Genome Sequencing of Streptomyces olivaceus SCSIO T05 and Activated Production of Lobophorin CR4 via Metabolic Engineering and Genome Mining

Marine-sourced actinomycete genus Streptomyces continues to be an important source of new natural products. Here we report the complete genome sequence of deep-sea-derived Streptomyces olivaceus SCSIO T05, harboring 37 putative biosynthetic gene clusters (BGCs). A cryptic BGC for type I polyketides was activated by metabolic engineering methods, enabling the discovery of a known compound, lobophorin CR4 (1). Genome mining yielded a putative lobophorin BGC (lbp) that missed the functional FAD-dependent oxidoreductase to generate the d-kijanose, leading to the production of lobophorin CR4 without the attachment of d-kijanose to C17-OH. Using the gene-disruption method, we confirmed that the lbp BGC accounts for lobophorin biosynthesis. We conclude that metabolic engineering and genome mining provide an effective approach to activate cryptic BGCs

Identification and utilization of two important transporters: SgvT1 and SgvT2, for griseoviridin and viridogrisein biosynthesis in Streptomyces griseoviridis

Abstract Background Griseoviridin (GV) and viridogrisein (VG, also referred as etamycin), both biosynthesized by a distinct 105 kb biosynthetic gene cluster (BGC) in Streptomyces griseoviridis NRRL 2427, are a pair of synergistic streptogramin antibiotics and very important in treating infections of many multi-drug resistant microorganisms. Three transporter genes, sgvT1–T3 have been discovered within the 105 kb GV/VG BGC, but the function of these efflux transporters have not been identified. Results In the present study, we have identified the different roles of these three transporters, SgvT1, SgvT2 and SgvT3. SgvT1 is a major facilitator superfamily (MFS) transporter whereas SgvT2 appears to serve as the sole ATP-binding cassette (ABC) transporter within the GV/VG BGC. Both proteins are necessary for efficient GV/VG biosynthesis although SgvT1 plays an especially critical role by averting undesired intracellular GV/VG accumulation during biosynthesis. SgvT3 is an alternative MFS-based transporter that appears to serve as a compensatory transporter in GV/VG biosynthesis. We also have identified the γ-butyrolactone (GBL) signaling pathway as a central regulator of sgvT1–T3 expression. Above all, overexpression of sgvT1 and sgvT2 enhances transmembrane transport leading to steady production of GV/VG in titers ≈ 3-fold greater than seen for the wild-type producer and without any notable disturbances to GV/VG biosynthetic gene expression or antibiotic control. Conclusions Our results shows that SgvT1–T2 are essential and useful in GV/VG biosynthesis and our effort highlight a new and effective strategy by which to better exploit streptogramin-based natural products of which GV and VG are prime examples with clinical potential

Enhancement of himastatin bioproduction via inactivation of atypical repressors in Streptomyces hygroscopicus

Three atypical regulatory genes, hmtABD have been discovered within the himastatin biosynthetic gene cluster (BGC) in Streptomyces hygroscopicus ATCC 53653 and the roles of their products have been identified. HmtA and HmtD do not show any structurally distinct features characteristic of regulatory function yet were shown to play important repressive and stimulatory roles, respectively, related to himastatin biosynthesis. HmtB encodes a conserved acetylglutamate kinase; new member of this family serves as repressor of secondary metabolism. Through repressive networks engineering, the limiting functions of HmtA and HmtB along with the activating functions of HmtD in the himastatin BGC have been identified for the first time by gene activation, qPCR, RT-PCR and HPLC studies of selected mutant strains; two of these mutant strains (ΔhmtA and ΔhmtB) produced himastatin in titers (19.02 ± 1.2 μg/mL, 9.9 folds and 30.40 ± 0.83 μg/mL, 15.8 folds) far exceeding those of the wild-type (WT) producer. Overall, this work provides significant insight into secondary metabolic regulatory mechanisms in Streptomyces. These efforts also highlight and validate a new strategy enabling expanded exploitation of cyclopeptidic natural products such as himastatin that demonstrate exciting antimicrobial and antitumor potentials. Keywords: Himastatin, Streptomyces hygroscopicus, Repressor, Biosynthesis, Bioproductio

Coordinated Action of NRPS, Baeyer-Villiger Monooxygenase, and Methyltransferase Ensures the Economical Biosynthesis of Bohemamines in Streptomyces sp. CB02009

Bohemamines (BHMs) are bacterial alkaloids containing a pyrrolizidine core with two unprecedented methyl groups. Herein we report the activation of BHMs biosynthesis in Streptomyces sp. CB02009 using a ribosome engineering approach. Identification and characterization of the bhm gene cluster reveals a coordinated action of nonribosomal peptide synthetase BhmJ, Baeyer-villiger monooxygenase BhmK and methyltransferase BhmG for BHMs biosynthesis. BhmG is responsible for the C-methylation on C-7, while the C-9 methyl group is from a non-proteinogenic amino acid (2S,5S)-5-methylproline, required for BHMs production in three model Streptomyces hosts. Our study shed light on the intricate interaction of BhmJ/BhmK/BhmG for the economical biosynthesis of BHMs in their native producer, and also unraveled that BhmJ and BhmK are competent biocatalysts in Streptomyce albus.</p

