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. 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.
Department of Chemistry, Duke University, Durham, NC 27708, USA.
Amanda J
AJ
Hoertz
James B
JB
Hamburger
David M
DM
Gooden
Maria M
MM
Bednar
Dewey G
DG
McCafferty
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D047630:Depsipeptides / Q000096:biosynthesis* / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis* / Q000494:pharmacology; D006094:Gram-Positive Bacteria / Q000187:drug effects; D007700:Kinetics; D008848:Micromonosporaceae / Q000235:genetics; D005810:Multigene Family; D010453:Peptide Synthases / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D047630:Depsipeptides; D006023:Glycoproteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; C416073:ramoplanin A2