Lysobacter harbors a plethora of cryptic biosynthetic gene clusters (BGCs), albeit only a limited number have been analyzed to date. In this study, we described the activation of a cryptic polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene cluster (lsh) in Lysobacter sp. DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp. S001. As a result of this methodology, we were able to isolate two novel linear lipopeptides, lysohexaenetides A (1) and B (2), from the recombinant strain S001-lsh. Furthermore, we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs. This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes, particularly in the absence of genetic manipulation tools.
Lysobacter/metabolism* ; Streptomyces/metabolism* ; Lipopeptides/metabolism* ; Polyketide Synthases/genetics* ; Multigene Family

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.
Amino Acids ; Bacillus subtilis/metabolism* ; Culture Media ; Fermentation ; Lipopeptides ; Peptides, Cyclic

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.
Anti-Bacterial Agents ; biosynthesis ; Chromatography, High Pressure Liquid ; Lipopeptides ; biosynthesis ; Paenibacillus ; metabolism ; Protoplasts ; metabolism ; Real-Time Polymerase Chain Reaction

Biosurfactants (BSs) are highlighted owing to their multiple advantages in diverse applications. To screen a superior strain that producing a blend-biosurfactant of lipopeptide and glycolipid, the hemolytic activity assay on blood agar plates, the modified oil-red spreading test and MALDI-TOF Mass Spectrometry identification of the purified products was carried out. Bacillus subtilis THY-7 was selected and its principal products were surfactin and dirhamnolipid. The medium component and culture conditions of THY-7 were optimized by both single factor and orthogonal experiments. After 48 h optimal batch culture in flask, the cell density (OD600) was 37.0 and the product titer was 2.4 g/L, which was 3.4 folds and 3.1 folds of that under original condition, respectively. A fed-batch culture in a 5 L fermentor was further performed coupling with in situ recovery of foam, in which the titer of blend-BS increased to 4.5 g/L at 25 h. Quantification by HPLC and anthrone colorimetry revealed that surfactin and dirhamnolipid accounted for 74% and 22% of the blend-BS, respectively.
Bacillus subtilis ; growth & development ; isolation & purification ; metabolism ; Chromatography, High Pressure Liquid ; Culture Media ; Glycolipids ; biosynthesis ; Industrial Microbiology ; Lipopeptides ; biosynthesis ; Surface-Active Agents ; metabolism

Isolation and idenfication of lipopeptides from Bacillus subtilis fmbJ was carried out in this paper. With HPLC method, it was determined that the antimicrobial substance was composed of many components, and one of them had the similar retention time similar to surfactin. In addition, the antimicrobial substance was proved to include the closed cycle peptide bind by TLC, and one of them had the migrating rate similar to surfactin. Furthermore, ESI-MS analysis showed that the antimicrobial substance contained five homologues of fengycin, such as m/z1449.9, m/zl1463.8, m/zl1477.8, m/z1491.9 and m/z1505.9, and three homologues of surfactin, such as m/z1008.8, m/z1022.8 and m/z1036.8.
Anti-Infective Agents ; chemistry ; isolation & purification ; Bacillus subtilis ; metabolism ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Lipopeptides ; chemistry ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization

Panax ginseng is one of the most important traditional Chinese herbal medicine, soil borne diseases influenced the yield and quality severely. In our previous work, endophytic Bacillus subtilis ge25 strain was isolated from ginseng root, and which showed significant antagonistic activity against several most destructive ginseng phytopathogens. In the present work, crude protein and lipopeptid extracts were prepared from LB and Landy supernate by salting out, acid precipitation methods respectively. The antagonistic activity of crude extracts and stability to temperature and protease digestion were examined by ginseng phytopathogen Alternaria panax. Results showed that, the antagonistic activity of crude protein extracts from LB culture was complete and partially lost when treated by high temperature and proteinase K. However, crude lipopeptid from Landy culture showed significant stabile antagonistic activity to them. Acid-hydrolyzation and TLC-bioautography analysis showed, that the crude lipopeptide contained at least one cyclic lipopeptide. In consideration of the stability and perfect antagonistic activity of ge25, further researches will promote the biocontrol of ginseng diseases in the field.
Alternaria ; drug effects ; physiology ; Bacillus subtilis ; metabolism ; physiology ; Bacterial Proteins ; isolation & purification ; metabolism ; pharmacology ; Endopeptidase K ; metabolism ; Endophytes ; metabolism ; physiology ; Fermentation ; Lipopeptides ; isolation & purification ; pharmacology ; Panax ; microbiology ; Plant Roots ; microbiology ; Temperature

