Engineering of Peptide Synthetases

Abstract

Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.Two mechanisms for the biosynthesis of peptides are known to exist in bacteria and fungi, ribosomal synthesis and production by peptide synthetases. These are large enzymatic complexes responsible for the synthesis of hundreds of types of peptides, some of which have immunoactive, antibiotic, antifungal, or surfactant properties. Whereas polypeptides produced by ribosomal synthesis typically contain only the amino acids directly specified by the triplets of the genetic code, peptides built on synthetases often contain unusual amino or hydroxy acids as building blocks that are not present in proteins. The amino acids can be modified by peptide synthetases either through methylation, hydroxylation, or enantiomerization. The peptides are typically short (up to about 20 residues) and can be linear, circular, or branched (1, 2). Peptide synthesis proceeds by the "multiple carrier thiotemplate mechanism" (3, 4), and many of the details of these systems remain to be investigated experimentally. According to this mechanism each domain recognizes a specific amino (or hydroxy) acid that is covalently bound to the cofactor via a thioester bond after activation to the corresponding acyladenylate derivative. The growth of the polypeptide chain thus occurs through a series of thioester bond cleavages and the simultaneous formation of amide or ester bonds in the peptide. At the end of synthesis of each peptide, the chain is thought to be released from the enzyme by a thioesterase (TE) 1 activity encoded in the synthetase gene (1). Peptide synthetases are interesting not only from a scientific and evolutionary point of view but also for their biotechnological potential. Many enzymatically synthesized peptides are in fact biologically active, and some of them are industrially produced, among which are cyclosporins, surfactin, and fungicides. A growing number of laboratories are involved in applied research projects focused on the isolation and characterization of new peptide synthetases and on the genetic manipulation of peptide synthetase genes to optimize peptide production and to genetically modify the sequence of the peptides produced. In fact, the structural organization...

Construction of hybrid peptide synthetases by module and domain fusions

“…The system TycA͞ ProCAT-LeuCATTe readily synthesized the tripeptides DTrpPro-Leu, DPhe-Sar-Leu, DPhe-Abu-Leu, DPhe-Pro-Ile, and DPhe-Pro-NVal (norvaline) as judged by the presence of products with the corresponding mass (Table 1) ine, and, fused to the Orn module, the hydrophilic Orn and Lys. Similar observations have been published for the Te-domain of the surfactin synthetase (21). It is important to note that the Te-domain in the natural tyrocidine synthetase most probably catalyzes the formation of the cyclic decapeptide, thus acting as a cyclase (9,23), whereas in the artificial systems described here it acts as a hydrolase.…”

Coordinate Transcription and Physical Linkage of Domains in Surfactin Synthetase Are Not Essential for Proper Assembly and Activity of the Multienzyme Complex

“…This might appear to contradict our recently published results on the engineering of surfactin synthetase in which, when the TE domain was moved next to either the fourth or the fifth domain of the enzyme, functional recombinant peptide synthetases were obtained that led to the synthesis of a four-amino acid and a five-amino acid lipopeptide, respectively (21).…”

Peptide Bond Formation in Nonribosomal Peptide Biosynthesis

“…The inherent potential to do so has been demonstrated in initial work (4,5,53). Further exploitation of this potential will depend on a better understanding of the genetics and enzymology of the peptide synthetases.…”