Insights into an unusual nonribosomal peptide synthetase biosynthesis: identification and characterization of the GE81112 biosynthetic gene cluster.
The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.
Binz
Tina M
TM
From the Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), and Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, Saarbrücken 66123, Germany.
Maffioli
Sonia I
SI
Naicons Scrl, Via G. Fantoli, 16/15, Milan 20138, Italy.
Sosio
Margherita
M
Naicons Scrl, Via G. Fantoli, 16/15, Milan 20138, Italy.
Donadio
Stefano
S
Naicons Scrl, Via G. Fantoli, 16/15, Milan 20138, Italy.
Müller
Rolf
R
From the Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), and Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, Saarbrücken 66123, Germany. Electronic address: rom@mx.uni-saarland.de.
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000502:physiology*; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010453:Peptide Synthases