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Nonribosomal Peptides Synthetases and Their Applications in Industry Mario Alberto Martínez‑Núñez1,2* and Víctor Eric López Y López3

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Nonribosomal Peptides Synthetases and Their Applications in Industry Mario Alberto Martínez‑Núñez1,2* and Víctor Eric López Y López3 Martínez‑Núñez and López Sustain Chem Process (2016) 4:13 DOI 10.1186/s40508-016-0057-6 REVIEW Open Access Nonribosomal peptides synthetases and their applications in industry Mario Alberto Martínez‑Núñez1,2* and Víctor Eric López y López3 Abstract Nonribosomal peptides are products that fall into the class of secondary metabolites with a diverse properties as toxins, siderophores, pigments, or antibiotics, among others. Unlike other proteins, its biosynthesis is independent of ribosomal machinery. Nonribosomal peptides are synthesized on large nonribosomal peptide synthetase (NRPS) enzyme complexes. NRPSs are defined as multimodular enzymes, consisting of repeated modules. The NRPS enzymes are at operons and their regulation can be positive or negative at transcriptional or post-translational level. The pres‑ ence of NRPS enzymes has been reported in the three domains of life, being prevalent in bacteria. Nonribosomal pep‑ tides are use in human medicine, crop protection, or environment restoration; and their use as commercial products has been approved by the U. S Food and Drug Administration (FDA) and the U. S. Environmental Protection Agency (EPA). The key features of nonribosomal peptides and NRPS enzymes, and some of their applications in industry are summarized. Keywords: Nonribosomal peptides, Nonribosomal peptides synthetases, Environmental restoration, Human healt Background half of the NRPS enzymes found in a genome-mining Nonribosomal peptides is a diverse family of natural study of 2699 genomes by Wang et al. [1] are nonmodular products fall into the class of secondary metabolites with NRPS enzymes. Nonmodular NRPS enzymes are found a diverse properties as toxins, siderophores, pigments, in siderophore biosynthetic pathways like EntE and antibiotics, cytostatics, immunosuppressants or antican- VibH in enterobactin, and VibE in vibriobactin [5] or as cer agents [1, 2]; and have a particularity: their synthesis a stand-alone peptidyl carrier protein such as BlmI from is independent of ribosomal machinery. Soil-inhabiting the bleomycin gene cluster [6]. The presence of NRPS microorganisms, such as Actinomycetes and Bacilli, and enzymes has been reported in the three domains of life, eukaryotic filamentous fungi are mostly producers of being prevalent in bacteria, less frequent in eukarya and nonribosomal peptides, but marine microorganisms have rare in archaea. Within bacteria domain, Proteobacteria, also emerged as a source for such peptides [3]. These Actinobacteria, Firmicutes, and Cyanobacteria were the peptides have a structural features such as contain amino phyla with major abundance of these enzymes, and there acids like ornithine or imino acids, and their structures has been observed a correlation between genome size are macrocyclic, branched macrocyclic, dimers or trim- and the number of NRPS’s clusters [1]. The key features ers of identical structural elements [4]. Usually nonribo- of the microbial biosynthesis of nonribosomal peptides, somal peptides are synthesized on large nonribosomal the structure and regulation of NRPS enzymes, and some peptide synthetase (NRPS) enzyme complexes, defined as applications in industry are summarized below. modular multidomain enzymes; nevertheless more than Biosynthesis of nonribosomal peptides The biosynthesis of nonribosomal peptides is done by *Correspondence: [email protected] 1 Laboratorio de Ecogenómica, Facultad de Ciencias, Universidad NRPSs which are modularly organized multi-enzyme Nacional Autónoma de México, Unidad Académica de Ciencias y complexes which serve as templates and biosynthetic Tecnología de Yucatán, Carretera Sierra Papacal‑Chuburna Km 5, machinery, via a thiotemplate mechanism independent 97302 Mérida, Yucatán, Mexico Full list of author information is available at the end of the article of ribosomes [2]. A module is defined as a section of the © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martínez‑Núñez and López Sustain Chem Process (2016) 4:13 Page 2 of 8 NRPS enzyme that incorporate in a specific manner one that can be considered as the “codons” of NRPS enzymes amino acid into the peptide backbone, and in turn the and are important to substrate specificity [12]. The sub- modules can be divided into domains, which catalyze strates that can be recognized by A-domain can include the individual