A new peucemycin derivative and impacts of peuR and bldA on peucemycin biosynthesis in Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is known to produce a variety of secondary metabolites, including two important antitumor anthracyclines: daunorubicin and doxorubicin. Identification of peucemycin and 25-hydroxy peucemycin (peucemycin A), as well as their biosynthetic pathway, has expanded its biosynthetic potential. In this study, we isolated a new peucemycin derivative and identified it as 19-hydroxy peucemycin (peucemycin B). Its antibacterial activity was lower than those of peucemycin and peucemycin A. On the other hand, this newly identified peucemycin derivative had higher anticancer activity than the other two compounds for MKN45, NCI-H1650, and MDA-MB-231 cancer cell lines with IC50 values of 76.97 µM, 99.68 µM, and 135.2 µM, respectively. Peucemycin biosynthetic gene cluster revealed the presence of a SARP regulator named PeuR whose role was unknown. The presence of the TTA codon in the peuR and the absence of global regulator BldA in S. peucetius reduced its ability to regulate the peucemycin biosynthetic gene cluster. Hence, different mutants harboring these genes were prepared. S. peucetius bldA25 harboring bldA produced 1.75 times and 1.77 times more peucemycin A (11.8 mg/L) and peucemycin B (21.2 mg/L), respectively, than the wild type. On the other hand, S. peucetius R25 harboring peuR produced 1.86 and 1.79 times more peucemycin A (12.5 mg/L) and peucemycin B (21.5 mg/L), respectively, than the wild type. Finally, strain S. peucetius bldAR25 carrying bldA and peuR produced roughly 3.52 and 2.63 times more peucemycin A (23.8 mg/L) and peucemycin B (31.5 mg/L), respectively, than the wild type. KEY POINTS: • This study identifies a new peucemycin derivative, 19-hydroxy peucemycin (peucemycin B). • The SARP regulator (PeuR) acts as a positive regulator of the peucemycin biosynthetic gene cluster. • The overexpression of peuR and heterologous expression of bldA increase the production of peucemycin derivatives.

Biosynthetic pathway of peucemycin and identification of its derivative from Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is a well-known producer of important anticancer compounds, daunorubicin and doxorubicin. In this study, we successfully identified a new macrolide, 25-hydroxy peucemycin, that exhibited an antibacterial effect on some pathogens. Based on the structure of a newly identified compound and through the inactivation of a polyketide synthase gene, we successfully identified its biosynthetic gene cluster which was considered to be the cryptic biosynthetic gene cluster. The biosynthetic gene cluster spans 51 kb with 16 open reading frames. Five type I polyketide synthase (PKS) genes encode eight modules that synthesize the polyketide chain of peucemycin before undergoing post-PKS tailoring steps. In addition to the regular starter and extender units, some modules have specificity towards ethylmalonyl-CoA and unusual butylmalonyl-CoA. A credible explanation for the specificity of the unusual extender unit has been searched for. Moreover, the enzyme responsible for the final tailoring pathway was also identified. Based on all findings, a plausible biosynthetic pathway is here proposed. KEY POINTS: • Identification of a new macrolide, 25-hydroxy peucemycin. • An FMN-dependent monooxygenase is responsible for the hydroxylation of peucemycin. • The module encoded by peuC is unique to accept the butylmalonyl-CoA as an unusual extender unit.

Genome mining Streptomyces sp. KCTC 0041BP as a producer of dihydrochalcomycin.

Streptomyces sp. KCTC 0041BP, which was isolated from a soil sample in Cheolwon, Republic of Korea, is a dihydrochalcomycin producer. In this study, we obtained the genome of S. sp. KCTC 0041BP with 7.54 Mb genome size. antiSMASH and the dbCAN2 meta server predicted that the genome would contain 26 secondary metabolite biosynthetic gene clusters (BGCs) and 285 carbohydrate-active enzymes. Besides dihydrochalcomycin, 21 compounds were successfully identified from S. sp. KCTC 0041BP, and among them, the structure of 8 compounds were proven by high-resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR). The identification of chalcomycin analogs led to a better understanding of the biosynthetic pathway of dihydrochalcomycin/chalcomycin. From the analysis of cluster 2 and solvent selection, linearmycins were determined. Linearmycins showed antibacterial activity with both Gram-positive and Gram-negative bacteria and antifungal activity. One strain many compounds (OSMAC) strategy was applied to activate the salicylic acid production in this strain. A salicylic acid biosynthetic pathway was also predicted, but not by antiSMASH. These results showed that this strain can produce many useful compounds and potentially produce novel compounds with most secondary BGCs yet to be experimentally identified.

Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Bioactivities of ß-mangostin and its new glycoside derivatives synthesized by enzymatic reactions.

Beta-mangostin is a xanthone commonly found in the genus Garcinia. Unlike α-mangostin, to date, there have only been a few studies on the biological activity and derivatization of ß-mangostin. In this study, two novel glycosylated derivatives of ß-mangostin were successfully synthesized via a one-pot enzymatic reaction. These derivatives were characterized as ß-mangostin 6-O-ß-d-glucopyranoside and ß-mangostin 6-O-ß-d-2-deoxyglucopyranoside by TOF ESI/MS and 1H and 13C NMR analyses. Beta-mangostin showed cytotoxicity against KB, MCF7, A549 and HepG2 cancer cell lines, with IC50 values ranging from 15.42 to 21.13 µM. The acetylcholinesterase and α-glucosidase inhibitory activities of ß-mangostin were determined with IC50 values of 2.17 and 27.61 µM, respectively. A strong anti-microbial activity of ß-mangostin against Gram-positive strains (Bacillus subtilis, Lactobacillus fermentum and Staphylococcus aureus) was observed, with IC50 values of 0.16, 0.18 and 1.24 µg ml-1, respectively. Beta-mangostin showed weaker activity against Gram-negative strains (Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa) as well as Candida albicans fungus, with IC50 and MIC values greater than the tested concentration (greater than 32 µg ml-1). The new derivatives of ß-mangostin showed weaker activities than those of ß-mangostin, demonstrating the important role of the hydroxyl group at C-6 of ß-mangostin in its bioactivity.

Identification and enhancing production of a novel macrolide compound in engineered Streptomyces peucetius.

Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines.

Recent Advances in Strategies for Activation and Discovery/Characterization of Cryptic Biosynthetic Gene Clusters in Streptomyces.

Streptomyces spp. are prolific sources of valuable natural products (NPs) that are of great interest in pharmaceutical industries such as antibiotics, anticancer chemotherapeutics, immunosuppressants, etc. Approximately two-thirds of all known antibiotics are produced by actinomycetes, most predominantly by Streptomyces. Nevertheless, in recent years, the chances of the discovery of novel and bioactive compounds from Streptomyces have significantly declined. The major hindrance for obtaining such bioactive compounds from Streptomyces is that most of the compounds are not produced in significant titers, or the biosynthetic gene clusters (BGCs) are cryptic. The rapid development of genome sequencing has provided access to a tremendous number of NP-BGCs embedded in the microbial genomes. In addition, the studies of metabolomics provide a portfolio of entire metabolites produced from the strain of interest. Therefore, through the integrated approaches of different-omics techniques, the connection between gene expression and metabolism can be established. Hence, in this review we summarized recent advancements in strategies for activating cryptic BGCs in Streptomyces by utilizing diverse state-of-the-art techniques.

Streptomyces sp. VN1, a producer of diverse metabolites including non-natural furan-type anticancer compound.

Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.