Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in either condition, which enabled precise determination of the 5'-UTR length of the corresponding transcripts as well as the identification of 2 consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including 3 examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster to support future regulatory studies, genetic manipulation, and industrial production.

Biosynthesis of the ß-Lactone Proteasome Inhibitors Belactosin and Cystargolide.

Belactosins and cystargolides are natural product proteasome inhibitors from Actinobacteria. Both feature dipeptidic backbones and a unique ß-lactone building block. Herein, we present a detailed investigation of their biosynthesis. Identification and analysis of the corresponding gene clusters indicated that both compounds are assembled by rare single-enzyme amino acid ligases. Feeding experiments with isotope-labeled precursors and in vitro biochemistry showed that the formation of the ß-lactone warhead is unprecedented and reminiscent of leucine biosynthesis, and that it involves the action of isopropylmalate synthase homologues.

Biosynthetic Origin of the Antibiotic Pseudopyronines†A and B in Pseudomonas putida BW11M1.

Within the framework of our effort to discover new antibiotics from pseudomonads, pseudopyronines A and B were isolated from the plant-derived Pseudomonas putida BW11M1. Pseudopyronines are 3,6-dialkyl-4-hydroxy-2-pyrones and displayed high in vitro activities against several human pathogens, and in our hands also towards the plant pathogen Pseudomonas savastanoi. Here, the biosynthesis of pseudopyronine B was studied by a combination of feeding experiments with isotopically labeled precursors, genomic sequence analysis, and gene deletion experiments. The studies resulted in the deduction of all acetate units and revealed that the biosynthesis of these α-pyrones occurs with a single PpyS-homologous ketosynthase. It fuses, with some substrate flexibility, a 3-oxo-fatty acid and a further unbranched saturated fatty acid, both of medium chain-length and provided by primary metabolism.

A membrane-bound prenyltransferase catalyzes the O-prenylation of 1,6-dihydroxyphenazine in the marine bacterium Streptomyces sp. CNQ-509.

Streptomyces sp. CNQ-509 produces the rare O-prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O-prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ-509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine-1-carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6-dihydroxyphenazine geranyltransferase, which catalyzes the C-O bond formation between C-1 of the geranyl moiety and O-6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O-prenyl transfer to a phenazine.

The Systematic Investigation of the Quorum Sensing System of the Biocontrol Strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 Unveils aurI to Be a Biosynthetic Origin for 3-Oxo-Homoserine Lactones.

The shoot endophytic biocontrol strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 produces a wide range of exoproducts, including enzymes and antibiotics. The production of exoproducts is commonly tightly regulated. In order to get a deeper insight into the regulatory network of PB-St2, the strain was systematically investigated regarding its quorum sensing systems, both on the genetic and metabolic level. The genome analysis of PB-St2 revealed the presence of four putative acyl homoserine lactone (AHL) biosynthesis genes: phzI, csaI, aurI, and hdtS. LC-MS/MS analyses of the crude supernatant extracts demonstrated that PB-St2 produces eight AHLs. In addition, the concentration of all AHL derivatives was quantified time-resolved in parallel over a period of 42 h during the growth of P. aurantiaca PB-St2, resulting in production curves, which showed differences regarding the maximum levels of the AHLs (14.6 nM- 1.75 µM) and the production period. Cloning and heterologous overexpression of all identified AHL synthase genes in Escherichia coli proved the functionality of the resulting synthases PhzI, CsaI, and AurI. A clear AHL production pattern was assigned to each of these three AHL synthases, while the HdtS synthase did not lead to any AHL production. Furthermore, the heterologous expression study demonstrated unequivocally and for the first time that AurI directs the synthesis of two 3-oxo-AHLs.

Complete Genome Sequence of the Sugar Cane Endophyte Pseudomonas aurantiaca PB-St2, a Disease-Suppressive Bacterium with Antifungal Activity toward the Plant Pathogen Colletotrichum falcatum.

The endophytic bacterium Pseudomonas aurantiaca PB-St2 exhibits antifungal activity and represents a biocontrol agent to suppress red rot disease of sugar cane. Here, we report the completely sequenced 6.6-Mb genome of P. aurantiaca PB-St2. The sequence contains a repertoire of biosynthetic genes for secondary metabolites that putatively contribute to its antagonistic activity and its plant-microbe interactions.

Genome-based discovery of a novel membrane-bound 1,6-dihydroxyphenazine prenyltransferase from a marine actinomycete.

Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated Streptomyces sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in E. coli and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms.