Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

NA4/NA6, an intermediate degradation product of ß-agarase, is a high value-added product with anticancer, anti-obesity, and anti-diabetic effects. Therefore, a method that enables the efficient production of NA4/NA6 would be useful from economic and medical perspectives. In this study, we aimed to generate a Streptomyces coelicolor A3(2) mutant M22-2C43 that produces NA4/NA6 as a final product; this method serves as a more efficient alternative to the enzymatic conversion of ß-agarase for the generation of these products. The M22-2C43 strain was generated through two rounds of mutagenesis and screening for increased ß-agarase activity and effective production of NA4/NA6. We assembled the complete genomes of two mutants, M22 and M22-2C43, which were identified following a two-round screening. Large and small genetic changes were found in these two mutants, including the loss of two plasmids present in wild-type S. coelicolor A3(2) and chromosome circularization of mutant M22-2C43. These findings suggest that mutant M22-2C43 can produce NA4/NA6 as a degradation product due to functional inactivation of the dagB gene through a point mutation (G474A), ultimately preventing further degradation of NA4/NA6 to NA2. To our knowledge, this is the first report of a microbial strain that can effectively produce NA4/NA6 as the main degradation product of ß-agarase, opening the door for the use of this species for the large-scale production of this valuable product.

Anti-obesity effect of Neoagaro-oligosaccharides with overweight and obese subjects: a 16-week, randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: This trial aimed to evaluate the anti-obesity effects and safety of Neoagaro-oligosaccharides (NAOs) in humans in a 16 week, randomized, double-blind, placebo-controlled clinical trial. METHODS: One hundred overweight or obese subjects with a body mass index of 23 to 34.9 kg/m2 and a percent body fat of > 25% for males or > 30% for females were enrolled. NAOs or placebo products were administered at 3 g (twice a day, four capsules once) each for 16 weeks. Efficacy and safety biomarkers were measured before and after intervention. RESULTS: After 16 weeks of intervention, the group administered with NAOs had statistically significant decreases in visceral fat area and visceral-subcutaneous fat area ratio compared to the placebo group. The NAOs group suppressed the increase in weight and BMI compared to the placebo group, which was significant between groups. High-density lipoprotein- cholesterol was increased in the group administered with NAOs, which showed a significant trend compared to the placebo group. Clinical changes were not observed for any safety biomarkers. CONCLUSIONS: These results suggest that NAOs have a beneficial effect on obesity. Thus, NAOs could be used as an anti-obesity supplement without side effects. TRIAL REGISTRATION: cris.nih.go.kr: (KCT0006640, 07/10/2021).

Neoagarooligosaccharide Protects against Hepatic Fibrosis via Inhibition of TGF-ß/Smad Signaling Pathway.

Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by ß-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-ß-treated LX-2 cells. Furthermore, downstream of TGF-ß, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl4)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl4-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-ß/Smad signaling pathway.

Hepatoprotective Effect of Neoagarooligosaccharide via Activation of Nrf2 and Enhanced Antioxidant Efficacy.

Neoagarooligosaccharides (NAOS) are generated by ß-agarases, which cleave the ß-1,4 linkage in agarose. Previously, we reported that NAOS inhibited fat accumulation in the liver and decreased serum cholesterol levels. However, the hepatoprotective effect of NAOS on acute liver injury has not yet been investigated. Thus, we examined whether NAOS could activate nuclear factor (NF)-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) and upregulates its target gene, and has hepatoprotective effect in vivo. In hepatocytes, phosphorylation and subsequent nuclear translocation of Nrf2 are increased by treatment with NAOS, in a manner dependent on p38 and c-Jun N-terminal kinase (JNK). Consistently, NAOS augmented ARE reporter gene activity and the antioxidant protein levels, resulting in increased intracellular glutathione levels. NAOS antagonized tert-butylhydroperoxide-induced reactive oxygen species (ROS) generation. Moreover, NAOS inhibited acetaminophen (APAP)-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and significantly decreased hepatocyte degeneration and inflammatory cell infiltration. Moreover, ROS production and glutathione depletion by APAP were reversed by NAOS. APAP-mediated apoptotic signaling pathways were also inhibited in NAOS-treated mice. Upregulalted hepatic expression of genes related to inflammation by APAP were consistently diminished by NAOS. Collectively, our results demonstrate that NAOS exhibited a hepatoprotective effect against APAP-mediated acute liver damage through its antioxidant capacity.

Safety evaluation of ß-agarase preparations from Streptomyces coelicolor A3(2).

Recent studies on neoagarooligosaccharides prepared by hydrolyzing agar with ß-agarase DagA produced from Streptomyces coelicolor A3(2) have enhanced our knowledge about the enzymatic utility of S. coelicolor. For safety evaluation, a crude extracellular protein containing DagA (crDagA) was prepared from the culture broth of S. coelicolor A3(2) M22-2C43, a highly productive strain of DagA. All genotoxicity tests, such as bacterial reverse mutation assay, eukaryotic chromosomal aberration assay, and in vivo micronucleus assay in mice showed no mutagenic activity of crDagA. No abnormalities were found in the appearance or behavior upon single oral administration up to 20,000 mg/kg body weight (BW) [318 mg TOS (Total Organic Solids)/kg BW] and long-term repeated oral administration toxicity tests up to 10,000 mg/kg BW/day (159 mg TOS/kg BW/day) in Sprague Dawley®™ rats. In addition, there were no statistically significant differences in the body weight change, food intake, hematology, blood biochemistry, organ weight, and clinical signs between the crDagA-administered and non-administered groups during the experimental period. This result showed that crDagA produced from S. coelicolor A3(2) is a safe, non-toxic substance, and therefore, can be used safely for manufacturing neoagarooligosaccharide, a functional substance effective in improving metabolic syndrome.

Non-targeted metabolomics unravels a media-dependent prodiginines production pathway in Streptomyces coelicolor A3(2).

The genus Streptomyces is the best-known source of therapeutic secondary metabolites, especially antibiotics with pharmaceutical applications. Here, we performed a comparative study based on the time-resolved metabolic disparity in S. coelicolor A3(2) subjected to fermentative cultivation in two different types of media (R2YE and RSM3) in order to investigate secondary metabolite production pathways. The relative abundance of secondary metabolites, such as prodiginines, indoles, germicidins, and selected diketopiperazines, was increased in S. coelicolor A3(2) cultivated in R2YE medium compared to that in RSM3 medium, variably at the late-log and stationary phases of fermentative growth. Correlation analysis indicated that "antibiotic prodiginines" contributed maximally to the absorption maxima (A530) of culture supernatants, indicating their optimal production at 96 hours in R2YE medium. A higher abundance of L-proline (48-72 hours) followed by prodiginines (96 hours) was evident, substantiating the intertwined links between precursor and activated prodiginines pathway. Similarly, the higher abundance of indoles was concurrent with tryptophan levels in the shikimate pathway, whereas diketopiperazines were synchronously abundant along with the levels of phenylalanine, leucine, and proline. Additionally, acetyl-CoA induced the acetate pathway, resulting in the production of germicidins. Thus, our results demonstrate that S. coelicolor A3(2) produces specific secondary metabolites by enhancing the dedicated metabolic pathway responsible for their production. In conclusion, our results from this study provide insight into the metabolic pathways of S. coelicolor A3(2), and can be applied to further optimize the production of prodiginines.

Toxicological evaluation of neoagarooligosaccharides prepared by enzymatic hydrolysis of agar.

Agar, a heterogeneous polymer of galactose, is the main component of the cell wall of marine red algae. It is well established as a safe, non-digestible carbohydrate in Oriental countries. Although neoagarooligosaccharides (NAOs) prepared by the hydrolysis of agar by ß-agarase have been reported to exert various biological activities, the safety of these compounds has not been reported to date. For safety evaluation, NAOs containing mainly neoagarotetraose and neoagarohexaose were prepared from agar by enzymatic hydrolysis using ß-agarase DagA from Streptomyces coelicolor. Genotoxicity tests such as the bacterial reverse mutation assay, eukaryotic chromosome aberration assay, and in vivo micronucleus assay all indicated that NAOs did not exert any mutational effects. The toxicity of NAOs in rat and beagle dog models was investigated by acute, 14-day, and 91-day repeated oral dose toxicity tests. The results showed that NAO intake of up to 5,000 mg/kg body weight resulted in no significant changes in body weight, food intake, water consumption, hematologic and blood biochemistry parameters, organ weight, or clinical symptoms. Collectively, a no-observed-adverse-effect level of 5,000 mg/kg body weight/day for both male and female rats was established for NAO. These findings support the safety of NAO for possible use in food supplements and pharmaceutical and cosmetic products.

Anti-Obesity and Anti-Diabetic Effect of Neoagarooligosaccharides on High-Fat Diet-Induced Obesity in Mice.

Neoagarooligosaccharides (NAOs), mainly comprising neoagarotetraose and neoagarohexaose, were prepared by hydrolyzing agar with ß-agarase DagA from Streptomyces coelicolor, and the anti-obesity and anti-diabetic effects of NAOs on high-fat diet (HFD)-induced obesity in mice were investigated after NAOs-supplementation for 64 days. Compared to the HFD group, the HFD-0.5 group that was fed with HFD + NAOs (0.5%, w/w) showed remarkable reduction of 36% for body weight gain and 37% for food efficiency ratios without abnormal clinical signs. Furthermore, fat accumulation in the liver and development of macrovesicular steatosis induced by HFD in the HFD-0.5 group were recovered nearly to the levels found in the normal diet (ND) group. NAOs intake could also effectively reduce the size (area) of adipocytes and tissue weight gain in the perirenal and epididymal adipose tissues. The increased concentrations of total cholesterol, triglyceride, and free fatty acid in serum of the HFD group were also markedly ameliorated to the levels found in serum of the ND group after NAOs-intake in a dose dependent manner. In addition, insulin resistance and glucose intolerance induced by HFD were distinctly improved, and adiponectin concentration in the blood was notably increased. All these results strongly suggest that intake of NAOs can effectively suppress obesity and obesity-related metabolic syndromes, such as hyperlipidemia, steatosis, insulin resistance, and glucose intolerance, by inducing production of adiponectin in the HFD-induced obese mice.

Development of anangiogenesis-focused cDNA chip and validation of its functionality.

DNA chip has been used as a powerful tool to study the genetic reprogramming of cells and its link to cellular phenotype such as angiogenesis. To evaluate the angiogenesis related genetic reprogramming more efficiently, we here developed an angiogenesis-focused cDNA chip containing 153 angiogenesis related genes arrayed in duplicate on a slide glass. In order to validate the functionality of the angiogenesis-focused cDNA chip, we examined gene expression profiles in HT1080 cells treated with either fetal bovine serum, a well known pro-angiogenic factor, or trichostatin A, a known angiogenesis inhibitor, using the cDNA chip. All duplicate data from the analysis are well matched with each other and gene expression profiles are well consistent with previously reported data. These results demonstrate that the angiogenesis-focused cDNA chip developed here can be a useful tool towards angiogenesis- related researches.

Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium.

A newly isolated rod-shaped, gram-negative anaerobic bacterium from human feces, named Julong 732, was found to be capable of metabolizing the isoflavone dihydrodaidzein to S-equol under anaerobic conditions. The metabolite, equol, was identified by using electron impact ionization mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectroscopy, and UV spectral analyses. However, strain Julong 732 was not able to produce equol from daidzein, and tetrahydrodaidzein and dehydroequol, which are most likely intermediates in the anaerobic metabolism of dihydrodaidzein, were not detected in bacterial culture medium containing dihydrodaidzein. Chiral stationary-phase high-performance liquid chromatography eluted only one metabolite, S-equol, which was produced from a bacterial culture containing a racemic mixture of dihydrodaidzein. Strain Julong 732 did not show racemase activity to transform R-equol to S-equol and vice versa. Its full 16S rRNA gene sequence (1,429 bp) had 92.8% similarity to that of Eggerthella hongkongenis HKU10. This is the first report of a single bacterium capable of converting a racemic mixture of dihydrodaidzein to enantiomeric pure S-equol.

Chromosome 4q;10q translocations; comparison with different ethnic populations and FSHD patients.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by the weakness of facial, shoulder-girdle and upper arm muscles. Most patients with FSHD have fewer numbers of tandem repeated 3.3-kb KpnI units on chromosome 4q35. Chromosome 10q26 contains highly homologous KpnI repeats, and inter-chromosomal translocation has been reported. METHODS: To clarify the influence on the deletion of the repeats, we surveyed three different ethnic populations and FSHD patients using the BglII/BlnI dosage test. RESULTS: The frequency of translocation in 153 Japanese, 124 Korean, 114 Chinese healthy individuals and 56 Japanese 4q35-FSHD patients were 27.5%, 29.8%, 19.3%, and 32.1%, respectively. The ratio of '4 on 10' (trisomy and quatrosomy of chromosome 4) was higher than that of '10 on 4' (nullsomy and monosomy of chromosome 4) in all populations. CONCLUSIONS: The inter-chromosomal exchange was frequently observed in all four populations we examined, and no significant difference was observed between healthy and diseased groups.