Dataset Information

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

ABSTRACT: Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products.

SUBMITTER: Chen Y

PROVIDER: S-EPMC4777305 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Chen Yunqiu Y McClure Ryan A RA Kelleher Neil L NL

Methods in molecular biology (Clifton, N.J.) 20160101

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separa ...[more]

PMID: 26831706

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Project description:Identification of novel bioactive compounds represents an important field in modern biomedical research. Microorganisms of the underexplored environments, such as deserts, hot springs, oceans, and caves are highly promising candidates for screening such metabolites. Screening for biosynthetic genes is the most effective strategy to characterize bioactivity in a certain environment. However, knowledge is either scant or non-existent about the expression of the biosynthetic genes encoding for various bioactive compounds in the microorganisms from the caves. The aim of the current study was to screen for the genes of polyketide synthases and non-ribosomal peptide synthetases in Krubera-Voronja Cave (43.4184 N 40.3083 E, Western Caucasus) bacterial isolates as well as to evaluate the expression of these genes under laboratory conditions. In total, 91 bacterial strains isolated from the cave were screened for the presence of polyketide synthase and non-ribosomal peptide synthetase genes. Phenotypically inactive strains were the main focus (the test group) of our study, while the strains with the identified antibacterial activity served as the control group. Our PCR-based screening clearly showed that the majority of the strains harbored at least one biosynthetic gene. Prediction of the putative products allowed us to identify bioactive compounds with antibacterial, anticancer, antifungal, anti-inflammatory, antimycoplasmic, antiviral, insecticidal, and thrombolytic activity. For most polyketide synthases and non-ribosomal peptide synthetases, putative products could not be predicted; they are unknown. Qualitative transcriptional analysis did not show substantial differences between the test group and the control group of the strains. One to four biosynthetic genes were constitutively expressed in all the tested strains, irrespective of the group. Quantitative transcriptional analysis of the constitutively expressed biosynthetic genes demonstrated that the expression of a particular gene could be affected by both the amount of the nutrients in the culture medium and the growth phase.

Dataset Information

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Publications

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Similar Datasets

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