Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-L-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo.

The polyoxin (POL) biosynthetic gene cluster (pol) was recently cloned from Streptomyces cacaoi subsp. asoensis. A 3.3 kb DNA fragment carrying an obvious open reading frame (polR), whose deduced product shows sequence similarity to SanG of Streptomyces ansochromogenes and PimR of Streptomyces natalensis, was revealed within the pol gene cluster. Disruption of polR abolished POL production, which could be complemented by the integration of a single copy of polR into the chromosome of the non-producing mutant. The introduction of an extra copy of polR in the wild-type strain resulted in increased production of POLs. The transcription start point (tsp) of polR was determined by S1 mapping. Reverse transcriptase PCR experiments showed that PolR is required for the transcription of 18 structural genes in the pol gene cluster. Furthermore, we showed that polC and polB, the respective first genes of two putative operons (polC–polQ2 and polA–polB) consisting of 16 and 2 of these 18 genes, have similar promoter structures. Gel retardation assays indicated that PolR has specific DNA-binding activity for the promoter regions of polC and polB. Our data suggest that PolR acts in a positive manner to regulate POL production by activating the transcription of at least two putative operons in the pol gene cluster.

Natural products are critical for drug discovery and development; however their discovery is challenged by the wide inactivation or silence of microbial biosynthetic pathways. Currently strategies targeting this problem are mainly concentrated on chromosome dissembling, transcription, and translation-stage regulations as well as chemical stimulation. In this study, we developed a novel approach to awake cryptic/silenced microbial biosynthetic pathways through augmentation of the conserved protein modification step-phosphopantetheinylation of carrier proteins. Overexpression of phosphopantetheinyl transferase (Pptase) genes into 33 Actinomycetes achieved a significantly high activation ratio at which 23 (70%) strains produced new metabolites. Genetic and biochemical studies on the mode-of-action revealed that exogenous PPtases triggered the activation of carrier proteins and subsequent production of metabolites. With this approach we successfully identified five oviedomycin and halichomycin-like compounds from two strains. This study provides a novel approach to efficiently activate cryptic/silenced biosynthetic pathways which will be useful for natural products discovery.

SummaryMinimycin (MIN) is a C-nucleoside antibiotic structurally related to pseudouridine, and indigoidine is a naturally occurring blue pigment produced by diverse bacteria. Although MIN and indigoidine have been known for decades, the logic underlying the divergent biosynthesis of these interesting molecules has been obscure. Here, we report the identification of a minimal 5-gene cluster (min) essential for MIN biosynthesis. We demonstrated that a non-ribosomal peptide synthetase (MinA) governs “the switch” for the divergent biosynthesis of MIN and the cryptic indigoidine. We also demonstrated that MinCN (the N-terminal phosphatase domain of MinC), MinD (uracil phosphoribosyltransferase), and MinT (transporter) function together as the safeguard enzymes, which collaboratively constitute an unusual self-resistance system. Finally, we provided evidence that MinD, utilizing an unprecedented substrate-competition strategy for self-resistance of the producer cell, maintains competition advantage over the active molecule MIN-5′-monophosphate by increasing the UMP pool in vivo. These findings greatly expand our knowledge regarding natural product biosynthesis.

BackgroundAurantimycin (ATM), produced by Streptomyces aurantiacus JA 4570, is a potent antimicrobial and antitumor antibiotic. Although the chemical structure of ATM is highly distinctive and features a cyclohexadepsipeptide scaffold attached with a C14 acyl side chain, little is known about its biosynthetic pathway and regulatory mechanism.ResultsIn this work, we report the identification and characterization of the ATM biosynthetic gene cluster from S. aurantiacus JA 4570. Targeted inactivation of artG, coding for a NRPS enzyme, completely abolished ATM production, thereof demonstrating the target gene cluster (art) is responsible for ATM biosynthesis. Moreover, four NRPS adenylation (A) domains including a freestanding enzyme ArtC have been characterized in vitro, whose substrate specificities are consistent with in silico analysis. Further genetic analysis of the two regulatory genes artB and artX unambiguously suggested both of them play positive roles in ATM biosynthesis, and ATM-A production was thus rationally enhanced to about 2.5 fold via tandem overexpression of artB and artX in S. aurantiacus JA 4570.ConclusionsThese results will provide the basis for the understanding of precise mechanisms for ATM biosynthesis, and open the way for both rational construction of high-production ATM producer and orient-directed generation of designer ATM derivatives via synthetic biology strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0559-7) contains supplementary material, which is available to authorized users.

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Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria and propose that their biosynthesis is likely initiated by a lysine N6-monooxygenase. Moreover, we show that forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of β-RFA-P (β-ribofuranosyl-aminobenzene 5ʹ-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics and also open the way for target-directed genome mining of PRF-A/FOR-A-related antibiotics. IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of a C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand the biochemical repertoire for novel enzymatic reactions but also permit target-oriented genome mining of FOR-A/PRF-A-related C-nucleoside antibiotics. Moreover, the availability of FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunAtunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.