A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria

Abstract

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.

Figure 1

(a) The work and logic flow for PrISM used in this study. High molecular weight proteins (>150 kDa) from a bacterial proteome are subjected to in-gel trypsin digestion and LC-MS/MS analysis. The data were analyzed by both database searching (top) and de novo peptide sequencing (bottom). Peptides identified as coming from NRPSs/PKSs were reverse translated into nucleotide sequences for PCR amplification of the target region. The PCR products guided the identification of biosynthetic gene clusters as well as discovery of the natural products. (b) Decision tree for LC-MS/MS data analysis. The LC-MS/MS data were first searched against NCBInr using OMSSA, and strains with NRPS/PKS identifications were divided into two categories based on their sequence similarity to known NRPS/PKS proteins, which was represented by the number of peptides identified and sequence coverage for each protein identification. Strains with low similarity to known NRPSs/PKSs were subjected to de novo peptide sequencing and homology-based searching by PEAKS Studio. Peptides identified by either search engines were used for PCR amplification. After confirming the specificity of the PCR products, they were used to guide the identification of biosynthetic gene clusters.

Figure 2

NRPS/PKS gene clusters detected by PrISM and their corresponding natural products. For each gene cluster, the NRRL number of strain, the type of biosynthetic enzymes and a diagram of the genes are shown. Gene sequences were based on the most homologous gene cluster as shown. The sequence of the NRPS gene cluster identified from strain F-6133 was based on shotgun genome sequencing and partially assembled into contigs. The natural product produced by each gene cluster is shown at right.

Figure 3. Association of foroxymithine with its biosynthetic pathway

(a) The foroxymithine gene cluster identified from strain F-6562, using the DNA sequence from Streptomyces pristinaespiralis ATCC 25486 as a template. Domain organization and substrate specificity of adenylation domains of the NRPS were predicted by bioinformatics. C, condensation domain; A, adenylation domain; T, thiolation domain; E, epimerization domain. (b) LC-MS Base peak chromatogram (top) of F-6562 culture supernatant produced by cells grown in 4×R2A medium for 4 days and selected ion chromatogram (SIC, bottom) of m/z 576.2631; inset, mass spectrum of species at m/z 576.2631 (iron free form) and 629.1726 (iron bound form). (c) Structure of foroxymithine with predicted fragment masses (top). The CID fragmentation spectrum on m/z 576.26 is shown at bottom. (d) ¹³C₆-arginine feeding of strain F-6562 shows three ornithines are incorporated into the species at m/z 576.2618 as evidenced by a mass shift of 15 Da. Arginine can be converted to ornithine in vivo by losing the terminal -C(NH)NH₂ group. (e) Predicted biosynthetic mechanism for foroxymithine based on the NRPS domain organization.

Figure 4. Discovery of the biosynthetic gene cluster for antibiotic S 213L in strain F-6133

(a) The partial gene cluster determined by shotgun genome sequencing of strain F-6133. orf 12 NRPS contains peptides identified by LC-MS/MS and two PCR products from F-6133. Gene annotations were based on BLAST analysis. (b) SIC for m/z 799.39 detected in a culture supernatant from strain F-6133. The mass spectrum of the SIC peak is shown as an inset. Two masses separated by 52.9 Da (mass difference of Fe and 3 × H⁺) were shown, suggesting iron free and iron bound forms of the same species. (c) Structure of antibiotic S 213L. The labeled fragment ions were observed by tandem MS of the species at m/z 799.39.