Biochemical and Biophysical Research Communications
Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666
Research highlights
► We cloned, sequenced and analyzed the tirandamycin gene cluster. ► Seven genes were disrupted to support the tirandamycin biosynthetic pathway. ► TrdH as a positive regulator and TrdK as a negative regulator were identified. ► Inactivation of TrdI resulted in accumulation of two products. ► A model for tirandamycin biosynthesis is proposed.
Introduction
The tirandamycins (1–4, Fig. 1) belong to a small group of naturally occurring tetramic acid (2, 4-pyrrolidinedione) antibiotics that exhibited broad biological activities, such as antiviral, antiulcerative, antitumor, and antibacterial properties [1]. The structures of tirandamycins are further featured with a conjugated dienoyltetramic acid chromophore and an intriguing bicyclic ketal scaffold. Other structurally closely related natural products include streptolydigin (6), tirandalydigin (7), Bu-2313A (8), Bu-2313B (Nocamycin I, 9) and Nocamycin II (10) (Fig. 1) [1]. This small focused group of antibiotics all displayed antimicrobial activity by targeting bacterial RNA polymerases (RNAPs) and blocking the transcriptional initiation and elongation process [1], [2].
Tirandamycin A (1) was first isolated in 1971 from Streptomyces tirandis[3] and was re-isolated from Streptomyces flaveolus in 1976 along with tirandamycin B (2) [4]. In recent years, 1 and 2 were again isolated from Streptomyces sp. 307–9 [5] and Streptomyces sp. SCSIO1666 [6], two marine-derived actinomycete bacteria isolated from samples collected in Salt Kay, US Virgin Islands and Sanya Island, South China Sea, respectively. Tirandamycins C (3) and D (4) were also purified as trace elements by using resin-capture method [5]. Our early work has revealed that 2 is the predominant product under normal fermentation conditions in Streptomyces sp. SCSIO 1666, and the yield of 1 was improved 250-fold when XAD-16 resin was added to the fermentation broth to trap biosynthetic intermediates, indicating that 2 is the final and most highly oxidized product of the tirandamycin biosynthetic pathway [6].
Recent years, the complex crystal structures of RNAPs from Escherichia coli and Thermus thermophilus with 6 have been solved, providing molecular evidence for the structural basis of the RNAP interaction mechanism [7]. To date, rifampicin is the only RNAP inhibitor currently used in clinical practice for the treatment of tuberculosis, requiring us to develop more effective RNAP inhibitors to combat the increasing drug resistant bacteria. Understanding the biosynthetic pathway of the structurally unique tetramic acid-containing antibiotics as well as the regulatory and resistant mechanisms would have great impact on guiding us to engineer and design novel analogs of this class as antibacterial agents. Due to the unusual structural features, unique mode of action and potential for drug development, we started to clone the tirandamycin gene cluster from Streptomyces sp. SCSIO1666. While the work was progressing, Salas et al. has reported the biosynthetic gene cluster for 6 from Streptomyces lydicus NRRL2433 and generated four glycosylated derivatives [8], and Sherman et al. has reported a tirandamycin gene cluster from Streptomyces sp. 307–9 [9]. In this paper, we report: (i) the cloning, sequencing, analyses and confirmation of the tirandamycin biosynthetic gene cluster from Streptomyces sp. SCSIO 1666; (ii) the identification of TrdH as a positive regulator and TrdK as a negative regulator involved in the tirandamycin biosynthesis; (iii) inactivation of TrdI resulting in accumulation of 3 and a trace amount of new product tirandamycin C2 (5); and (iv) a proposed model for tirandamycin biosynthesis that is supported by bioinformatics analyses and gene inactivation experiments.
Section snippets
Bacterial strains, plasmids, medium and culture conditions
See Supplementary materials for details.
Construction and screening of genomic library
See Supplementary materials for experimental procedures.
DNA sequencing and assembly of the gene cluster
See Supplementary materials for experimental procedures.
Generation of Streptomyces sp. SCSIO1666 mutant strains
Seven double crossover mutant strains that showed ApramycinRKanmycinS phenotype, Δksor ΔtrdAI (Ju1002), ΔtrdD (Ju1003), ΔtrdB (Ju1004), ΔtrdH (Ju1005), ΔtrdF (Ju1006), ΔtrdK (Ju1007) and ΔtrdI (Ju1008) were obtained, which were further verified by PCR for correction. Detailed experimental procedures were described in Supplementary materials.
