This is corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner., Conclusions: Variability in cytokine production after BCG vaccination is associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia is found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination., (© 2021. The Author(s).)

The enzyme carbon monoxide dehydrogenase is capable of efficiently converting [Formula: see text] to CO and, therefore, can enable an affordable [Formula: see text] recycling strategy. The reduction of [Formula: see text] occurs at a peculiar nickel-iron-sulfur cluster, following a mechanism that remains little understood. In this study, we have used ab initio molecular dynamics simulations to explore the free energy landscape of the reaction. We predict the existence of a COOH ligand that strongly interacts with the surrounding protein residues and favours a mechanism where a [Formula: see text] molecule is eliminated before CO. We have taken advantages of the insights offered by our simulations to revisit the catalytic mechanism and the role of the residues surrounding the active centre in particular, thus assisting in the design of inorganic catalysts that mimic the enzyme., (© 2021. Society for Biological Inorganic Chemistry (SBIC).)

Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Rice (Oryza sativa L.) straw, an agricultural waste of high yield, is a sustainable source of fermentable sugars for biofuel and other chemicals. However, it shows recalcitrance to microbial catalysed depolymerization. We herein describe development of thermotolerant microbial consortium (RSV) from vermicompost with ability to degrade rice straw and analysis of its metagenome for bacterial diversity, and lignocellulolytic carbohydrate active enzymes (CAZymes) and their phylogenetic affiliations. RSV secretome exhibited cellulases and hemicellulases with higher activity at 60 °C. It catalysed depolymerization of chemical pretreated rice straw as revealed by scanning electron microscopy and saccharification yield of 460 mg g -1 rice straw. Microbial diversity of RSV was distinct from other compost habitats, with predominance of members of phyla Firmicutes, Proteobacteria and Bacteroidetes; and Pseudoclostridium, Thermoanaerobacterium, Chelatococcus and Algoriphagus being most abundant genera. RSV harboured 1389 CAZyme encoding ORFs of glycoside hydrolase, carbohydrate esterase, glycosyl transferase, carbohydrate binding module and auxiliary activity functions. Microorganisms of Firmicutes showed central role in lignocellulose deconstruction with importance in hemicellulose degradation; whereas representatives of Proteobacteria and Bacteroidetes contributed to cellulose and lignin degradation, respectively. RSV consortium could be a resource for mining thermotolerant cellulolytic bacteria or enzymes and studying their synergism in deconstruction of chemically pretreated rice straw.

The microbial H 2 -producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe 4 S 4 ] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Ni p , the nickel proximal to [Fe 4 S 4 ]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Ni p randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Ni p -bound methyl and CO, forming Ni p -acetyl. The substrate CoA-S - then binds to Ni p , allowing C-S bond formation between the Ni p -bound acetyl and CoA-S - . Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Ni p 2+ and Ni p 1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe 4 S 4 ] cluster. The [Fe 4 S 4 ] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Ni p (0) state could not be obtained. Optimizations always led to a Ni p 1+ state coupled with [Fe 4 S 4 ] 1+ . The calculations show a comparable activity for Ni p 1+ /[Fe 4 S 4 ] 1+ , Ni p 1+ /[Fe 4 S 4 ] 2+ , and Ni p 2+ /[Fe 4 S 4 ] 2+ . The results here give significant insights into the chemistry of the important ACS reaction.

Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H 2  + CO 2 by the only pathway that combines CO 2 fixation with ATP synthesis, the Wood-Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP + , an electron carrier in the Wood-Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP + oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe-Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.

Cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, are well known for their role in organisms' primary and secondary metabolism. Research on eukaryotes such as animals, plants, oomycetes and fungi has shown that P450s profiles in these organisms are affected by their lifestyle. However, the impact of lifestyle on P450 profiling in bacteria is scarcely reported. This study is such an example where the impact of lifestyle seems to profoundly affect the P450 profiles in the bacterial species belonging to the phylum Firmicutes. Genome-wide analysis of P450s in 972 Firmicutes species belonging to 158 genera revealed that only 229 species belonging to 37 genera have P450s; 38% of Bacilli species, followed by 14% of Clostridia and 2.7% of other Firmicutes species, have P450s. The pathogenic or commensal lifestyle influences P450 content to such an extent that species belonging to the genera Streptococcus, Listeria, Staphylococcus, Lactobacillus, Lactococcus and Leuconostoc do not have P450s, with the exception of a handful of Staphylococcus species that have a single P450. Only 18% of P450s are found to be involved in secondary metabolism and 89 P450s that function in the synthesis of specific secondary metabolites are predicted. This study is the first report on comprehensive analysis of P450s in Firmicutes.

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases ( Cb PL3 and Cb PL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii , belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two Cb PLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module ( Cb CBM66) located in the N terminus of the two Cb PLs shares 100% amino acid identity. A Cb CBM66-truncated mutant of Cb PL9 showed lower activities than the wild type, whereas Cb PL3 with a Cb CBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of Cb CBM66. Prediction by the I-TASSER server revealed that Cb CBM66 is structurally close to Bs CBM66 from Bacillus subtilis ; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between Cb CBM66 and Bs CBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two Cb PLs had strong affinities for pectate-related substrates, but Cb CBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two Cb PLs are impressive sources for the degradation of plant biomass. IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, Cb PL3 and Cb PL9, from a thermophilic bacterium, Caldicellulosiruptor bescii The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module ( Cb CBM66) was identified in the two Cb PLs, sharing 100% amino acid identity. The deletion of Cb CBM66 dramatically decreased the activity of Cb PL9 but increased the activity and thermostability of Cb PL3, suggesting different roles of Cb CBM66 in the two enzymes. Moreover, the degradation products of the two Cb PLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries., (Copyright © 2020 American Society for Microbiology.)

β-Glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity on p-nitrophenyl D-glucopyranoside stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that, however, does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, functional sugars, such as d-mannose, have attracted considerable attention due to their excellent physiological benefits for human health and wide applications in food and pharmaceutical industries. Therefore, d-mannose production using a sugar isomerase such as d-lyxose isomerase (d-LIase) has emerged as a research hotspot owing to its advantages over plant extraction and chemical synthesis methods. In this study, a putative d-LIase gene from Caldanaerobius polysaccharolyticus was cloned and expressed in Escherichia coli. Then, a biochemical characterization of the recombinant d-LIase was carried out and its potential use in d-mannose production also assessed. Results showed that d-LIase exhibited its maximum activity under these optimal conditions: temperature of 65 °C, a pH of 6.5, and the Mn 2+ metal ion. The d-LIase was active at pH 6.0-8.0; it was also quite thermostable up to 60 °C and approximately 85 % of its maximum activity was retained after incubating for 4 h. Further, our Nano-DSC analysis determined that its melting temperature (T m ) was 70.74 °C. Using 100, 300, and 500 g L -1 of d-fructose as substrate, 25.6, 74.4, and 115 g L -1 of d-mannose were produced respectively, corresponding to a conversion rate of 25.6 %, 24.8 %, and 23.0 % under optimal conditions. Taken together, our results provide evidence for a promising candidate d-LIase for producing d-mannose directly from d-fructose., Competing Interests: Declaration of Competing Interest The authors also declared no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGCs could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans . Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multigene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12a D156R -VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10 -6 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted β-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (k cat /K M ) of TnoGH1 and HorGH1 towards β-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M -1  s -1 ) and HorGH1-M229R (13,260 M -1  s -1 ) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce β-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of β-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted β-glycosidases. •β-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.