MOESM1 of Identification and utilization of two important transporters: SgvT1 and SgvT2, for griseoviridin and viridogrisein biosynthesis in Streptomyces griseoviridis

Additional file 1: Figure S1. The multiple alignment of SgvT1/T3 with other transporters. Figure S2. The multiple alignment of SgvT2 with other transporters. Figures S3–S5. The inactivation of sgvT1-T3. Figure S6–S8. HPLC analyses of the fermentation extract of Wild-type & ΔsgvT1-T3. Figure S9. HPLC analyses of the fermentation extract of WT::sgvT1–T2. Figure S10. The HPLC standard curve of GV/ VG. Figure S11. HPLC analyses of fermentation extract of complemented mutants. Table S1. Primer pairs used for PCR-targeting of sgvT1–T3. Table S2. Primers used for PCR confirmation of double-crossover mutants. Table S3. Primer pairs used for complementation of sgvT1–T3. Table S4. Primer pairs used for RT-PCR. Table S5. Primer pairs used for qPCR. Table S6. Quantitative analysis of GV/VG production

Syntheses, Structures, and Properties of Four Metal–Organic Frameworks Based on a N‑Centered Multidentate Pyridine-Carboxylate Bifunctional Ligand

Four new metal–organic frameworks have been synthesized under hydrothermal conditions by the self-assembly of a N-centered multidentate bifunctional ligand (H₂L: 4,4′-((4-(pyridin-4-yl)­phenyl)­azanediyl)­dibenzoic acid) with different N-containing heterocyclic coligands (BIMB = 4,4′-bis­((imidazol-1-yl)­methyl)­biphenyl, BPY = 4,4′-bipyridine, BIP = 1,4-bis­(imidazol-1-yl)­phenyl) and transition metal salts (Co­(NO₃)₂·6H₂O, Zn­(NO₃)₂·6H₂O). They are [Co­(L)­(BIMB)_0.5­(H₂O)]·2DMF (<b>1</b>), [Co­(L)­(BPY)_0.5­(H₂O)₂]­·3DMF­·2H₂O (<b>2</b>), [Zn₂(L)₂]­·4DMF­·3H₂O (<b>3</b>), and [Zn₂(L)₂(BIP)]­·2DMF­·2H₂O (<b>4</b>). Compound <b>1</b> is an infrequent 3,4-connected self-penetration three-dimensional (3D) architecture. Compound <b>2</b> has been classified as a trinodal (3,4,4)-connected 3D <b>sqc69</b> framework. Compound <b>3</b> displays a 2-fold interpenetrating 3D net based on the [Zn₂(CO₂)₄] units which are connected by L^2– ligands. For compound <b>4</b>, the larger potential voids lead to a 4-fold interpenetration, which can be classified as type <b>IIIa</b>. In addition, their thermal stability, the adsorption isotherms, and optical properties have been studied in detail

Discovery of a New Family of Dieckmann Cyclases Essential to Tetramic Acid and Pyridone-Based Natural Products Biosynthesis

Bioinformatic analyses indicate that TrdC, SlgL, LipX₂, KirHI, and FacHI belong to a group of highly homologous proteins involved in biosynthesis of actinomycete-derived tirandamycin B, streptolydigin, α-lipomycin, kirromycin, and factumycin, respectively. However, assignment of their biosynthetic roles has remained elusive. Gene inactivation and complementation, <i>in vitro</i> biochemical assays with synthetic analogues, point mutations, and phylogenetic tree analyses reveal that these proteins represent a new family of Dieckmann cyclases that drive tetramic acid and pyridone scaffold biosynthesis

The Isolation of Pyrroloformamide Congeners and Characterization of Its Biosynthetic Gene Cluster from Streptomyces sp. CB02980 Revealed a Unified Mechanism for Dithiolopyrrolone Biosynthesis

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. In the current study, two new dithiolopyrrolones, pyrroloformamide C (3) and pyrroloformamide D (4), were isolated from Streptomyces sp. CB02980, together with the known pyrroloformamides 1 and 2. The biosynthetic gene cluster for pyrroloformamides was identified from S. sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a non-ribosomal peptide synthetase, abolished the production of 1－4. Overexpression of pyfN, a type II thioesterase gene, increased the production of 1 and 2. The structure elucidation and biosynthetic characterization of pyrroloformamides 1 - 4 may inspire future efforts to discover new dithiolopyrrolones, which are promising drug leads for the treatment of infectious diseases or cancer

Molecular characterization of hybrid virulence plasmids in ST11-KL64 KPC-2-producing multidrug-resistant hypervirulent Klebsiella pneumoniae from China

IntroductionCarbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aim of this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids.MethodsThe resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids.ResultsTwo ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC−2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC−2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid.ConclusionOur studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed