Production of the siderophore lysochelin in rich media through maltose-promoted high-density growth of Lysobacter sp. 3655.

Siderophores are produced by bacteria in iron-restricted conditions. However, we found maltose could induce the biosynthesis of the siderophore lysochelin in Lysobacter sp. 3655 in rich media that are not compatible with siderophore production. Maltose markedly promoted cell growth, with over 300% increase in cell density (OD600) when LB medium was added with maltose (LBM). While lysochelin was not detectable when OD600 in LBM was below 5.0, the siderophore was clearly produced when OD600 reached 7.5 and dramatically increased when OD600 was 15.0. Coincidently, the transcription of lysochelin biosynthesis genes was remarkably enhanced following the increase of OD600. Conversely, the iron concentration in the cell culture dropped to 1.2 µM when OD600 reached 15.0, which was 6-fold lower than that in the starting medium. Moreover, mutants of the maltose-utilizing genes (orf2677 and orf2678) or quorum-sensing related gene orf644 significantly lowered the lysochelin yield. Transcriptomics analysis showed that the iron-utilizing/up-taking genes were up-regulated under high cell density. Accordingly, the transcription of lysochelin biosynthetic genes and the yield of lysochelin were stimulated when the iron-utilizing/up-taking genes were deleted. Finally, lysochelin biosynthesis was positively regulated by a TetR regulator (ORF3043). The lysochelin yield in orf3043 mutant decreased to 50% of that in the wild type and then restored in the complementary strain. Together, this study revealed a previously unrecognized mechanism for lysochelin biosynthetic regulation, by which the siderophore could still be massively produced in Lysobacter even grown in a rich culture medium. This finding could find new applications in large-scale production of siderophores in bacteria.

An Antifungal Polycyclic Tetramate Macrolactam, Heat-Stable Antifungal Factor (HSAF), Is a Novel Oxidative Stress Modulator in Lysobacter enzymogenes.

Polycyclic tetramate macrolactams (PoTeMs) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeMs have been investigated for potential physiological functions in their producers. Here, we used heat-stable antifungal factor (HSAF), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeMs form complexes with iron ions, with an association constant (Ka ) of 2.71 × 106 M-1 The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stress and exhibited markedly increased production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and restored normal growth in the mutants under the oxidative stress. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damage rather than as an iron-acquisition siderophore. The characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. The study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells.IMPORTANCE PoTeMs are a family of structurally distinct metabolites that have been found in a large number of bacteria. Although PoTeMs exhibit diverse therapeutic properties, the physiological function of PoTeMs in the producer microorganisms had not been investigated. HSAF from Lysobacter enzymogenes is an antifungal PoTeM that has been subjected to extensive studies for mechanisms of biosynthesis, regulation, and antifungal activity. Using HSAF as a model system, we here showed that the characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. In L. enzymogenes, HSAF functions as a small-molecule modulator for oxidative damage caused by iron, H2O2, and UV light. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells. HSAF represents the first member of the fast-growing PoTeM family of microbial metabolites whose potential biological function has been studied.

Identification of the Biosynthetic Gene Cluster for the anti-MRSA Lysocins through Gene Cluster Activation Using Strong Promoters of Housekeeping Genes and Production of New Analogs in Lysobacter sp. 3655.

The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotic biosynthesis using synthetic biology strategies.

LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in Lysobacter enzymogenes OH11.

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf-orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13-14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent.

Numerical study on the impact response of aircraft fuselage structures subjected to large-size tire fragment.

By applying finite element software ANSYS/LS-DYNA, finite element models of front bulkhead and main cabin are established, which aims to assess the dynamic response of fuselage structures impacted by tire fragment under bursting mode. Besides, dynamic characteristics of the two fuselage structures impacted by tire fragment are simulated and critical damage velocities of each working condition are obtained. The results show that composite front bulkhead cannot bear the impact load of front tire fragment at the velocity of 100 m/s, but aluminum alloy front bulkhead can. Main cabin with two properties both can bear the impact loads of front and main tire fragments. When impacted by front tire fragment, critical damage velocity of front bulkhead is approximately half of that of main cabin, while critical damage velocity of aluminum alloy fuselage is larger than that of composite fuselage. However, when impacted by main tire fragment, critical damage velocity of aluminum alloy main cabin is less than that of composite main cabin. Furthermore, maximum contact pressure of composite fuselage is 3-3.3 times than that of aluminum alloy fuselage. The difference in concave deformation is not significant when impacted by front tire fragment, but the difference is great when impacted by main tire fragment.