The development of drug-resistant bacteria and the necessity for unique antimicrobial agents, directed to the search of new habitats to screen the production of anti-infective substances. Culture-dependent studies of heterotrophic bacteria from the intertidal macroalgae thriving along the Southern coast of India resulted in the isolation of 148 strains, which were assayed for antibacterial activities against wide spectrum of pathogens including drug-resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Two of the most active strains with a zone of inhibition ≥ 30 mm on spot over lawn assay, belonging to the phyla Firmicutes and Gamma-proteobacteria, isolated from a  Rhodophycean marine macroalga, Hypnea valentiae, were selected for bioprospecting studies. They were further characterized as Shewanella algae MTCC 12715 and Bacillus amyloliquefaciens MTCC 12716, based on integrated phenotypic and genotypic analysis. The bacterial extracts exhibited significant antibacterial activities against MRSA and VRE with minimum inhibitory concentrations of 6.25-12.5 µg/mL. Time kill kinetic profiles of these bacteria revealed rapid bactericidal activity against both MRSA and E. coli, showing a ≥ 3log 10 decline in viable cell count compared to the initial. In BacLight™ live/dead staining technique, the propidium iodide uptake results appropriately attributed that the components in the B. amyloliquefaciens extract might compromise the integrity of the cytoplasmic membrane of the pathogenic bacteria. Type-1 pks gene (MH157093) of S. algae and hybrid nrps/pks gene (MH157092) of B. amyloliquefaciens could be amplified. Antibacterial activity study combined with the results of amplified genes coding for polyketide synthase and nonribosomal peptide synthetase showed that these marine symbiotic bacteria had a promising broad-spectrum activity, and therefore, could be used against the emerging dilemma of antibiotic-resistant bacterial infections.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > β-glucosidase > aminopeptidase, while it was in the order of β-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

The present work aimed to design a degenerate primer pair to target a large part of the hppd soil bacterial community, possibly affected by herbicides. We validated these primers by qPCR and high-throughput sequencing analysis of soil samples., Competing Interests: Declaration of Competing Interest The research project was done within the framework of the PhD studies of Miss Clémence Thiour-Mauprivez under the co-supervision of Pr Lise Barthelmebs and of myself. The PhD is funded by public funds of the Région Occitanie, European Funds for Regional Development (FEDER) and of the Perpignan University Via Domitia (France). All authors are aware of and accept responsibility for the manuscript and declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg -1 . The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn 2+ at 55 °C and pH 6.5 with a melting temperature (T m ) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with K m , k cat and K cat /k m values of 94.2 mM, 23854 min -1 and 253.3 min -1 mM -1 , respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L -1 L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods., Competing Interests: Declaration of Competing Interest The authors declare that they do not have any conflict of interest regarding the publication of this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: To determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics., Methods: The resistant H. kunzii clinical isolate UCN99 was challenged with subinhibitory concentrations of a wide range of ribosome-targeting drugs. The methylation status of the H. kunzii ribosomal RNA at the MLSB binding site was then determined using an MS approach and was correlated with any increase in resistance to the drugs., Results: The H. kunzii erm(47) gene encodes a monomethyltransferase. Expression is induced by subinhibitory concentrations of the macrolide erythromycin, as is common for many erm genes, and surprisingly also by 16-membered macrolide, lincosamide, streptogramin, ketolide, chloramphenicol and linezolid antibiotics, all of which target the 50S ribosomal subunit. No induction was detected with spectinomycin, which targets the 30S subunit., Conclusions: The structure of the erm(47) leader sequence functions as a hair trigger for the induction mechanism that expresses resistance. Consequently, translation of the erm(47) mRNA is tripped by MLSB compounds and also by drugs that target the 50S ribosomal subunit outside the MLSB site. Expression of erm(47) thus extends previous assumptions about how erm genes can be induced., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p < .05) and T2 diets (p < .05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca., (© 2019 Blackwell Verlag GmbH.)

Thermophilic microbes are an attractive bioproduction platform due to their inherently lower contamination risk and their ability to perform thermostable enzymatic processes which may be required for biomass processing and other industrial applications. The engineering of microbes for industrial scale processes requires a suite of genetic engineering tools to optimize existing biological systems as well as to design and incorporate new metabolic pathways within strains. Yet, such tools are often lacking and/or inadequate for novel microbes, especially thermophiles. This chapter focuses on genetic tool development and engineering strategies, in addition to challenges, for thermophilic microbes. We provide detailed instructions and techniques for tool development for an anaerobic thermophile, Caldanaerobacter subterraneus subsp. tengcongensis, including culturing, plasmid construction, transformation, and selection. This establishes a foundation for advanced genetic tool development necessary for the metabolic engineering of this microbe and potentially other thermophilic organisms.

d-Ribulose and l-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of d-arabinose and l-fucose byDd-arabinose and l-fucose by d-arabinose isomerase from bacterial sources for the production of d-ribulose and l-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable d-ribulose- and l-fuculose producing d-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The recombinant d-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both d-arabinose and l-fucose were observed to be 98.08 and 85.52 U mg -1 , respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn 2+ was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both d-arabinose and l-fucose at 75 °C after 10 h. The Michaelis-Menten constant (K m ), the turnover number (k cat ) and catalytic efficiency (k cat /K m ) for d-arabinose/l-fucose were 111/81.24 mM, 18,466/10,688 min -1 , and 166/132 mM -1  min -1 , respectively. When the reaction reached to equilibrium, the conversion rates of d-ribulose from d-arabinose and l-fuculose from l-fucose were almost 27% (21 g L -1 ) and 24.88% (19.92 g L -1 ) from 80 g L -1 of d-arabinose and l-fucose, respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm&#95;Ram106B, Tn&#95;Ram106B, Cb&#95;Ram106B and Ts&#95;Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as K m , V max and k cat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC 50 ) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.

Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

The Calvin-Benson-Bassham (CBB) cycle assimilates CO 2 for the primary production of organic matter in all plants and algae, as well as in some autotrophic bacteria. The key enzyme of the CBB cycle, ribulose-bisphosphate carboxylase/oxygenase (RubisCO), is a main determinant of de novo organic matter production on Earth. Of the three carboxylating forms of RubisCO, forms I and II participate in autotrophy, and form III so far has been associated only with nucleotide and nucleoside metabolism. Here, we report that form III RubisCO functions in the CBB cycle in the thermophilic chemolithoautotrophic bacterium Thermodesulfobium acidiphilum, a phylum-level lineage representative. We further show that autotrophic CO 2 fixation in T. acidiphilum is accomplished via the transaldolase variant of the CBB cycle, which has not been previously demonstrated experimentally and has been considered unlikely to occur. Thus, this work reveals a distinct form of the key pathway of CO 2 fixation., Competing Interests: The authors declare no conflict of interest.

The ginsenoside 20- O -β-glucopyranosyl-20( S )-protopanaxadiol or compound K is an essential ingredient in functional food, cosmetics, and traditional medicines. However, no study has reported the complete conversion of all protopanaxadiol (PPD)-type ginsenosides from ginseng extract into compound K using whole-cell conversion. To increase the production of compound K from ginseng extract using whole recombinant cells, the β-glucosidase enzyme from Caldicellulosiruptor bescii was coexpressed with a chaperone expression system (pGro7), and the cells expressing the coexpression system were permeabilized with ethylenediaminetetraacetic acid. The permeabilized cells carrying the chaperone coexpression system showed a 2.6-fold increase in productivity and yield as compared with nontreated cells, and completely converted all PPD-type ginsenosides from ginseng root extract into compound K with the highest productivity among the results reported so far. Our results will contribute to the industrial biological production of compound K.

