ABSTRACT
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
Subject(s)
Biosynthetic Pathways/genetics , Evolution, Molecular , Genetic Variation , Multigene Family , Bioengineering , Polyketide Synthases/genetics , Sirolimus/chemistry , Sirolimus/metabolismABSTRACT
Hormaomycin produced by Streptomyces griseoflavus is a structurally highly modified depsipeptide that contains several unique building blocks with cyclopropyl, nitro, and chlorine moieties. Within the genus Streptomyces, it acts as a bacterial hormone that induces morphological differentiation and the production of bioactive secondary metabolites. In addition, hormaomycin is an extremely potent narrow-spectrum antibiotic. In this study, we shed light on hormaomycin biosynthesis by a combination of feeding studies, isolation of the biosynthetic nonribosomal peptide synthetase (NRPS) gene cluster, and in vivo and in vitro functional analysis of enzymes. In addition, several nonnatural hormaomycin congeners were generated by feeding-induced metabolic rerouting. The NRPS contains numerous highly repetitive regions that suggest an evolutionary scenario for this unusual bacterial hormone, providing new opportunities for evolution-inspired metabolic engineering of novel nonribosomal peptides.
Subject(s)
Bacterial Proteins/genetics , Depsipeptides/biosynthesis , Peptide Synthases/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biocatalysis , Depsipeptides/chemistry , Multigene Family , Peptide Biosynthesis, Nucleic Acid-Independent , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Signal Transduction , Streptomyces/enzymology , Streptomyces/geneticsABSTRACT
A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.
Subject(s)
Benzoquinones/pharmacology , Biological Products/pharmacology , Genetic Engineering , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Benzoquinones/chemistry , Benzoquinones/metabolism , Biological Products/chemistry , Biological Products/metabolism , HSP90 Heat-Shock Proteins/metabolism , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/metabolism , Molecular Sequence Data , Molecular StructureABSTRACT
A new octaketide named fogacin (1) was isolated from Streptomyces sp. (strain Tü 6319). Furthermore two shunt metabolites, SEK4b (2) and anhydroSEK4b (3), were detected and identified as non-enzymatically cyclized products of polyketide intermediates built during the biosynthesis of actinorhodin. SEK4b (2) as well as anhydroSEK4b (3) were previously described as metabolites of genetically engineered strains.
Subject(s)
Anti-Bacterial Agents/chemistry , Macrolides/chemistry , Streptomyces/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Circular Dichroism , Macrolides/isolation & purification , Macrolides/pharmacology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Optical Rotation , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, UltravioletABSTRACT
The macrocyclic compound hormaomycin has been investigated by NMR spectroscopy and by restrained molecular-dynamics simulations. Measurement of residual dipolar couplings induced by dissolving the depsipeptide in a polyacrylamide gel compatible with DMSO and their incorporation into the structure calculation of the title compound improved the precision of the family of structures. In DMSO the macrocyclic ring shows two beta-turns, whose positions in the sequence differ from those found in the CDCl3 solution structure and in the crystal structure obtained from hexylene glycol/H2O (50:50). The bulky side chain consisting of a 3-(2-nitrocyclopropyl)alanine and a chlorinated N-hydroxypyrrole moiety is flexible in DMSO.