Initial Crack Propagation and the Influence Factors of Aircraft Pipe Pressure.

In aircraft engineering, an increase of internal pressure in a hydraulic pipe increases the probability of pipe damage, leading to crack propagation becoming a serious issue. In this study, the extended finite element method (XFEM) is applied to simulate initial crack propagation in hydraulic pipes and to investigate the influence factors. Stress intensity factors are extracted to verify the mesh independence of XFEM, which is based on the level set method and unit decomposition method. A total of 30 finite element models of hydraulic pipes with cracks are established. The distribution of von Mises stress under different initial crack lengths and internal pressures is obtained to analyze the change of load-carrying capacity in different conditions. Then, a total of 300 finite element models of hydraulic pipes with different initial crack sizes and locations are simulated under different working conditions. The relationship between the maximum opening displacement and crack length is analyzed by extracting the opening displacement under different initial crack lengths. The length and depth of the initial crack are changed to analyze the factors affecting crack propagation. The opening size and crack propagation length are obtained in different directions. The results show that radial propagation is more destructive than longitudinal propagation for hydraulic pipes in the initial stage of crack propagation.

Systematic optimization for production of the anti-MRSA antibiotics WAP-8294A in an engineered strain of Lysobacter enzymogenes.

WAP-8294A is a group of cyclic lipodepsipeptides and considered as the first-in-class new chemical entity with potent activity against methicillin-resistant Staphylococcus aureus. One of the roadblocks in developing the WAP-8294A antibiotics is the very low yield in Lysobacter. Here, we carried out a systematic investigation of the nutritional and environmental conditions in an engineered L. enzymogenes strain for the optimal production of WAP-8294A. We developed an activity-based simple method for quick screening of various factors, which enabled us to optimize the culture conditions. With the method, we were able to improve the WAP-8294A yield by 10-fold in small-scale cultures and approximately 15-fold in scale-up fermentation. Additionally, we found the ratio of WAP-8294A2 to WAP-8294A1 in the strains could be manipulated through medium optimization. The development of a practical method for yield improvement in Lysobacter will facilitate the ongoing basic research and clinical studies to develop WAP-8294A into true therapeutics.

Spermidine-Regulated Biosynthesis of Heat-Stable Antifungal Factor (HSAF) in Lysobacter enzymogenes OH11.

Heat-Stable Antifungal Factor (HSAF) and its analogs are antifungal natural products produced by the biocontrol agent Lysobacter enzymogenes. The production of HSAF is greatly influenced by environmental stimuli and nutrients, but the underlying molecular mechanism is mostly unclear. Here, we found that HSAF production in L. enzymogenes OH11 is strictly controlled by spermidine, which is the most prevalent triamine in bacteria. When added into OH11 cultures, spermidine regulated the production of HSAF and analogs in a concentration-dependent manner. To verify the role of spermidine, we deleted LeSDC and LeADC genes, encoding S-adenosylmethionine decarboxylase and arginine decarboxylase, respectively, that are the key enzymes for spermidine biosynthesis. Both deletion mutants produced barely detectable spermidine and HSAF including its analogs, whereas the antifungals production was restored by exogenous spermidine. The results showed that the OH11 cells must maintain a proper spermidine homeostasis for the antifungals production. Indeed, the expression level of the key HSAF biosynthetic genes was significantly impaired in LeSDC and LeADC mutants, and exogenous spermidine restored the gene expression level in the mutants. Ornithine is a key substrate for HSAF biosynthesis, and OH11 genome contains arg1 and arg2 genes, encoding arginases that convert arginine to ornithine. While the expression of arg1 and arg2 was affected slightly upon mutation of LeSDC and LeADC, exogenous spermidine significantly increased the arginase gene expression in LeSDC and LeADC mutants. Together, the data revealed a previously unrecognized mechanism, in which spermidine controls antibiotic production through controlling both the biosynthetic genes and the substrate-production genes.

Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in Lysobacter enzymogenes OH11.

The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five cotranscribed genes (orf1-5) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon's promoter region. This led to the transcription of the genes increased by 5-48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (orf6, orf7, orf9 and orf10) by reorganizing them into an operon under the control of a strong Lysobacter promoter, PHSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20-60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2-0.8 µg/mL) and specific activity against S. aureus among a battery of clinically relevant Gram-positive pathogens (54 isolates).

Genetic variant of miR-146a rs2910164 C>G and gastric cancer susceptibility.

The single nucleotide polymorphism (SNP) rs2910164 G>C within miR-146a has been reported that is associated with the increased risk of gastric cancer (GCa). However, the results are inconclusive, espicially among Asian populations, which probably due to small sample size in each single study. To validate this association and get a more precise estimation, we conducted a large GCa study including 1,125 cases and 1,196 controls in an eastern Chinese population. Our results showed that this SNP was not associated with GCa risk in either of the three genetic models [co-dominant model: CG vs. CC, odds ratio (OR) = 0.99, 95% confidence interval (95%CI): 0.83-1.19; GG vs. CC, OR = 1.03, 95%CI: 0.81-1.32; dominant model: (CG+GG) vs. CC, OR = 1.00, 95%CI = 0.84-1.19; recessive model: GG vs. (CG+CC), OR = 1.04, 95%CI = 0.83-1.29]. Stratified analysis by age, gender, smoking status, drinking status, or tumor location confirmed this non-significant association. In summary, these results suggest that the miR-146a SNP rs2910164 may not be a risk factor for GCa in this Chinese population. Larger and well-designed, preferably prospective studies are needed to further confirm our findings.

A Regulatory Gene SCO2140 is Involved in Antibiotic Production and Morphological Differentiation of Streptomyces coelicolor A3(2).

Streptomyces coelicolor is the soil-dwelling bacterium with a complex life cycle and a strong ability to produce plenty of secondary metabolites which are strictly regulated by a variety of regulators. Amino acid alignment shows that the deduced protein of SCO2140 belongs to the family of Leucine-responsive regulatory proteins (Lrps). Disruption of SCO2140 significantly decreased the yields of actinorhodin and calcium-dependent antibiotics, and the complemented strain restored the antibiotic productions to the wild-type level. In contrast, overexpression of SCO2140 increased the actinorhodin production. In agreement with it, the transcriptions of actII-ORF4 and cdaR remarkably reduced in the SCO2140 disruption mutant. The aerial mycelium formation of the SCO2140 disruption mutant was clearly delayed in R2YE medium due to the decrease of ramS expression while its complemented strain could restore the normal formation of aerial mycelia. These results indicated that SCO2140 was involved in antibiotic biosynthesis and morphological differentiation of Streptomyces coelicolor A3(2).

GntR family regulator SCO6256 is involved in antibiotic production and conditionally regulates the transcription of myo-inositol catabolic genes in Streptomyces coelicolor A3(2).

SCO6256 belongs to the GntR family and shows 74% identity with SCO6974, which is the repressor of myo-inositol catabolism in Streptomyces coelicolor A3(2). Disruption of SCO6256 significantly enhanced the transcription of myo-inositol catabolic genes in R2YE medium. The purified recombinant SCO6256 directly bound to the upstream regions of SCO2727, SCO6978 and SCO6985, as well as its encoding gene. Footprinting assays demonstrated that SCO6256 bound to the same sites in the myo-inositol catabolic gene cluster as SCO6974. The expression of SCO6256 was repressed by SCO6974 in minimal medium with myo-inositol as the carbon source, but not in R2YE medium. Glutathione-S-transferase pull-down assays demonstrated that SCO6974 and SCO6256 interacted with each other; and both of the proteins controlled the transcription of myo-inositol catabolic genes in R2YE medium. These results indicated SCO6256 regulates the transcription of myo-inositol catabolic genes in coordination with SCO6974 in R2YE medium. In addition, SCO6256 negatively regulated the production of actinorhodin and calcium-dependent antibiotic via control of the transcription of actII-ORF4 and cdaR. SCO6256 bound to the upstream region of cdaR and the binding sequence was proved to be TTTCGGCACGCAGACAT, which was further confirmed through base substitution. Four putative targets (SCO2652, SCO4034, SCO4237 and SCO6377) of SCO6256 were found by screening the genome sequence of Strep. coelicolor A3(2) based on the conserved binding motif, and confirmed by transcriptional analysis and electrophoretic mobility shift assays. These results revealed that SCO6256 is involved in the regulation of myo-inositol catabolic gene transcription and antibiotic production in Strep. coelicolor A3(2).

Nanocomposite of Au Nanoparticles/Helical Carbon Nanofibers and Application in Hydrogen Peroxide Biosensor.