Background: Consuming red and processed meat has been associated with an increased risk of colorectal cancer (CRC), which is partly attributed to exposure to carcinogens such as heterocyclic amines (HCA) formed during cooking and preservation processes. The interaction of gut microbes and HCA can result in altered bioactivities and it has been shown previously that human gut microbiota can transform mutagenic HCA to a glycerol conjugate with reduced mutagenic potential. However, the major form of HCA in the colon are glucuronides (HCA-G) and it is not known whether these metabolites, via stepwise microbial hydrolysis and acrolein conjugation, are viable precursors for glycerol conjugated metabolites. We hypothesized that such a process could be concurrently catalyzed by bacterial beta-glucuronidase (B-GUS) and glycerol/diol dehydratase (GDH) activity. We therefore investigated how the HCA-G PhIP-N2-β-D-glucuronide (PhIP-G), a representative liver metabolite of PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), which is the most abundant carcinogenic HCA in well-cooked meat, is transformed by enzymatic activity of human gut microbial representatives of the phyla Firmicutes, Bacteroidetes, and Proteobacteria., Results: We employed a combination of growth and enzymatic assays, and a bioanalysis approach combined with metagenomics. B-GUS of Faecalibacterium prausnitzii converted PhIP-G to PhIP and GDH of Flavonifractor plautii, Blautia obeum, Eubacterium hallii, and Lactobacillus reuteri converted PhIP to PhIP-M1 in the presence of glycerol. In addition, B-GUS- and GDH-positive bacteria cooperatively converted PhIP-G to PhIP-M1. A screen of genes encoding B-GUS and GDH was performed for fecal microbiome data from healthy individuals (n = 103) and from CRC patients (n = 53), which revealed a decrease in abundance of taxa with confirmed GDH and HCA transformation activity in CRC patients., Conclusions: This study for the first time demonstrates that gut microbes mediate the stepwise transformation of PhIP-G to PhIP-M1 via the intermediate production of PhIP. Findings from this study suggest that targeted manipulation with gut microbes bearing specific functions, or dietary glycerol supplementation might modify gut microbial activity to reduce HCA-induced CRC risk.

The nitrogen (N) cycle is closely related to the stability of marine ecosystems. Microbial communities have been directly linked to marine N-cycling processes. However, systematic research on the bacterial community composition and diversity involved in N cycles in different seas is lacking. In this study, microbial diversity in the Bohai Sea (BHS), Yellow Sea (YS) and South China Sea (SCS) was surveyed by targeting the hypervariable V4 regions of the 16S rRNA gene using next-generation sequencing (NGS) technology. A total of 2,505,721 clean reads and 15,307 operational taxonomic units (OTUs) were obtained from 86 sediment samples from the three studied China seas. LEfSe analysis demonstrated that the SCS had more abundant microbial taxa than the BHS and YS. Diversity indices demonstrated that Proteobacteria and Planctomycetes were the dominant phyla in all three China seas. Canonical correspondence analysis (CCA) indicated that pH (P = 0.034) was the principal determining factors, while the organic matter content, depth and temperature had a minor correlated with the variations in sedimentary microbial community distribution. Cluster and functional analyses of microbial communities showed that chemoheterotrophic and aerobic chemoheterotrophic microorganisms widely exist in these three seas. Further research found that the cultivable protease-producing bacteria were mainly affiliated with the phyla Proteobacteria, Firmicutes and Bacteroidetes. It was very clear that Pseudoalteromonadaceae possessed the highest relative abundance in the three sea areas. The predominant protease-producing genera were Pseudoalteromonas and Bacillus. These results shed light on the differences in bacterial community composition, especially protease-producing bacteria, in these three China seas., Competing Interests: The authors have the following interests: Ming Chen, Xinwu Guo and Jun Wang are employee of Sanway Gene Technology. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report 1 H, 13 C and 15 N NMR chemical shift assignments for this carbohydrate binding module (CBM).

d-Allose, a rare sugar, is an ideal table-sugar substitute and has many advantageous physiological functions. l-Rhamnose isomerase (l-RI) is an important d-allose-producing enzyme, but it exhibits comparatively low catalytic activity on d-allulose. In this study, an array of hydrophobic residues located within β1-α1-loop were solely or collectively replaced with polar amino acids by site-directed mutagenesis. A group of mutants was designed to weaken the hydrophobic environment and strengthen the catalytic behavior on d-allulose. Compared with that of the wild-type enzyme, the relative activities of the V48N/G59N/I63N and V48N/G59N/I63N/F335S mutants toward d-allulose were increased by 105.6 and 134.1%, respectively. Another group of mutants was designed to enhance thermostability. Finally, the t 1/2 values of mutant S81A were increased by 7.7 and 1.1 h at 70 and 80 °C, respectively. These results revealed that site-directed mutagenesis is efficient for improving thermostability and catalytic behavior toward d-allulose.

