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United States Department of Agriculture

Agricultural Research Service

Title: The Genomisotopic Approach: A Systematic Method to Isolate the Products of Orphan Biosynthetic Gene Clusters

Authors
item Gross, Harald - SCRIPPS INSTITUTE
item Stockwell, Virginia - OREGON STATE UNIVERSITY
item Henkels, Marcella
item Nowak-Thompson, Brian - NORTHLAND COLLEGE
item Loper, Joyce
item Gerwick, William - SCRIPPS INSTITUTE

Submitted to: Chemistry and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 3, 2006
Publication Date: January 3, 2007
Repository URL: http://www.ars.usda.gov/SP2UserFiles/person/3432/PDF/Grossetalchembiol07.pdf
Citation: Gross, H., Stockwell, V.O., Henkels, M.D., Nowak-Thompson, B., Loper, J.E., Gerwick, W.H. 2007. The genomisotopic approach: a systematic method to isolate the products of orphan biosynthetic gene clusters. Chemistry and Biology. 14(1):53-64.

Interpretive Summary: This manuscript describes a new approach to identify unknown chemicals produced by a microorganism from the genomic sequence of that microorganism. The approach uses methods from both molecular biology and natural products chemistry to identify unknown chemical metabolites. It begins with a thorough analysis of nucleotide sequences to predict components of the final chemical structure, proceeds by identifying conditions where the genes are expressed, and incorporates labelling methods, used in natural products chemistry, to purify the unknown chemical. The efficiency of the new approach was demonstrated in this study by identifying an unknown chemical predicted from the recently available genomic sequence of Pseudomonas fluorescens Pf-5. This manuscript describes a new group of biosurfactants called orfamides that are produced by Pf-5 and have the capacity to lyse oospores produced by Oomycete plant pathogens.

Technical Abstract: A new approach for the exploitation of orphan biosynthetic pathways is presented. This method uses a combination of genomic sequence analysis and isotope guided fractionation. Analysis of the Pseudomonas fluorescens Pf-5 genome led to the identification of an orphan gene cluster which was predicted to code for a new lipopeptide natural product. Application of a systematic approach to isolate the product of this gene cluster, which employed a selective isotope-labeled precursor feeding, resulted in the discovery of orfamide A, founder of a new group of bioactive cyclic lipopeptides. The planar structure was determined by extensive spectroscopic analysis (NMR, MS, IR), while the absolute configuration was determined by chiral GC-MS and chiral Marfey´s analysis. The correlation of the orphan gene sequence and the final natural product orfamide A was verified by gene disruption experiments. This study established that orfamide A acts as a surfactant, lowering surface tension and enhancing swarming motility of bacterial cells, and has the capacity to lyse zoospores of an Oomycete plant pathogen.

Last Modified: 10/22/2014
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