Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.

The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.

Utilization of dietary mixed-linkage ß-glucans by the Firmicute Blautia producta.

The ß-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - ß-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16MLG cleaved various types of ß-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94MLG and ß-glucosidases (BpGH3-AR8MLG and BpGH3-X62MLG). Using targeted deletion, we demonstrated BpSBPMLG is essential for B. producta growth on barley ß-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16MLG. Disentangling the ß-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells.

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges.

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4-4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen-bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

Discovery of novel glycoside hydrolases from C-glycoside-degrading bacteria using sequence similarity network analysis.

C-Glycosides are an important type of natural product with significant bioactivities, and the C-glycosidic bonds of C-glycosides can be cleaved by several intestinal bacteria, as exemplified by the human faeces-derived puerarin-degrading bacterium Dorea strain PUE. However, glycoside hydrolases in these bacteria, which may be involved in the C-glycosidic bond cleavage of C-glycosides, remain largely unknown. In this study, the genomes of the closest phylogenetic neighbours of five puerarin-degrading intestinal bacteria (including Dorea strain PUE) were retrieved, and the protein-coding genes in the genomes were subjected to sequence similarity network (SSN) analysis. Only four clusters of genes were annotated as glycoside hydrolases and observed in the genome of D. longicatena DSM 13814T (the closest phylogenetic neighbour of Dorea strain PUE); therefore, genes from D. longicatena DSM 13814T belonging to these clusters were selected to overexpress recombinant proteins (CG1, CG2, CG3, and CG4) in Escherichia coli BL21(DE3). In vitro assays indicated that CG4 efficiently cleaved the O-glycosidic bond of daidzin and showed moderate ß-D-glucosidase and ß-D-xylosidase activity. CG2 showed weak activity in hydrolyzing daidzin and pNP-ß-D-fucopyranoside, while CG3 was identified as a highly selective and efficient α-glycosidase. Interestingly, CG3 and CG4 could be selectively inhibited by daidzein, explaining their different performance in kinetic studies. Molecular docking studies predicted the molecular determinants of CG2, CG3, and CG4 in substrate selectivity and inhibition propensity. The present study identified three novel and distinctive glycoside hydrolases, highlighting the potential of SSN in the discovery of novel enzymes from genomic data.

Enzymatic Synthesis of l-threo-ß-Hydroxy-α-Amino Acids via Asymmetric Hydroxylation Using 2-Oxoglutarate-Dependent Hydroxylase from Sulfobacillus thermotolerans Strain Y0017.

ß-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of ß-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-ß-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-ß-hydroxy-His and 150 mM l-threo-ß-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-ß-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of ß-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for ß-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As ß-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Characterization of a Recombinant D-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in D-Allulose Production.

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. D-Allulose, the C-3 epimer of D-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. D-Allulose 3-epimerase (DAEase) is indispensable in D-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co2+. The half-life (t1/2) at 50 °C was 13.6 h, and the melting temperature (Tm) was 62.4 °C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm. Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L D-allulose from 500 g/L D-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of D-allulose.

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria.

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.

Fucosidases from the human gut symbiont Ruminococcus gnavus.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

Computational modeling and small-angle X-ray scattering based structure analysis and identifying ligand cleavage mechanism by processive endocellulase of family 9 glycoside hydrolase (HtGH9) from Hungateiclostridium thermocellum ATCC 27405.

The cellulases of family 9 glycoside hydrolase with subtle difference in amino acid sequence have shown different types of catalytic activities such as endo-, exo- or processive endocellulase. However, the reason behind the different types of catalytic activities still unclear. In this study, the processive endocellulase, HtGH9 of family 9 GH from Hungateiclostridium thermocellum was modeled by homology modeling. The catalytic module (HtGH9t) of HtGH9 modeled structure displayed the (α/α)6 barrel topology and associated family 3 carbohydrate binding module (HtCBM3c) displayed ß-sandwich fold. Ramachandran plot of HtGH9 modeled structure displayed all the amino acid residues in allowed region except Asn225 and Asp317. Secondary structure analysis of modeled HtGH9 showed the presence of 41.3% α-helices and 11.0% ß-strands which was validated through circular dichroism analysis that showed the presence of 42.6% α-helices and 14.5% ß-strands. Molecular Dynamic (MD) simulation of HtGH9 structure for 50 ns showed Root Mean Square Deviation (RMSD), 0.84 nm and radius of gyration (Rg) 3.1 nm. The Small-angle X-ray scattering of HtGH9 confirmed the monodisperse state. The radius of gyration for globular shape (Rg) was 5.50 ± 0.15 nm and for rod shape (Rc) by Guinier plot was 2.0 nm. The loop formed by amino acid residues, 264-276 towards one end of the catalytic site of HtGH9 forms a barrier, that blocks the non-reducing end of the cellulose chain causing the processive cleavage resulting in the release of cellotetraose. The position of the corresponding loop in cellulases of family 9 GH is responsible for different types of cleavage patterns.

Cloning and characterization of a L-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6.

Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel ß-N-acetylhexosaminidase from Tyzzerella nexilis.

ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.

Integration of logic gates to CRISPR/Cas12a system for rapid and sensitive detection of pathogenic bacterial genes.

For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 103 CFU/mL, and the dynamic range was 103-107 CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection.

A thermophilic and thermostable xylanase from Caldicoprobacter algeriensis: Recombinant expression, characterization and application in paper biobleaching.

A novel xylanase gene xynBCA, encoding a polypeptide of 439 residues (XynBCA), was cloned from Caldicoprobacter algeriensis genome and recombinantly expressed in Escherichia coli BL21(DE3). The amino acid sequence analysis showed that XynBCA belongs to the glycoside hydrolase family 10. The purified recombinant enzyme has a monomeric structure of 52 kDa. It is active and stable in a wide range of pH from 3 to 10 with a maximum activity at 6.5. Interestingly, XynBCA was highly thermoactive with an optimum temperature of 80 °C, thermostable with a half-life of 20 min at 80 °C. The specific activity was 117 U mg-1, while the Km and Vmax were 1.247 mg ml-1, and 114.7 µmol min-1 mg-1, respectively. The investigation of XynBCA in kraft pulp biobleaching experiments showed effectiveness in releasing reducing sugars and chromophores, with best achievements at 100 U g-1 of pulp and 1 h of incubation. The comparative molecular modeling studies with the less thermostable Xylanase B from Clostridium stercorarium, revealed extra charged residues at the surface of XynBCA potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability.

Computer-aided search for a cold-active cellobiose 2-epimerase.

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (kcat), Michaelis constant (Km), and catalytic efficiency (kcat/Km) using lactose as a substrate were 117.0 ± 7.7 s-1, 429.9 ± 57.3 mM, and 0.27 mM-1s-1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.

Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.