Fermentation and analysis of Streptomyces sp. SCSIO1666 and mutants strains
See
Cloning, sequencing and characterizing the tirandamycin biosynthetic gene cluster
Based on the structure feature of tirandamycin and other gene clusters identified in tetramic acid antibiotics [8], [10], it was likely that the tirandamycin is biosynthesized by hybrid polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS). Hence, we used a pair of degenerated primer targeting the conserved region of KS domains of PKS to probe the tirandamycin biosynthetic gene cluster from Streptomyces sp. SCSIO1666 [11]. A single PCR product with the expected size was amplified
Discussion
In this study, we have cloned the tirandamycin biosynthetic gene cluster from marine-derived Streptomyces sp. SCSIO1666. Inactivation of seven representative genes and analyses of their metabolic profiles, coupled with bioinformatics analyses led to a proposed model for tirandamycin biosynthesis.
Tirandamycin is biosynthesized by hybrid PKS–NRPS genes. And the overall PKS–NRPS domain organizations in the tirandamycin gene cluster abide by the colinearity rule. However, two unusual structural
Acknowledgments
We thank Professor Meifeng Tao, Shanghai Jiaotong University, for critical reading and helpful discussion of the manuscript. We thank analytical facility center of South China Sea Institute of Oceanology for recording NMR data. This work is supported in part by Grants from the National Basic Research Program of China (2010CB833805), and the Knowledge Innovation Programs of the Chinese Academy of Sciences (KZCX2-YW-JC202, KSCX2-YW-G-065, LYQY200805, KSCX2-YW-G-073, and KZCX2-EW-G-12). J.J. is a
References (26)
- et al.
Structural basis of transcription inhibition by antibiotic streptolydigin
Mol. Cell
(2005) - et al.
Deciphering biosynthesis of the RNA polymerase inhibitor streptolydigin and generation of glycosylated derivatives
Chem. Biol.
(2009) - et al.
Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety
Chem. Biol.
(2008) - et al.
Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin
J. Biol. Chem.
(2002) - et al.
A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus
J. Biol. Chem.
(2006) - et al.
Characterization of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus unveiling new insights into dialkylmaleic anhydride and polyketide biosynthesis
J. Biol. Chem.
(2008) - et al.
Spiroketal polyketide formation in sorangium: identification and analysis of the biosynthetic gene cluster for the highly cytotoxic spirangienes
Chem. Biol.
(2007) Naturally-occurring tetramic acids – structure, isolation, and synthesis
Chem. Rev.
(1995)Tirandamycin, an inhibitor of bacterial ribonucleic-acid polymerase
Antimicrob. Agents Chemother.
(1976)Tirandamycin, a new antibiotic isolation and characterization
J. Antibiot.
(1971)
Metabolic products of microorganisms .158. Tirandamycin-B
Arch. Microbiol.
Isolation and characterization of tirandamycins from a marine-derived Streptomyces sp.
J. Nat. Prod.
Fermentation optimization, isolation and identification of tirandamycins A and B from marine-derived Streptomyces sp. SCSIO 1666
Chin. J. Mar. Drugs
Cited by (41)
Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi
2022, Natural Product ReportsBiosynthetic strategies for tetramic acid formation
2021, Natural Product ReportsCis double bond formation in polyketide biosynthesis
2021, Natural Product ReportsExpansion of chemical space for natural products by uncommon P450 reactions
2017, Natural Product ReportsCytochromes P450 for natural product biosynthesis in: Streptomyces: sequence, structure, and function
2017, Natural Product ReportsGenerate a bioactive natural product library by mining bacterial cytochrome P450 patterns
2016, Synthetic and Systems BiotechnologyCitation Excerpt :Similarly, the 99% identity between CYP KALB_3295 (in GC10) and Asm30 (CYP in ansamitocin biosynthesis gene cluster94) implies that GC10 may produce epoxide compounds similar to ansamitocin. Similar analysis suggests that tirandamycin-like compounds may be produced through GC19 based on the 66% identity between KALB_3945 and TamI.98,135 Unfortunately, the 27% identity between KALB_5792 (in GC33) and AziB1 (CYP in the azinomycin B biosynthetic gene cluster74), and the 19% identity between KALB_5804 (in GC33) and EpoK (CYP in the epothilone biosynthesis gene cluster63) make it unpredictable for the natural products biosynthesized by GC33.