Strain Prioritization for Natural Product Discovery by a High-Throughput Real-Time PCR Method

Abstract

Natural products offer unmatched chemical and structural diversity compared to other small-molecule libraries, but traditional natural product discovery programs are not sustainable, demanding too much time, effort, and resources. Here we report a strain prioritization method for natural product discovery. Central to the method is the application of real-time PCR, targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds in a high-throughput format. The practicality and effectiveness of the method were showcased by prioritizing 1911 actinomycete strains for diterpenoid discovery. A total of 488 potential diterpenoid producers were identified, among which six were confirmed as platensimycin and platencin dual producers and one as a viguiepinol and oxaloterpin producer. While the method as described is most appropriate to prioritize strains for discovering specific natural products, variations of this method should be applicable to the discovery of other classes of natural products. Applications of genome sequencing and genome mining to the high-priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered.

New isofuranonaphthoquinones and isoindolequinones from Streptomyces sp. CB01883

“…It grows and sporulates well on ISP4 agar medium, and was classified as a Streptomyces species on the basis of a phylogenetic analysis using the concatenated partial sequences of three housekeeping genes 16 S rRNA, rpoB and trpB (Genebank accession number KT722854, KT736417 and KT793843, respectively). [14][15][16] In our continued effort Isofuranonaphthoquinones from Streptomyces sp. CB01883 Z Guo et al to study the biosynthesis of hybrid peptide-polyketide natural products, [19][20][21] we sequenced the genome of Streptomyces sp.…”

“…The NPLI biases natural products from Actinomycetales that are isolated from unexplored or underexplored ecological niches and unavailable in public strain collections. [7][8][9][10][11][12][13][14][15][16][17] The current library at TSRI consists of (i) purified natural products with fully assigned structures, (ii) C-18 mediumpressure liquid chromatography fractions, and (iii) crude extracts of microbial fermentation. Typically, strains were fermented in multiple media and subjected to HPLC and LC-MS analysis.…”

Structure, Bioactivity, and Resistance Mechanism of Streptomonomicin, an Unusual Lasso Peptide from an Understudied Halophilic Actinomycete

“…bioassay-guided isolation) increasingly unappealing and economically disadvantageous (Baltz, 2006). In response, a number of modern natural product discovery strategies have been developed, including genome-guided discovery (Doroghazi et al, 2014), antibiotic resistance-mediated isolation (Thaker et al, 2014), reactivity-based screening (Cox et al, 2014), PCR-based strain prioritization (Hindra et al, 2014), mass spectrometry-based network analysis (Nguyen et al, 2013), heterologous expression (Feng et al, 2010), and metagenomics (Kang and Brady, 2013) with the general aim of reducing the burden of rediscovery and thereby accelerating the drug discovery process.…”

A genetically amenable platensimycin- and platencin-overproducer as a platform for biosynthetic explorations: a showcase of PtmO4, a long-chain acyl-CoA dehydrogenase

“…2). 8 Comparison of the ptm and ptn gene clusters revealed high conservation in both sequence and organization; however, a striking difference between them was the absence of a 5.4 kb cassette containing five open reading frames (ORFs) in the PTN gene cluster. 7 The five ORFs include ptmO3 , ptmO4 , ptmT3 , ptmO5 , and ptmR3 .…”

“…Unfortunately, both SB12001 and SB12002 were unamenable to further genetic modifications, which led us to discover alternative PTM and PTN producers. 8 Inactivation of ptmR1 by gene replacement in one of the six new producers, S. platensis CB00739, afforded SB12026, a dual PTM-PTN overproducer with titers comparable to those of SB12002 and more importantly, genetic amenability. 8, 12 This mutant circumvented the technical difficulties of the original wild-type and overproducing strains and established a system to investigate the biosynthesis of the highly functionalized bacterial diterpenoids PTM and PTN.…”