Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 µg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)-signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

Lipid mediators derived from DHA alleviate DNCB-induced atopic dermatitis and improve the gut microbiome in BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that primarily results from immune dysregulation. We determined the potential therapeutic benefits of lipid mediators (LM, 17S-monohydroxy DHA, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA. The underlying molecular mechanisms involved in TNF-α/IFN-γ-stimulated HaCaT cells as well as its effect in an AD mouse model induced by DNCB in BALB/c mice were examined. The results indicated that LM effectively attenuates the production of inflammatory cytokines (IL-6 and IL-1ß) and chemokines (IL-8 and MCP-1) by inhibiting the NF-κB signaling pathway in TNF-α/IFN-γ-stimulated HaCaT cells. The oral administration of LM at 5 or 10 µg/kg/day significantly reduced skin lesions, epidermal thickness, and mast cell infiltration in AD mice. Furthermore, LM reduced the production of IgE and inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in the serum, modulated gut microbiota diversity, and restored the microbial composition. Overall, our findings suggest that LM represents a potential therapeutic agent for improving AD symptoms through its ability to suppress inflammatory cytokines and alter the composition of gut microbiota.

Complete genome sequence of pectin-degrading Flavobacteriaceae bacterium GSB9.

Pectic oligosaccharides, which are considered to be potential prebiotics, may be generated by pectin-degrading enzymes. Here, we report the complete genome sequence of the pectin-degrading marine bacterium, Flavobacteriaceae bacterium GSB9, which was isolated from seawater of South Korea. The complete genome sequence revealed that the chromosome was 3,630,376 bp in size, had a G + C content of 36.6 mol%, and was predicted to encode 3100 protein-coding sequences (CDSs), 40 tRNAs, and six 16S-23S-5S rRNAs. Genome sequence analysis revealed that this strain possesses multiple genes predicted to encode pectin-degrading enzymes. Our analysis may facilitate the future application of this strain against pectin in various industries.

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5'-coding region variant of the cadA gene.

Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72-h culture after induction with isopropyl ß-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. © 2015 Wiley Periodicals, Inc.

Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region.

Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria.

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 µM and 1.55 × 10(-)(4 )µmol min(-1), respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-ß-d-cellobioside (0.3 U mg(-1)), CMC (105.9 U mg(-1)), birch wood xylan (132.3 U mg(-1)), oat spelt xylan (67.9 U mg(-1)), and 2-hydroxyethyl-cellulose (26.3 U mg(-1)). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.

Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system.

Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-ß-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10(4) clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.

A rapid and simple method for preparing an insoluble substrate for screening of microbial xylanase.

Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil.

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium.

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 x 10⁻² U mg⁻¹, respectively. The Michaelis-Menten constant (K (m) values) and the maximal reaction velocities (V(max) values) of CelEdx16 were 47.1 µM and 9.6 x 10⁻³ µmole min⁻¹ when endocellulase activity was measured and 106.3 µM and 2.1 x 10⁻5 µmol min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40 °C.

Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.

A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.