steps of nonribosomal peptide synthe- D and L forms of the 20 amino acids used in ribosomal sis. Each module consist of three domains, adenylation proteins synthesis, as well as non-proteino-genic amino (A) domain, peptidyl carrier protein (PCP) or thiolation acids such as ornithine, imino acids, and hydroxy acids (T) domain, and condensation (C) domain which carry such as α-aminoadipic and β-butyric acids [2]. The reac- out the synthesis of nonribosomal peptides (Fig. 1) [7]. tion carried out by the A-domain shares the same chem- The order of modules is usually co-linear to the product istry as that performed by aminoacyl-tRNA synthetases peptide sequences [8, 9]. Nonribosomal peptides syn- (AARSs), which act in the initial step of ribosome-driven thesis proceeds in a N- to C-terminal direction, produc- protein synthesis [8, 13]. The second step is performs by ing peptides that are usually about 3–15 amino acids in the PCP-domain of ~80 amino acids [12], which cova- length, and the released peptides can be linear, cyclic, or lently binds the activated amino acid to their cofactor branched-cyclic [3]. There is a sequence motif conserved 4′-phosphopantetheine (PP) arm via a thioester bond and in domains that facilitates the identification of these transfer the activated substrate and the elongation inter- using sequences search tools like BLAST, as in the study mediates to C-domain [14]. The final step is carried out done by Etchegaray et al. [10] to identified NRPSs in the by the C-domain of ~450 amino acids, which catalyzes genome of Xanthomonas axonopodis and X. campestri. peptide bond formation between the carboxyl group of The first step in the biosynthesis is done by the A-domain, the nascent peptide and the amino acid carried by the which recognizes and performs activation of amino acid flanking module, allowing the translocation of the grow- substrate via adenylation using Mg-ATP, result in an ami- ing chain to the following module [12, 13]. After conden- noacyl adenylated intermediate [11]. The A-domain con- sation step, the linear intermediate peptide is release with sist of ~550 amino acids, and has ten amino acid residues the help of thioesterase (TE) domain by either hydrolysis Fig. 1 Structural features of nonribosomal peptide synthetase enzymes. Nonribosomal peptide synthetase enzymes can be subdivided in modules, each incorporating one amino acid. Each module consist of three domains: adenylation (A) domain, peptidyl carrier protein (PCP) domain, and condensation (C) domain, which carry out the synthesis of nonribosomal peptides; the epimerization (E) domain and thioesterase (TE) domain is also represents. In this figure, surfactin biosynthesis was exemplified Martínez‑Núñez and López Sustain Chem Process (2016) 4:13 Page 3 of 8 or internal cyclization in bacterias, and less frequent in Regulation of NRPS genes NRPSs of fungi. In fungi the chain release is carried out Global regulators such as DegU and Comp/ComA two- by a variety of mechanisms, two of which are (1) a termi- component system can act at transcriptional level, nal C domain, which catalyzes release by inter- or intra- regulating positively the expression of srfA, bac, and molecular amide bond formation, and (2) a thioesterase bmy cluster genes of Bacillus spp., which encodes sur- NADP(H) dependent reductase (R) domain which cata- factin (Fig. 1), bacilysin and bacillomycin, respectively lyzes reduction with NADPH to form an aldehyde [2]. [18–20]. In the case of srfA genes that encoding for the The primary product of this synthesis may be post-syn- surfactin, the transcriptional initiation is through ComX thetically modified to achieve its mature form by addi- pheromone that can activated ComP, causing ComP to tional tailoring enzymes which are not part of NRPS by autophosphorylate and, subsequently, donate a phos- N-, C- and O-methylation, glycosylation, hydroxylation, phate group to ComA. Upstream of the srfA promoter acylation, halogenation or heterocyclic ring formation [3, there are ComA boxes, which are recognized by phos- 15, 16]. These tailoring enzymes and their reactions con- phorylated ComA and binding to them as a tetramer tribute to generate the structural diversity of nonriboso- initializing the transcription of srfA [18, 19, 21]. Another mal peptides [10]. positive regulator of transcription of srfA is PerR protein, which positively modulates srfA expression by binding to Structure of nonribosomal peptide synthetase regions located upstream of ComA boxes, known as PerR (NRPS) enzymes boxes [22]. Expression of the bac operon is dependent on NRPSs are defined as multimodular enzymes, consisting a σA-dependent
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