Strain prioritization and genome mining for enediyne natural products

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family

An experimental study on the flow characteristics during the leakage of high pressure CO 2 pipelines

High pressure pipeline transportation is dominant mode for economically transporting large amounts of CO 2 . However, potential leakage is one of the main risks associated with pressurized CO 2 pipeline transportation. Thus, to investigate the leakage behavior of high pressure CO 2 and to de-risk such transportations, a new laboratory scale experimental setup (total length of 14.85 m and the inner diameter of 15 mm) was constructed. Leakage experiments for pure CO 2 and CO 2 mixtures containing various N 2 concentrations were carried out for different initial phase states (supercritical, liquid and gaseous respectively). The pressure and temperature characteristics and phase transitions of high pressure CO 2 were then studied following pipeline leakage through small diameter nozzles. The results show that a minimum temperature occurs during the leakage process of various initial phase states; while the temperature characteristics of supercritical and gaseous CO 2 leakage were rather different from that of liquid CO 2 leakage. Impacts of impurity (N 2 ), initial inner pressure and nozzle sizes on the leakage behavior were investigated, and two minimum temperature lines, based on N 2 concentrations and initial inner pressures were obtained, respectively. These findings will ascertain that the pipeline is operated above the ductile-brittle transition temperature. © 2019 Institution of Chemical Engineer

Genome Mining of <i>Streptomyces mobaraensis</i> DSM40847 as a Bleomycin Producer Providing a Biotechnology Platform To Engineer Designer Bleomycin Analogues

<i>Streptomyces mobaraensis</i> DSM40847 has been identified by genome mining and confirmed to be a new bleomycin (BLM) producer. Manipulation of BLM biosynthesis in <i>S. mobaraensis</i> has been demonstrated, as exemplified by the engineered production of 6′-deoxy-BLM A2, providing a biotechnology platform for BLM biosynthesis and engineering. Comparison of DNA cleavage efficiency and kinetics among 6′-deoxy-BLM A2 and selected analogues supports the wisdom of altering the disaccharide moiety to fine-tune BLM activity

Type VI secretion system contributes to Enterohemorrhagic <i>Escherichia coli</i> virulence by secreting catalase against host reactive oxygen species (ROS)

<div><p>Enterohemorrhagic <i>Escherichia coli</i> (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of <i>katN</i> is promoted by RpoS and OxyR and repressed by H-NS, and <i>katN</i> contributes to bacterial growth under oxidative stress <i>in vitro</i>. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of <i>katN</i> had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.</p></div

Angucyclines and Angucyclinones from <i>Streptomyces</i> sp. CB01913 Featuring C‑Ring Cleavage and Expansion

Angucyclines and angucyclinones are aromatic polyketides with a tetracyclic benz­[<i>a</i>]­anthracene skeleton. The benz­[<i>a</i>]­anthracene scaffold is biosynthesized by type II polyketide synthases that catalyze the decarboxylative condensation of a short acyl-CoA starter and nine extender units. Angucyclines and angucyclinones, the largest group of polycyclic aromatic polyketides, achieve structural diversity via subsequent oxidation, ring cleavage, amino acid incorporation, and glycosylation. We here report the discovery of 14 angucyclinones and two angucyclines (<b>1</b>–<b>16</b>) from <i>Streptomyces</i> sp. CB01913, identifying 12 new compounds featuring various oxidations on rings A and C (<b>1</b>, <b>2</b>, and <b>4</b>), different sugar moieties attached to rings A and B (<b>3</b> and <b>6</b>), and C-ring cleavage (<b>5</b> and <b>10</b>–<b>14</b>) and expansion (<b>8</b>). These new structural features, highlighted by C-ring cleavage and expansion, enrich the structural diversity of angucyclines and angucyclinones. All compounds were tested for cytotoxicity and antibacterial activities, with <b>1</b>, <b>5</b>, <b>15</b>, and <b>16</b> showing moderate activities against selected cancer cell lines or bacterial strains

The T6SS of EHEC does not show antibacterial activity <i>in vitro</i>.

<p>(A) The positive control of competition assays. <i>A</i>. <i>baylyi</i> ADP1 was used as a prey strain to be killed by the predator strains (<i>P</i>. <i>aeruginosa</i> PAO1, Δ<i>retS</i> or Δ<i>ppkA</i>). The predator strains were individually mixed with <i>A</i>. <i>baylyi</i> ADP1 in 20:1 ratio, and 5 μl of the mixture was spotted on LB agar plate. After incubated at 37°C for 2.5 h, bacterial spots were cut out and the cells were resuspended in 1 ml 1× PBS. The suspensions were diluted serially in 1× PBS, and 5 μl of the suspensions was spotted on the selective LB agar plates, followed by 16 h incubation at 30°C. PAO1 indicates <i>P</i>. <i>aeruginosa</i> PAO1; Δ<i>retS</i> indicates <i>P</i>. <i>aeruginosa</i> PAO1 Δ<i>retS</i>, a T6SS-activated (T6SS⁺) strain; Δ<i>ppkA</i> indicates <i>P</i>. <i>aeruginosa</i> PAO1 Δ<i>ppkA</i>, a T6SS-inactivated (T6SS^-) strain. EHEC T6SS⁺ (B) and T6SS^- (C) can not be killed by T6SS⁺ <i>P</i>. <i>aeruginosa</i>. (D) EHEC T6SS⁺ can not kill T6SS⁺ <i>P</i>. <i>aeruginosa</i>. Δ<i>hns</i> indicates EHEC Δ<i>hns</i>, a T6SS-activated strain. (E) T6SS⁺ EDL933 can not kill T6SS^- <i>E</i>. <i>coli</i> strain MG1655.</p

T6SS contributes to EHEC virulence in mouse model.

<p>(A) ΔT6SS has lower virulence in mice model compared with the WT. The streptomycin pre-treated BALB/c mice were intragastrically infected with the WT or its derived mutants at the inoculation of 10¹⁰ CFU. The percentage of surviving animals (8 mice per group) on each day was calculated. ***, P<0.001, Log-rank analysis. (B) Body weight changes of BALB/c mice infected by the WT, ΔT6SS or <i>ΔkatN</i>. The average body weight change percentage of surviving animals (8 mice per group) on each day was calculated. Error bars represented SD from at least three independent experiments. ***, P<0.001; ns, Not significant, ANOVA analysis. (C) The colonization of the WT, ΔT6SS or <i>ΔkatN</i> in mice gut. At the indicated times, fecal samples were collected, homogenized, diluted and plated on SMAC agar plates to determine the numbers of the WT or its derived mutants. The log₁₀ means of CFU per gram of feces for each group of 8 mice are presented for each time point. Error bars represented SD from at least three independent experiments. ***, P<0.001; ns, Not significant, ANOVA analysis.</p