Studies on the biosynthesis of the lipodepsipeptide antibiotic Ramoplanin A2

Ramoplanin, a non-ribosomally synthesized peptide antibiotic, is highly effective against several drug-resistant Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Rece...

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Bibliographic Details
Published in:Bioorganic & medicinal chemistry 2012-01, Vol.20 (2), p.859-865
Main Authors: Hoertz, Amanda J., Hamburger, James B., Gooden, David M., Bednar, Maria M., McCafferty, Dewey G.
Format: Article
Language:English
Subjects:
Actinoplanes
Anti-Bacterial Agents - biosynthesis
Anti-Bacterial Agents - pharmacology
Antibiotic
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
biosynthesis
Chaperonin 10 - genetics
Chaperonin 10 - metabolism
Chaperonin 60 - genetics
Chaperonin 60 - metabolism
chemistry
Depsipeptides - biosynthesis
Depsipeptides - pharmacology
drug resistance
Enduracidin
Enterococcus faecium
enzymes
fatty acids
Glycoproteins - biosynthesis
Glycoproteins - pharmacology
Gram-positive bacteria
Gram-Positive Bacteria - drug effects
hosts
humans
Kinetics
loci
Micromonosporaceae - genetics
Multigene Family
Non-ribosomal peptide synthesis
pathogens
Peptide
peptide antibiotics
Peptide Synthases - genetics
Ramoplanin
Staphylococcus aureus
system optimization
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Summary:Ramoplanin, a non-ribosomally synthesized peptide antibiotic, is highly effective against several drug-resistant Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Recently, the biosynthetic cluster from the ramoplanin producer Actinoplanes ATCC 33076 was sequenced, revealing an unusual architecture of fatty acid and non-ribosomal peptide synthetase biosynthetic genes (NRPSs). The first steps towards understanding how these biosynthetic enzymes cooperatively interact to produce the depsipeptide product are expression and isolation of each enzyme to probe its specificity and function. Here we describe the successful production of soluble enzymes from within the ramoplanin locus and the confirmation of their specific role in biosynthesis. These methods may be broadly applicable to the production of biosynthetic enzymes from other natural product biosynthetic gene clusters, especially those that have been refractory to production in heterologous hosts despite standard expression optimization methods.
ISSN:0968-0896
1464-3391
DOI:10.1016/j.bmc.2011.11.062