Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

A DasA family sugar binding protein Ste2 links nutrient and oxidative stress to exopolysaccharides production in Streptomyces sp. 139.

BACKGROUND: Ebosin is an exopolysaccharide produced by Streptomyces sp. 139, and its biosynthetic gene cluster (ste) has been previously described. Ste234 has high homology to the well-known ATP-binding cassette transport system DasABC, which has been linked to the regulation of morphological differentiation, antibiotics biosynthesis and aminosugars utilization in Streptomycetes. This study was conducted to evaluate the effect of the DasA family sugar binding protein Ste2 on Streptomyces sp. 139. RESULTS: The disruption of ste2 results in the upregulation of transcription of genes within Ebosin biosynthetic gene cluster and a two-fold increase in Ebosin production. RNA sequencing data suggests that the disruption of ste2 results in the decreased utilization of carbon and nitrogen sources, increased sensitivity to oxidative stress, as well as differed strain morphology, all of which have been experimentally proven. CONCLUSIONS: Taken together, Ste2 controls Ebosin yields, aminosugars uptake, sensitivity to oxidative stress, and morphological differentiation of Streptomyces sp. 139.

Gestational Weight Gain and Overweight in Children Aged 3-6 Years.

OBJECTIVE: To determine whether gestational weight gain (GWG) was associated with increased odds of childhood overweight after accounting for pre-pregnancy BMI. METHODS: In a prospective cohort study based on a premarital and perinatal health care system in China, data of 100 612 mother-child pairs were obtained. The main exposure was GWG as both a continuous and categorical variable. The outcome measure was overweight, defined by age- and sex-specific cutoff values for body mass index (BMI) in children aged 3-6 years. RESULTS: A 1-kg increase in maternal GWG was associated with an increase of 0.009 (95% confidence interval [CI]: 0.007-0.010, P < 0.001) in children's mean BMI; in the subgroup of pre-pregnancy overweight/obese mothers, the increase in children's BMI was 0.028 (95% CI, 0.017-0.039, P < 0.001). Excessive GWG played an important role in childhood overweight when adequate GWG was used as the reference, with an odds ratio (OR) of 1.21 (95% CI, 1.12-1.29). The risk was highest (OR 2.22; 95% CI, 1.79-2.76) in the children of mothers who were overweight/obese before pregnancy and gained excessive weight during pregnancy. CONCLUSIONS: Greater maternal GWG was associated with greater offspring BMI, and the risk of overweight was doubled in children whose mothers were overweight/obese before pregnancy and gained excessive weight during pregnancy. As a result, maintenance of appropriate weight gain during pregnancy and prophylaxis of maternal overweight/obesity before pregnancy should be a strategy for preventing childhood overweight/obesity.

Characterization of an Ebosin derivative produced by heterologous gene replacement in Streptomyces sp. 139.

BACKGROUND: Ebosin is a novel exopolysaccharide (EPS) produced by Streptomyces sp. 139 and evidenced to possess an anti-rheumatic arthritis activity in vivo. The Ebosin biosynthesis gene cluster (ste) consists of 27 ORFs and ste7 has previously been demonstrated to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugar units during Ebosin production. Aiming to generate derivatives of Ebosin for better activity, we replaced ste7 with a gene encoding for a glucosyltransferase (gtf) from Streptococcus thermophilus. RESULTS: This alteration resulted in a novel Ebosin derivative (EPS-7 g) with its monosaccharide composition dramatically changed, especially in the proportion of glucose which increased from 1.1% (Ebosin) to 84.01% (EPS-7 g). In an ELISA analysis, EPS-7 g exhibited a higher binding activity for IL-1R, as a competitor of interleukin-1, than that of Ebosin. It also exhibited a higher inhibitory effect on the activity of IL-1ß-converting enzyme and production of IL-1ß in fibroblast-like synoviocytes (FLS). In addition, experiments with acute inflamed mice induced by croton oil showed a significantly higher anti-inflammatory activity of EPS-7 g compared with Ebosin. CONCLUSIONS: The new Ebosin derivative EPS-7 g is more bioactive than Ebosin evaluated by a series of experiments. This is the first report demonstrating a modification of EPS structure via heterologous gene replacement in Streptomyces.

The synergistic effect of polyphenols and polypeptides for plant-based bioplastic film - Enhanced UV resistance, antioxidant and antibacterial performance.

The exceptional biodegradability and active biological functions of bio-based packaging materials have attracted increasing interest. In this study, a bioplastic film was developed by introducing simultaneously polyphenols (tea polyphenols, TPs) and peptides (nisin) into a soy protein isolate/sodium alginate (SPI/SA) based film-forming matrix. The research results revealed that the dynamic coordinated interaction between TPs and nisin enhanced mechanical properties, UV-resistance, and thermal stability of bioplastic films. Furthermore, the bioplastic film exhibited antibacterial activity and antioxidant properties. Significantly, biofilm growth of Staphylococcus aureus treated with TPs-5/Nisin-5 bioplastic film was inhibited by 91.12% compared to the blank group. The shelf life of beef with TPs-5/Nisin-5 bioplastic film was prolonged by 2 days because of the synergistic effect of TPs and nisin. Additionally, the bioplastic film biodegraded in the natural environment about 21 days. This environmentally friendly regeneration strategy and the integration of advantageous functions provided ideas for the development of active food packaging.

A new GntR family regulator Ste1 in Streptomyces sp. 139.

The novel exopolysaccharide Ebosin produced by Streptomyces sp. 139 has remarkable in vivo antirheumatic arthritis activity, and its biosynthesis gene cluster (ste) consisting of 27 ORFs has been identified. The present investigation focused on the function of ste1 gene. Database searching revealed that Ste1 is homologous to the GntR family regulator originated from microbes. To confirm its function in Ebosin biosynthesis, the gene was disrupted. The mutant strain Streptomyces sp. 139 D1 was found to have a much higher Ebosin production than that of the wild-type strain, whilst the complementation strain Streptomyces sp. 139 C1 showed a decrease in the exopolysaccharide produced. Real-time qPCR analysis indicated that in the mutant strain Streptomyces sp. 139 D1, transcription levels of gene ste5, ste8, and ste17 increased significantly compared with those in the wild-type strain. The electrophoretic mobility shift assay demonstrated that Ste1 binds with higher affinity to the promoter 1 and 3 regions in the ste gene cluster. It is concluded that ste1 plays the negative regulation as a transcription repressor during Ebosin biosynthesis. Growing on minimal agar medium supplemented with glucose and R2YE agar medium, the mutant strain Streptomyces sp. 139 D1 exhibited a Bld phenotype.

Characterization of and functional evidence for Ste27 of Streptomyces sp. 139 as a novel spermine/spermidine acetyltransferase.

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has remarkable anti-rheumatoid arthritis activity in vivo and its biosynthesis gene cluster (ste) consists of 27 ORFs (open reading frames). The present paper reports our study of the protein product encoded by ste27. Database searching reveals the homology of Ste27 with some spermidine/spermine acetyltransferases. To confirm the prediction, the ste27 gene was cloned and expressed in Escherichia coli BL21(DE3) cells and recombinant Ste27 was purified. The following enzymatic analysis revealed its ability of transferring the acetyl group from acetyl-CoA to spermidine and spermine, with spermidine being the preferred substrate. Ste27 can acetylate the N1, N4 and N8 positions on spermidine. The Km values of Ste27 were determined for spermidine and spermine, as well as for acetyl-CoA, poly-L-lysine and glucosamine 6-phosphate. Upon gene knockout, the exopolysaccharide-27m produced by the mutant strain Streptomyces sp. 139 (ste27-), compared with Ebosin, exhibited a significantly reduced binding activity to the interleukin-1 receptor. After gene complementation, the binding activity was partially restored. This demonstrated that the ste27 gene is involved in the biosynthesis of Ebosin. Molecular modelling was also carried out to predict the binding mode of Ste27 with acetyl-CoA, spermidine or spermine.

[Effect of Ebosin on the inflammatory cytokines in THP-1 cells].

Ebosin is a novel exopolysaccharide produced by Streptomyces sp.139 with remarkable activity against rheumatic arthritis in vivo. In this paper, we reported effects of Ebosin on the inflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in THP-1 cells. With the special fluorogenic peptide as substrates, the enzymatic activities of interleukin-1beta converting enzyme (ICE) and TNFalpha-converting enzyme (TACE) were inhibited by Ebosin separately. Using the real-time reverse transcription polymerase chain reaction (real-time PCR), the mRNA synthesis of the three cytokines were identified decline separately by Ebosin. The secretion quantum of three cytokines in THP-1 cells with Ebosin was lower than that of normal THP-1 cells determined by ELISA assay and Western blotting. All of these results showed that Ebosin has remarkably suppressed synthesis of the three cytokines in THP-1 cells through different pathways. The primary study of Ebosin on anti-inflammation mechanism was promoted developing the new drugs treating rheumatic arthritis.

Predictive value of serum human epididymis protein 4 for heart failure in patients with chronic kidney disease.

AIM: To investigate the predictive value of human epididymis protein 4 (HE4) for heart failure in patients with chronic kidney disease (CKD). METHODS: This study retrospectively analyzed the data of 241 patients with CKD admitted to Zhangjiakou First Hospital from January 2019 to January 2021. The subjects were divided into a heart failure (HF) group (n=117) and a non-HF group (n=124) according to whether heart failure occurred. The baseline data and laboratory hematologic indicators (complete set of HE4, blood routine and biochemistry) were collected and analyzed by univariate analysis. Subsequently, the variables that were significant in the correlation analysis were included in a multi-factor logistic regression analysis. RESULTS: The HF group exhibited higher serum creatinine, HE4, hemoglobin, total cholesterol, triglycerides (TG), high-density lipoprotein (P<0.05), as well as higher B-type natriuretic peptide (BNP), creatine kinase, and creatine kinase-MB than the non-HF group, with significant differences (P<0.05). Spearman's rank correlation analysis revealed that age, HE4, calcium, TG, BNP and left ventricle ejection fraction were associated with the occurrence of heart failure (P<0.05). Multivariate analysis demonstrated that HE4 was a significant factor that could predict the development of heart failure in CKD patients (P<0.01), and the risk of heart failure was higher when HE4>27.2368 pmol/L. CONCLUSIONS: HE4 is an important factor for predicting the occurrence of heart failure in CKD patients. A higher HE4 level predicts greater possibility of heart failure.

[Study of a novel compound 2460A with activities produced by fungus].

With IL-6R as target, a new compound 2460A was identified from fungus using HTS screening model. The taxonomics of the produced strain was confirmed to be Trichoderma hazianum rifai after sequencing analysis of rDNA-ITS (internal transcribed spacer). Results showed that this compound has a binding activity on IL-6R competed with IL-6, thus it is a new ligand of IL-6R originating from microbe. With MTT assay, the anti-tumor activities of 2460A were demonstrated on CM126 and HT-29 cell lines separately, the IC50 are 2.17 x 10(-5) mol x L(-1) and 1.8 x 10(-5) mol x L(-1) respectively. The compound affected lightly the HT-29 cell cycle at S phase. Studies for the anti-tumor activity of 2460A in vivo are in progress in our lab.

A new compound Trichomicin exerts antitumor activity through STAT3 signaling inhibition.

Trichomicin, a novel small-molecule compound isolated from the fungus Trichoderma harzianum and identified as new structure compound, exhibited antitumor activities in various human cancer cell lines and reversed drug resistance activity in the multidrug-resistant cancer cell line KBV. The underlying cellular and molecular mechanism was illuminated. Trichomicin can significantly induce cancer cell apoptosis and reduced IL-6 expression and phosphorylation of STAT3 were found in response to Trichomicin treatment. The blockade of IL-6 mediated JAK-STAT3 signaling pathway by Trichomicin was confirmed using reporter gene system. As a promising antitumor-activity compound, Trichomicin is presented in this study.

Gloss phenomena and image analysis of atomic force microscopy in molecular and cell biology.

Proper sample preparation, scan setup, data collection and image analysis are key factors in successful atomic force microscopy (AFM), which can avoid gloss phenomena effectively from unreasonable manipulations or instrumental defaults. Fresh cleaved mica and newly treated glass cover were checked first as the substrates for all of the sample preparation for AFM. Then, crystals contamination from buffer was studied separately or combined with several biologic samples, and the influence of scanner, scan mode and cantilever to data collection was also discussed intensively using molecular and cellular samples. At last, images treatment and analysis with off-line software had been focused on standard and biologic samples, and artificial glosses were highly considered for their high probability. SCANNING 31: 49-58, 2009. (c) 2009 Wiley Periodicals, Inc.

Biochemical characteristics and function of a fucosyltransferase encoded by ste7 in Ebosin biosynthesis of Streptomyces sp. 139.

A novel exopolysaccharide named Ebosin was produced by Streptomyces sp. 139, with medicinal activity. Its biosynthesis gene cluster (ste) has been previously identified. For the functional study of the ste7 gene in Ebosin biosynthesis, it was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS- 7m produced by the mutant strain Streptomyces sp. 139 (ste7(-)) was found altered from that of Ebosin, with fucose decreasing remarkably. For biochemical characterization of Ste7, the ste7 gene was cloned and expressed in Escherichia coli BL21. With a continuous coupled spectrophotometric assay, Ste7 was demonstrated to have the ability of catalyzing the transfer of fucose specifically from GDP-beta- L-fucose to a fucose acceptor, the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 (ste7(-)). Therefore, the ste7 gene has been identified to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugars units during Ebosin biosynthesis.

[The effects of genes ste7-stel5 double disruption in ebosin biosynthesis].

OBJECTIVE: To study the effects of genes ste7-ste15 double disruption in biosynthesis of Ebosin. METHODS: The ste7 gene was disrupted with a double crossover via homologous recombination in the mutant strain Streptomyces sp. The 139 (ste15-) and the mutant strain Streptomyces sp.139 (ste7- ste15-) were identified by Southern blot. Gene complementation of the knock-out mutant was done. Monosaccharide composition of the exopolysaccharides EPS15-7m, EPS15-7c producing by Streptomyces sp.139 (ste7- ste15-) and Streptomyces sp. 139 (pKC7-15c) separately were analyzed by Gas Chromatography,while their Mw and the antagonist activity for IL-1R were determined comparing with Ebosin. RESULTS: The mutant strain Streptomyces sp.139 (ste7- ste15-) and complementary strain Streptomyces sp. 139 (pKC7-15c) were constructed respectively. The analysis results showed that both of fucose and glucose decreased remarkably in EPS15-7m and its Mw and the antagonist activity for IL-1R were much lower than Ebosin. The composition of fucose and glucose were recovered notablely in EPS15-7c compared with EPS15-7m. CONCLUSION: The genes ste7 and ste15 played essential roles in the synthesis process of sugar repeating unit during biosynthesis of Ebosin. The activities of Ebosin new derivative produced by the mutant will be studied further.

Expression and purification of human vascular-endothelial-growth-factor-receptor-2 tyrosine kinase in Streptomyces for inhibitor screening.

VEGF (vascular endothelial growth factor) is a critical regulator in angiogenesis through binding to its specific receptors, including VEGFR-2 (VEGF receptor 2), a kinase insert domain-containing receptor, on the surface of endothelial cells. As angiogenesis has been shown to be essential for malignancy of tumours, VEGFR-2 is a potential therapeutic target for the treatment of cancers. To explore this potential, VEGFR-2-CD (the protein tyrosine kinase catalytic domain of VEGFR-2) was cloned and expressed in Streptomyces lividans TK24, a prokaryotic expression system. The recombinant protein was purified, and correlations between its activity and enzyme concentration, ATP concentration, substrate concentration and bivalent cations were characterized. An ELISA-based screening system was then established and used to search for inhibitors acting on the tyrosine kinase part of VEGFR-2. More than 600 compounds originating from a variety of microbes have been screened so far, and a number of them have been demonstrated to be potential inhibitors of VEGFR-2 TK.

4849F, a new metabolite produced by the Streptomyces sp. 4849 as an inhibitor of IL-4 receptor.

A new compound named 4849F was isolated from the Streptomyces sp. 4849. The structure of 4849F was elucidated by spectroscopic analyses. The immobilized ligand-binding assay showed that 4849F can competitively inhibit the binding of IL-4 with IL-4 receptor in a dose dependent manner with an IC50 value of 6.7 microM.

Effect of ebosin on modulating interleukin-1ß-induced inflammatory responses in rat fibroblast-like synoviocytes.

The interleukin-1ß-mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways are involved in the pathogenesis of rheumatoid arthritis. Ebosin, a novel exopolysaccharide (EPS), exhibits anti-inflammatory activity in rat collagen-induced arthritis by suppressing the production of tumor necrosis factor-α, interleukin-6 and interleukin-1ß. The aim of the present study was to assess the effects of ebosin on NF-κB and MAPK signaling pathways mediated through interleukin-1ß in rat fibroblast-like synoviocytes (FLSs). Western blotting showed decreased production of phosphorylated p38, JNK1, JNK2, IKKα, IKKß and IκB in the cytoplasm and NF-κB in the nucleus upon ebosin treatment. The DNA-binding activity of NF-κB in the cell nucleus was also inhibited by ebosin treatment, as demonstrated using an electrophoresis mobility gel shift assay. Analysis of the results of the immunofluorescence assay also showed a reduced amount of NF-κB in the nucleus of cells affected by ebosin. These results provided evidence for the effects of ebosin on both interleukin-1ß-mediated MAPK and NF-κB signaling pathways in rat FLSs. In addition, enzyme-linked immunosorbent assay demonstrated that ebosin reduces the levels of matrix metalloproteinases MMP-1 and MMP-3 and the chemokines, interleukin-8 and RANTES. Thus, the results of the present study provide further evidence for understanding the medicinal activity of ebosin at a molecular level, therefore nominating this EPS as a potential therapeutic candidate for the treatment of rheumatic arthritis.Cellular & Molecular Immunology advance online publication, 4 May 2015; doi:10.1038/cmi.2015.36.

[Cloning and identification of the priming glycosyltransferase gene involved in exopolysaccharide 139A biosynthesis in Streptomyces].

Recently in our laboratory, Streptomyces sp. 139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity. The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway, i.e. the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer. Degenerate primers-based PCR approach was adopted to isolate the putative priming glycosyltransferase gene in Streptomyces sp. 139. According to the genes encoding the priming glycosyltransferases that have been identified in several microorganisms, a multiple alignment of the amino acid sequences of these genes was used to identify regions conserved between all genes. To clone the priming glycosyltransferase gene in Streptomyces sp. 139, degenerate primers were designed from these conserved regions taking into account information on Streptomyces codon usage to amplify an internal DNA fragment of this gene. A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces sp. 139 total genomic DNA. Sequence analysis showed that it is part of a putative priming glycosyltransferase gene and contains the predicted conserved domain B. To isolate the complete priming glycosyltransferase gene, a Streptomyces sp. 139 genomic library was constructed in the E. coli--Streptomyces shuttle vector pOJ446. Using the 0.3 kb PCR product of priming glycosyltransferase gene as a probe, 17 positive colonies were isolated by colony hybridization. A 4.0 kb BamHI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed the complete priming glycosyltransferase gene. The priming glycosyltransferase gene ste5 (GenBank under accession number AY131229) most likely begins with GTG, preceded by a probable ribosome binding site (RBS), GGGGA. It encodes a 492-amino-acid protein with molecular weight of 54 kDa and isoelectric point of 10.6. The G + C content of ste5 is 73%, close to the average of G + C content (74%) for Streptomyces. Moreover, the preference usage of G or C as third base of codons are found in the ste5, which is in accordance with the Streptomyces codon usage. A BlastP search showed that the C-terminal region of Ste5 shows highly homology with a number of priming glycosyltransferases from many different organisms. Ste5 contains two putative catalytic residues, Glu and Asp (residues 423 and 474) with a spacing of approximately 50 amino acids that conserved in various beta-glycosyltransferases. Moreover, the C-terminal one third of Ste5 contains three domains, A, B and C that is reported to be common to glycosyltransferases. By hydrophilicity plot prediction, the N-terminal two thirds of Ste5 exhibits 5 putative transmembrane domains. To investigate the involvement of the identified polysaccharide gene cluster in EPS 139A biosynthesis, the gene ste5 encoding priming glycosyltransferase was insertionally disrupted by a single-crossover homologous recombination event. A 0.85 kb internal fragment of ste5 was cloned into vector pKC1139 to yield pLY5015 that was transduced into Streptomyces sp. 139. Correct integration in Streptomyces LY1001 ste5- mutant strain was confirmed by Southern hybridization. After fermentation, no EPS 139A could be detected in the cultures of ste5- mutant strain Streptomyces LY1001. Therefore, the gene ste5 identified in this work is involved in the synthesis of the Streptomyces sp. 139 EPS.

Identification and functional analysis of the gene ste9 involving in Ebosin biosynthesis from Streptomyces sp. 139.

Ebosin is a novel exopolysaccharide produced by Streptomyces sp. 139 with remarkable antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste) consisting of 27 ORFs has been identified. For functional analysis, one of the ste genes, ste9, was disrupted and then the gene complementation was performed. The resultant mutant Streptomyces sp. 139 (ste9(-)) produced polysaccharides with molecular weights of about 4.153 × 10(5) which is much smaller than that of Ebosin (9.03 × 10(5)). The complemented strain Streptomyces sp. 139 (pKC9c) showed recovery in the molecular weights of EPS produced (8.004 × 10(5)). As the theoretical protein product of ste9 is a chain length determinant (Wzz) homologue by sequence similarity, ste9 was cloned and expressed in E. coli 086:H2 (wzz(-)) for a complementation test. SDS-PAGE analysis showed that E. coli 086:H2 (wzz(-)) (pET30a-ste9) produced a modal chain length lipid polysaccharide (LPS) similar to that of the wild-type E. coli 086:H2. In addition, the expression of ste9 was able to restore the serum resistance of E. coli 086:H2 (wzz(-)) to almost the level of the wild-type strain. These results indicate that the ste9 gene is coding for a chain length determinant which plays an important role in Ebosin biosynthesis.

Identification and functional analysis of the chain length determinant gene ste8 involved in the biosynthesis of ebosin by Streptomyces sp. 139.

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (Δwzz). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (Δwzz) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 (ste8(-)) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.