A Genomic Screening Approach to the Structure‐Guided Identification of Drug Candidates from Natural Sources

Abstract

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.

Isolation and antimicrobial activities of actinobacteria closely associated with liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza inflate BAT. in Xinjiang, China

“…Primers K1F (5¢-GTSCCSGTSCCRTGSSCYTCSAC-3¢) and M6R (5¢-GCSATGGAYCCSCARCARCGSVT-3¢) (Schirmer et al, 2005), KSaF (5¢-TSGCSTGCTTGGAYGCSATC-3¢) and KSaR (5¢-TSGCSTGC-TTGGAYGCSATC-3¢) (Mets€ a-Ketel€ a et al, 1999), A3F (5¢-GCSTACSY-SATSTACACSTCSGG-3¢) and A7R (5¢-SASGTCVCCSGTSCGCTAS-3¢) (Ayuso-Sacido & Genilloud, 2005), and B4F (5¢-TTCCCSCGSTAC-CASATCGGSGAG-3¢) and B7R (5¢-GSGGGATSWMCCAGWAC-CASCC-3¢) (Hornung et al, 2007) were employed to amplify polyketide synthase gene type I PKS, the KSa gene in the type II PKS polyketide synthase gene cluster, the non-ribosomal peptide synthetase gene (NRPS) and the FADH 2 -dependent halogenase gene, respectively. PCR amplification was carried out in 20 µl PCR reaction mixture consisting of 1Âbuffer with 1.5 mM Mg 2+ , 0.5 mM dNTP mixture, 1 µM of primers, 1 µl of template DNA, 1 U Taq polymerase and 1.25 µg µl À1 BSA.…”

Genome-wide survey of polyketide synthase and nonribosomal peptide synthetase gene clusters in Streptomyces turgidiscabies NBRC 16081

“…This cluster also contained a halogenase gene next to the PKS/NRPS hybrid gene (data not shown). The products of this pathway may therefore be halogen-containing polyketide-peptide hybrid compounds (Gao and Huang, 2009;Hornung et al, 2007).…”

Mining microbial genomes for new natural products and biosynthetic pathways

“…5), which can be purified and structurally characterized as in the first approach. CBS40 is an example of a novel metabolite that has been identified by this approach (Hornung et al, 2007). One potential obstacle which often has to be overcome in the Fig.…”