A combined sol-gel/hydrogen reduction method has been developed for the mass production of helical carbon nanofibers (HCNFs) by the pyrolysis of acetylene at 425 degrees C in the presence of NiO nanoparticles. The synthesized HCNFs were characterized with scanning electron microscopy (SEM), X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). The helical-structured carbon nanofibers have a large specific surface area and excellent biocompatibility. A novel enzymatic hydrogen peroxide sensor was then successfully fabricated based on the nanocomposites containing HCNFs and gold nanoparticles (AuNPs). The results indicated that the Au/HCNFs nanocomposites exhibited excellent electrocatalytic activity to the reduction of H2O2, offering a wide linear range from 1.0 µM to 3157 µM with a detection limit as low as 0.46 µM. The apparent Michaelis-Menten constant of the biosensor was 0.61 mM. The as-fabricated biosensor showed a rapid and sensitive amperometric response to hydrogen peroxide with acceptable preparation reproducibility and excellent stability. Because of their low cost and high stability, these novel HCNFs represent seem to be a kind of promising biomaterial and may find wide new applications in scopes such as biocatalysis, immunoassay, environmental monitoring and so on.

Regulation of myo-inositol catabolism by a GntR-type repressor SCO6974 in Streptomyces coelicolor.

Myo-inositol is important for Streptomyces growth and morphological differentiation. Genomic sequence analysis revealed a myo-inositol catabolic gene cluster in Streptomyces coelicolor. Disruption of the corresponding genes in this cluster abolished the bacterial growth on myo-inositol as a single carbon source. The transcriptions of these genes were remarkably enhanced by addition of myo-inositol in minimal medium. A putative regulatory gene SCO6974, encoding a GntR family protein, is situated in the cluster. Disruption of SCO6974 significantly enhanced the transcription of myo-inositol catabolic genes. SCO6974 was shown to interact with the promoter regions of myo-inositol catabolic genes using electrophoretic mobility shift assays. DNase I footprinting assays demonstrated that SCO6974 recognized a conserved palindromic sequence (A/T)TGT(A/C)N(G/T)(G/T)ACA(A/T). Base substitution of the conserved sequence completely abolished the binding of SCO6974 to the targets demonstrating that SCO6974 directly represses the transcriptions of myo-inositol catabolic genes. Furthermore, the disruption of SCO6974 was correlated with a reduced sporulation of S. coelicolor in mannitol soya flour medium and with the overproduction of actinorhodin and calcium-dependent antibiotic. The addition of myo-inositol suppressed the sporulation deficiency of the mutant, indicating that the effect could be related to a shortage in myo-inositol due to its enhanced catabolism in this strain. This enhanced myo-inositol catabolism likely yields dihydroxyacetone phosphate and acetyl-CoA that are indirect or direct precursors of the overproduced antibiotics.

Disruption of rimP-SC, encoding a ribosome assembly cofactor, markedly enhances the production of several antibiotics in Streptomyces coelicolor.

BACKGROUND: Ribosome assembly cofactor RimP is one of the auxiliary proteins required for maturation of the 30S subunit in Escherichia coli. Although RimP in protein synthesis is important, its role in secondary metabolites biosynthesis has not been reported so far. Considering the close relationship between protein synthesis and the production of secondary metabolites, the function of ribosome assembly cofactor RimP on antibiotics production was studied in Streptomyces coelicolor and Streptomyces venezuelae. RESULTS: In this study, the rimP homologue rimP-SC was identified and cloned from Streptomyces coelicolor. Disruption of rimP-SC led to enhanced production of actinorhodin and calcium-dependent antibiotics by promoting the transcription of actII-ORF4 and cdaR. Further experiments demonstrated that MetK was one of the reasons for the increment of antibiotics production. In addition, rimP-SC disruption mutant could be used as a host to produce more peptidyl nucleoside antibiotics (polyoxin or nikkomycin) than the wild-type strain. Likewise, disruption of rimP-SV of Streptomyces venezuelae also significantly stimulated jadomycin production, suggesting that enhanced antibiotics production might be widespread in many other Streptomyces species. CONCLUSION: These results established an important relationship between ribosome assembly cofactor and secondary metabolites biosynthesis and provided an approach for yield improvement of secondary metabolites in Streptomyces.

Synthesis of four natural prenylflavonoids and their estrogen-like activities.

Four prenylflavonoids, bavachin 1, isobavachin 2, 7,4'-dihydroxy-8-prenylflavone 3, and 8-prenylapigenin 4 were synthesized and recognized for possessing estrogen-like activity in MCF-7/BOS cells, as evaluated by an estrogen-screening assay. All compounds significantly stimulated the proliferation of MCF-7/BOS cells in a dose-dependent manner. Isobavachin 2 showed the most potent activity, while bavachin 1 was the weakest. The estrogenic potency of these compounds is ranked as follows: 2 > 4 > 3 > 1.