Lignocellulosic biomass is a valuable raw material. As technology has evolved, industrial interest in new ways to take advantage of this raw material has grown. Biomass is treated with different microbial cells or enzymes under ideal industrial conditions to produce the desired products. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are excellent sources of industrially relevant thermostable enzymes that can withstand the harsh conditions of industrial processing. Thermostable xylanases display high-specific activity at elevated temperatures and distinguish themselves in biochemical properties, structures, and modes of action from their mesophilic counterparts. Natural xylanases can be further improved through genetic engineering. Rapid progress with genome editing, writing, and synthetic biological techniques have provided unlimited potential to produce thermophilic xylanases in their natural hosts or cell factories including bacteria, yeasts, and filamentous fungi. This review will discuss the biotechnological potential of xylanases from thermophilic microorganisms and the ways they are being optimized and produced for various industrial applications.

Enzymes in the S-adenosyl-l-methionine (AdoMet) radical enzyme superfamily are metalloenzymes that catalyze a wide variety of complex radical-mediated transformations with the aid of a [4Fe-4S] cluster, which is required for activation of AdoMet to generate the 5'-deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, some enzymes share an additional domain, the SPASM domain, that houses auxiliary FeS clusters whose functional significance is not clearly understood. The AdoMet radical enzyme Tte1186, which catalyzes a thioether cross-link in a cysteine rich peptide (SCIFF), has two auxiliary [4Fe-4S] clusters within a SPASM domain that are required for enzymatic activity but not for the generation of the 5'-deoxyadenosyl radical intermediate. Here we demonstrate the ability to measure independently the midpoint potentials of each of the three [4Fe-4S] clusters by employing Tte1186 variants for which only the first, second, or AdoMet binding cluster is bound. This allows, for the first time, assignment of reduction potentials for all clusters in an AdoMet radical enzyme with a SPASM domain. Our results show that the clusters have midpoint potentials that are within 100 mV of each other, suggesting that their electrochemical properties are not greatly influenced by the presence of the nearby clusters.

Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell 1 . Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms 2-4 but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.

Six multidomain glycoside hydrolases (GHs), CelA (Athe&#95;1867), CelB (Athe&#95;1859), CelC (Athe&#95;1857), CelD (Athe&#95;1866), CelE (Athe&#95;1865), and CelF (Athe&#95;1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe&#95;1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation., (© 2018 Wiley Periodicals, Inc.)

Background: d-Mannose is not only the epimer of d-glucose at the C-2 position, but also the aldose isomer of d-fructose. Because of its physiological properties and health benefits, d-mannose has attracted public interest. It has been confirmed that d-mannose has broad applications in food, cosmetics, and pharmaceutical industries. According to the Izumoring strategy, d-glucose isomerase (d-GI) and d-lyxose isomerase (d-LI) play important roles in the conversions of d-fructose from d-glucose and of d-mannose from d-fructose respectively. In this study, a one-step enzyme process of d-mannose production from d-glucose has been constructed by co-expression of the d-GI from Acidothermus cellulolyticus and d-LI from Thermosediminibacter oceani in Escherichia coli BL21(DE3) cells., Results: The co-expression system exhibits maximum activity at pH 6.5 and 65 °C with Co 2+ supplement. It is relatively thermostable at less than 65 °C. When the reaction reaches equilibrium, the ratio of d-glucose, d-fructose, and d-mannose is approximately 34 : 49.6 : 16.4. By using this co-expression system, about 60.0 g L -1 d-mannose is obtained from 400 g L -1 d-glucose in 8 h., Conclusion: This co-expression of d-GI and d-LI system provides a novel and efficient approach for d-mannose production. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, and EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT ( SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-free EutT homologue from Listeria monocytogenes ( LmEutT) has ACAT activity and that, unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin, but not cobinamide, showed specificity for ATP and 2'-deoxyATP and released a triphosphate byproduct. Bioinformatics analyses suggest that metal-free EutT ACATs are also present in other Firmicutes.

