Strain-specific proteogenomics accelerates the discovery of natural products via their biosynthetic pathways

Abstract

The use of proteomics for direct detection of expressed pathways producing natural products has yielded many new compounds, even when used in a screening mode without a bacterial genome sequence available. Here we quantify the advantages of having draft DNA-sequence available for strain-specific proteomics using the latest in ultrahigh-resolution mass spectrometry (MS) for both proteins and the small molecules they generate. Using the draft sequence of Streptomyces lilacinus NRRL B-1968, we show a >10-fold increase in the number of peptide identifications vs. using publicly available databases. Detected in this strain were six expressed gene clusters with varying homology to those known. To date, we have identified three of these clusters as encoding for the production of griseobactin (known), rakicidin D (an orphan NRPS/PKS hybrid cluster), and a putative thr and DHB-containing siderophore produced by a new non-ribosomal peptide sythetase gene cluster. The remaining three clusters show lower homology to those known, and likely encode enzymes for production of novel compounds. Using an interpreted strain-specific DNA sequence enables deep proteomics for the detection of multiple pathways and their encoded natural products in a single cultured bacterium.

Proteogenomics: concepts, applications and computational strategies

“…These developments, coupled with improvements in protein separation and enrichment methods for proteomics applications, have resulted in a substantial increase in the depth of protein detection, approaching that of global transcriptome profiling studies 1 . Significant advances have also been achieved in the area of top-down proteomics – a technology that offers complementary information useful for proteogenomic characterization 89 . A substantial challenge in proteogenomics has been lack of a sufficient amount of proteomic data in the public domain necessary to make a significant contribution to genome annotation efforts, in part due to the “data hoarding” mentality prevalent in the proteomics community in the early days.…”

Microbial genome mining for accelerated natural products discovery: is a renaissance in the making?

“…However, many significant technical and practical barriers must be addressed in order to realize the full potential of genome mining. Arguably, improvements in methods for isolation and structure elucidation of secondary metabolites from complex extracts have not kept pace with genomic advances and it is likely that these will become the rate limiting step in metabolite discovery [2, 56]. With the current state of the art, isolation and elucidation of new natural products at moderate abundance (1 mg/L) require weeks to months or longer per compound for full characterization.…”

The amido-pentadienoate-functionality of the rakicidins is a thiol reactive electrophile – development of a general synthetic strategy

“…10 Although the mechanistic foundation for the biological activity of RakA is still obscure, the mc-APD has recently been demonstrated to be a critical functionality. 13 In our hands, however, related transformations have turned out to be highly substratedependent and rather unpredictable. The mc-APD is expected to act as a Michael acceptor at both the band d-carbons (Fig.…”