BACKGROUNDHepatitis B virus (HBV) x protein (HBx) in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 amino acid residues of HBx has been identified. In this study, a peptide chain derived from the Kunitz domain (PKD) was used to study its effect on the cell cycle and apoptosis of HepG2 cells, and investigated the function of PKD on the activities of proteasomes and AAA-ATPase p97, which involves in the ubiquitin-proteasome protein degradation pathway.

METHODSThe PKD peptide (Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys) was chemically synthesized. MTT assays were used to determine the effects of PKD on HepG2 cell growth. Mouse anti-p97 antibody was developed for Western blotting to detect the expression of p97. ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes were analyzed with various peptidase-specific fluorogenic peptide substrates. Flow cytometry was used to determinate cell cycle phase and apoptosis.

RESULTSViability of HepG2 cells decreased in a PKD-dose-dependent manner. Cells exhibited significant cytotoxicity in the presence of 15 mmol/L of PKD. Western blotting analysis showed that expression of p97 was suppressed in HepG2 cells treated with PKD compared to untreated cells. The ATPase activity of proteasomes from immunoprecipitates of HepG2 cells pretreated with PKD was apparently decreased. Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10 mmol/L PKD; tryptic activity and peptidylglutamyl peptide hydrolase activity of proteasomes were less inhibited by PKD than chymotryptic activity. The cell cycle phase of HepG2 cells treated with PKD for 36 hours was blocked largely at the G(0)-G(1) phase, while untreated control cells were mainly in S phase. PKD also significantly induced apoptosis.

CONCLUSIONSThe peptide derived from Kunitz domain of HBx protein induces HepG2 cell growth arrest and apoptosis, which may result from down-regulation of p97 expression, and decrease of both the ATPase and chymotryptic activities of proteasomes.

Adenosine Triphosphatases ; metabolism ; Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Lipopeptides ; chemistry ; pharmacology ; Mice ; Nuclear Proteins ; metabolism ; Trans-Activators ; chemistry ; Viral Regulatory and Accessory Proteins ; chemistry

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C(13)-lipopeptide < C(14)-lipopepitde < C(15)-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC(50) values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.
Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; Bacillus subtilis ; Cell Cycle ; Cell Membrane ; chemistry ; metabolism ; Cell Survival ; drug effects ; Fatty Acids ; chemistry ; metabolism ; HeLa Cells ; Humans ; Lipopeptides ; chemistry ; pharmacology ; Ploidies ; Tumor Cells, Cultured

OBJECTIVETo investigate whether signaling through Toll-like receptor-2 (TLR-2) can affect the expression of some cytokines in human gingival fibroblasts.

METHODSThe gingival fibroblasts were isolated and cultured in vivo, divided into blank control group, lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) group and Escherichia coli (Ec) group. mRNA expression levels were measured by real-time polymerase chain reaction (PCR). The protein expression levels were detected by the enzyme linked immunosorbent assay (ELISA). The data was statistically analyzed by SPSS16.0 software package.

RESULTSLPS from Pg could stimulate the expression of interleukin (IL)-6 and leukemia inhibitory factor (LIF) mRNA and protein, which reached the peak (5.87 ± 0.83) at 10 h, and the expression level increased with the increase of the Pg concentration. IL-11 or oncostatin-M (OSM) mRNA expression was not affected by LPS. After treated with Pg for 48 h, the protein expression of IL-6 and LIF was up-regulated, (962 ± 57) ng/L and (47 ± 18) ng/L respectively.

CONCLUSIONSSignaling through TLR-2 controls the expression of cytokines of IL-6 family in human gingival fibroblasts.

Adolescent ; Adult ; Cells, Cultured ; Child ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Humans ; Interleukin-11 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia Inhibitory Factor ; genetics ; metabolism ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; administration & dosage ; isolation & purification ; pharmacology ; Oncostatin M ; genetics ; metabolism ; Porphyromonas gingivalis ; chemistry ; RNA, Messenger ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; agonists ; Young Adult