High-efficiency lactulose-producing enzyme of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (WT- CsCE) was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Conditions for enzyme aggregation and cross-linking were optimized, and a sugar-assisted strategy with less damage to enzyme secondary structures was developed to improve the activity yield of CLEAs up to approximately 65%. The resulting CLEAs with multiple-layer network structures exhibited an enlarged optimal temperature range (70-80 °C) and maintained higher activity at 50-90 °C. Besides, CLEAs retained more than 95% of their initial activity after 10 successive batches at 60 °C, demonstrating superior reusability. Moreover, CLEAs displayed an equivalent or higher catalytic ability to free WT- CsCE in lactulose biosynthesis, and the final sugar ratios were similar, lactulose 58.8-61.7%, epilactose 9.3-10.2%, and lactose 27.8-30%, with a constant isomerization selectivity of 0.84-0.87 regardless of enzymes used and temperature applied. The proposed strategy is the first trial for enzymatic synthesis of lactulose catalyzed by CLEAs of WT- CsCE.

Dtur&#95;0462 gene from the hypertermophilic bacterium Dictyoglomus turgidum, encoding a β-glucosidase, was synthetically produced and expressed in Escherichia coli BL21(DE3)-RIL. DturβGlu was purified to homogeneity by affinity chromatography and its homotetrameric structure was determined by gel filtration. The monomer is composed by 418 amino acidic residues and showed high sequence similarity with Glycoside Hydrolases (GHs) belonging to GH1 family. The maximum activity of DturβGlu was observed at 80°C and at pH5.4. DturβGlu was stable in the range of pH5-8 and retained 70% of its activity after 2h of incubation at 70°C. Metal ions and chemical reagents differently influenced the β-glucosidase activity; furthermore, DturβGlu displays a good ethanol and glucose tolerance (K i 750mM). The enzyme is active on p-nitrophenyl-β-d-glucopyranoside (pNPGlu) (K m 0.84mM) and p-nitrophenyl-β-d-galactopyranoside (pNPGal) (K m 1.36mM) and shows a broad substrate specificity towards natural compounds as salicin, cellobiose and genistin. The ability to hydrolyze different substrates, the activation in the presence of surfactants, the good thermal resistance, and finally the high glucose and ethanol tolerance make this enzyme a good candidate for industrial applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

d-Allulose is a low-calorie sweetener and has broad applications in the food, cosmetics, and pharmaceutical industries. Recently, most studies focus on d-allulose production from d-fructose by d-allulose 3-epimerase (DAEase). However, the major blocker of industrial production of d-allulose is the poor thermostability. In this study, site-directed mutagenesis at the interface regions of Dorea sp. DAEase was carried out, and the F154Y/E191D/I193F mutation was obtained. The mutant protein displayed much higher thermostability, with a t 1/2 value of 20.47 h (50 °C) and a T m value of 74.18 °C. Compared with the wild-type DAEase, the t 1/2 value at 50 °C increased by 5.4-fold, and the T m value increased by 17.54 °C. In the d-allulose production from 500 g/L d-fructose, 148.2 g/L d-allulose could be obtained by F154Y/E191D/I193F mutant protein. The results suggest that site-directed mutagenesis at the interface regions is an efficient approach for improving the thermostability of DAEase.

Background: l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized., Results: The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively., Conclusion: The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The ginsenoside 20- O-β-glucopyranosyl-20( S)-protopanaxadiol, compound K, has attracted much attention in functional food, traditional medicine, and cosmetic industries because of diverse pharmaceutical activities. The effective production of compound K from ginseng extracts has been required. However, an enzyme capable of completely converting all protopanaxadiol (PPD)-type ginsenosides to compound K has not been reported until now. In this study, unlike other enzymes, β-glucosidase from Caldicellulosiruptor bescii was able to hydrolyze sugar moieties such as l-arabinofuranose as well as d-glucose and l-arabinopyranose as the C-20 outer sugar in ginsenosides. Thus, ginsenoside Rc containing l-arabinofuranose can be converted to compound K by only this enzyme. Under the optimized reaction conditions, the enzyme completely converted PPD-type ginsenosides in ginseng extracts to compound K with the highest productivity among the reported results. This is the first report of the enzyme capable of completely converting all PPD-type ginsenosides into compound K.