Abstract
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.
Highlights
The GE81112 tetrapeptides [1,2,3] represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis
Isolation of the GE81112 Biosynthetic Gene Cluster—To capture the GE81112 biosynthetic gene cluster a cosmid library containing 2304 clones was generated from the genomic DNA of the GE81112 producer strain
As we expected nonribosomal peptide synthetase (NRPS) to be encoded by the identified cosmids, we used the degenerate NRPS primers to amplify and sequence A-domain segments from these cosmids
Summary
The emergence of multi-drug resistant microbial pathogens is driving the search for novel antibiotics with new mechanisms of action and natural products continue to provide original scaffolds affecting essential bacterial targets [1, 2]. As it is currently understood, most antibiotics act in three basic ways; 1) inhibition of DNA replication and repair, 2) inhibition of cell wall biosynthesis, and 3) inhibition of protein biosynthesis. We expressed five A-domains from the corresponding NRPS genes and characterized them in vitro These results together with the assignment of the configuration of most chiral centers have. GE81112 Biosynthetic Gene Cluster allowed us to delineate this unusual NRPS pathway and propose a biosynthetic model for the GE81112 antibiotics
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