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Pet?í?ková K,
Chro?áková A,
Zelenka T,
Chrudimský T,
Pospíšil S,
Pet?í?ek M,
Krištůfek V,
( 2015 ) Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques. PMID : 26300877 : DOI : 10.3389/fmicb.2015.00814 PMC : PMC4525017 Abstract >>
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.
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2. |
Johnston CW,
Skinnider MA,
Wyatt MA,
Li X,
Ranieri MR,
Yang L,
Zechel DL,
Ma B,
Magarvey NA,
( 2015 ) An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products. PMID : 26412281 : DOI : 10.1038/ncomms9421 PMC : PMC4598715 Abstract >>
Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC-MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.
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3. |
Zhu XM,
Hackl S,
Thaker MN,
Kalan L,
Weber C,
Urgast DS,
Krupp EM,
Brewer A,
Vanner S,
Szawiola A,
Yim G,
Feldmann J,
Bechthold A,
Wright GD,
Zechel DL,
( 2015 ) Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA-Specified Leu-tRNA(UUA) Molecule. PMID : 26374477 : DOI : 10.1002/cbic.201500402 Abstract >>
Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA-encoded Leu-tRNA(UUA) molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by (19) F NMR spectroscopy, HPLC-ESI-MS, and HPLC-continuum source molecular absorption spectroscopy for fluorine-specific detection. The molecule was purified from a large-scale culture and definitively characterized by NMR spectroscopy and high-resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5'-O-sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60-year-old mystery.
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4. |
Kalan L,
Gessner A,
Thaker MN,
Waglechner N,
Zhu X,
Szawiola A,
Bechthold A,
Wright GD,
Zechel DL,
( 2013 ) A cryptic polyene biosynthetic gene cluster in Streptomyces calvus is expressed upon complementation with a functional bldA gene. PMID : 24120331 : DOI : 10.1016/j.chembiol.2013.09.006 Abstract >>
Streptomyces calvus is best known as the producer of the fluorinated natural product nucleocidin. This strain of Streptomycetes is also unusual for displaying a "bald" phenotype that is deficient in the formation of aerial mycelium and spores. Genome sequencing of this organism revealed a point mutation in the bldA gene that is predicted to encode a misfolded Leu-tRNA(UUA) molecule. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a polyeneoic acid amide, 4-Z-annimycin, and a minor amount of the isomer, 4-E-annimycin. Bioassays reveal that these compounds inhibit morphological differentiation in other Actinobacteria. The annimycin gene cluster encoding a type 1 polyketide synthase was identified and verified through disruption studies. This study underscores the importance of the bldA gene in regulating the expression of cryptic biosynthetic genes.
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