|Chen, Yunqiu -|
|Ntai, Ioanna -|
|Ju, Kou-San -|
|Unger, Michelle -|
|Zamdborg, Leonid -|
|Robinson, Sarah -|
|Doroghazi, James -|
|Metcalf, William -|
|Kelleher, Neil -|
Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 8, 2011
Publication Date: January 1, 2012
Citation: Chen, Y., Ntai, I., Ju, K., Unger, M., Zamdborg, L., Robinson, S.J., Doroghazi, J.R., Labeda, D.P., Metcalf, W.W., Kelleher, N.L. 2012. A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria. Journal of Proteome Research. 11(1):85–94. Interpretive Summary: The class of bacteria called Actinobacteria, typically found in soils throughout the world, are widely recognized for their ability to produce a wide array of natural products, such as antibiotics, that are important to human and animal health and biotechnology. A significant percentage of antibiotics used in the treatment of human infections are produced by these bacteria. However, it has become increasingly difficult to discover new compounds produced by these microorganisms using traditional activity based screening. In this study, we evaluated a new method, called PrISM (Proteomic Investigation of Secondary Metabolism), to detect the production of enzymes that are essential in the biosynthesis of a wide array of important compounds. We were able to identify strains containing these enzymes which would suggest that they might produce potentially novel compounds under the proper growth conditions. This information will be extremely useful to pharmaceutical and clinical scientists searching for new compounds for the treatment of human or animal diseases caused by microorganisms that are resistant to currently available antibiotics.
Technical 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 (Nature Biotechnology, 2009, 27, 951 - 956), e.g. improved de novo peptide sequencing, have enabled the discovery of ten NRPS/PKS gene clusters from six 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.