Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure. Received: 10 July 1997 / Accepted: 11 September 1997     

Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.     

Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis.

Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin (TEII(srf)), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEII(srf) were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEII(srf) belongs to the alpha/beta-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribosomal peptide synthetase TEII.     

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.     

The sypA,sypS, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.     

Amino acid selectivity in the aminoacylation of coenzyme A and RNA minihelices by aminoacyl-tRNA synthetases

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TpsiC helix of tRNA(Ile) or tRNA(Val) were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.     

海绵共生萎缩芽孢杆菌C89 PPTase bap基因的异源表达

【背景】磷酸泛酰巯基乙胺基转移酶(PPTase)催化非核糖体肽合成酶(NRPS)中肽酰载体蛋白(PCP)从无活性的脱辅基形态转化为有活性的全辅基形态,从而启动非核糖体肽类化合物的生物合成。【目的】鉴定贪婪倔海绵共生萎缩芽孢杆菌C89中Sfp型PPTase Bap,验证Bap激活NRPS中PCP的能力。【方法】通过BLAST和氨基酸多序列比对鉴定萎缩芽孢杆菌C89中Sfp型PPTase Bap。将bap基因在sfp基因突变株枯草芽孢杆菌168中异源表达,通过重组菌枯草芽孢杆菌168-bap的代谢物检测非核糖体肽类化合物Surfactin。【结果】Bap为Sfp型PPTase,检测到重组菌枯草芽孢杆菌168-bap中Surfactin的产生。【结论】本研究为海洋萎缩芽孢杆菌中NRPS基因簇的异源表达奠定了基础。     

生物表面活性剂Surfactin生产菌株的定向改造策略

表面活性素(Surfactin)是芽胞杆菌属(Bacillussp.)代谢产生的脂肽类生物表面活性剂,是由非核糖体肽合成酶(NRPS)催化而得的一种次级代谢产物。由于surfactin具有稳定性好、可被降解、表面活性好等理化性质以及抑菌、抗肿瘤等生物活性,在医药、农业、食品、化妆品、石油开采等方面都具有很大的应用潜力。但是,天然菌株产率低、生产成本高等特点限制了surfactin的规模化应用。本文对surfactin的合成机理进行了简要阐述,并针对目前提升surfactin产量和改变结构组分的4种定向改造策略(启动子工程、强化外排分泌、改造NRPS结构域和脂肪酸链合成酶系)进行了综述,最后对surfactin的研究方向进行了展望。     

Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas

The distinguishing structural and functional domains of plant acyl-acyl carrier protein (ACP) thioesterases and their complex interaction with the ACP-linked fatty acid substrate complex have remained elusive. E. coli based heterologous expression and characterisation of many plant thioesterases reported so far have not been extended and linked to in silico modelling studies to explain the diversity in plant thioesterase substrate specificities. In this study, a thioesterase cDNA isolated from immature seed tissues of Jatropha curcas was found to be type B and specific to stearoyl acyl ACP when expressed in E. coli K27fadD88, a lipid utilisation mutant. Homology modelling and molecular docking of a selected region of the isolated JcFatB protein predicted that it had high affinity towards both stearate (18:0) and palmitate (16:0). Structural analysis of the sequence confirmed the presence of a transit peptide that is processed in multiple steps. The enzyme is localised in the chloroplasts and has an N-terminal inner chloroplast transmembrane domain characteristic of type B plant thioesterases. Docking of ligands with JcFatB and its comparison with a modelled Jatropha thioesterase type A provided further evidence for native substrate preferences of Jatropha thioesterases. This study provides essential clues to develop future methods for large-scale bacterial production of free fatty acids and for design of strategies to modulate the seed oil composition in this important non-edible, seed oil plant.     

Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp–hydroxamate–Fe³⁺ complex and the formation of l-Trp–l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.     

Recombinant acylheptapeptide lichenysin: high level of production by Bacillus subtilis cells

Peptide synthetases are multi-domain proteins that catalyze the assembly, from amino acids and amino acid derivatives, of peptides and lipopeptides, some of which exhibit activities (pharmaceutical, surfactant, etc.) of considerable biotechnological importance. Although there is substantial interest in the generation of greater peptide diversity, in order to create new biotechnologically interesting products, attempts reported so far to exchange amino acid-activating minimal modules between enzymes have only yielded hybrid catalysts with poor activities. We report here the replacement of an entire first, L-Glu-, and fifth, L-Asp-incorporating modules of surfactin synthetase, to create a fully active hybrid enzyme that forms a novel peptide in high yields. Whole encoding regions of lichenysin A synthetase modules were introduced into surfactin biosynthesis operon between His140/His1185 of SrfAA and His1183/His2226 of SrfAB, the amino acid residues of a proposed active-site motif (HHXXXDG) of the condensation domains which is involved in the catalysis of nonribosomal peptide bond formation (Stachelhaus et al., 1998). When the lipopeptides produced by the recombinant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical by their fatty acid profiles. We thereby demonstrate the utility of whole module swapping for designing novel peptides, for creating peptide diversity, and for redesigning existing peptides produced in performant production strains in high yields to correspond to desired peptides produced in low yields, or from strains unsuitable for production purposes.     

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.     

Nucleotide sequence of 5' portion of srfA that contains the region required for competence establishment in Bacillus subtilus.

The nucleotide sequence of the 20,535 base pairs of the 5' end of the srfA operon, containing the region required for competence development, was determined. This included the srfA promoter region, the first open reading frame, srfAA, encoding surfactin synthetase I and part of the second open reading frame, srfAB, encoding surfactin synthetase II. Three amino acid-activating domains characteristic of those found in peptide synthetases could be discerned in both srfAA (activating Glu, Leu and D-Leu) and srfAB (activating Val, Asp, and D-Leu). The presence of a conserved spacer motif in the amino-terminal end of srfAA suggests that the srfAA product may not initiate surfactin synthesis. The portion of srfA that contains the region required for competence is composed of srfAA and the first amino acid-activating domain of srfAB.     

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.     

Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.

Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.     

Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides.

Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed.