Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol In silico identifcation and evolutionary analysis of candidate genes involved in the biosynthesis methylproline genes in cyanobacteria strains of Iran Bahareh Nowruzi a, ⁎ , Saúl Blanco b,1 a Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran b Departamento de Biodiversidad y Gestión Ambiental, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071, León, España ARTICLE INFO Keywords: Adenylation domains NRPS 4-Methylprolines PKS Natural bioactive compounds Horizontal gene transfer ABSTRACT Cyanobacteria have the ability to produce a wide variety of natural bioactive compounds. In order to reveal the diversity of Cyanobacteria in Iran, and their potential to synthesise various natural products, we isolated 25 strains from diferent climatic/geographic regions and habitats, and screened the genes that are responsible for the synthesis of natural products (such as nonribosomal peptide synthetase or polyketide synthase and 4- methylproline) by means of PCR. In addition, we analysed adenylation domain substrate specifcity from screened NRPS fragments in order to identify peptidic molecules that are produced by the studied strains. The phylogenetic analysis showed the widespread, but sporadic, occurrence of the methylproline biosynthetic pathways in these strains. Our results do not only reveal the chemical diversity that is occurring in these Cyanobacteria of Iran that we isolated, but they also indicate that 4-methylproline biosynthetic genes have a complex evolutionary history in Cyanobacteria, which has been punctuated by a series of ancient horizontal gene transfer events. 1. Introduction Cyanobacteria are well-known producers of a variety of secondary metabolites with diverse chemical structures and biological properties (Nowruzi et al., 2018; Dittmann et al., 2015). Many of the bioactive secondary metabolites that are known to exist in Cyanobacteria are NRPS/PKS hybrid peptides, which are the end-products of non- ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways (Balskus and Walsh, 2010). PKSs and NRPSs represent a super family of complex biosynthetic pathway-associated enzymes that are found in prokaryotes and fungi (Jenke-Kodama et al., 2006). Although they are functionally conserved, PKSs and NRPSs are involved in the production of a structurally diverse range of metabolites with linear, cyclic, and branched conformations that are produced on mixed com- plexes of peptide synthetases and modifying enzymes (Nowruzi et al., 2017; Neilan et al., 1999; Silva-Stenico et al., 2011; Christiansen et al., 2001; Moftt and Neilan, 2001; Zhao et al., 2008). Nonribosomal peptides are composed of both proteinogenic and non-proteinogenic amino acids, which carry modifcations, such as heterocylization, gly- cosylation, acylation, methylation, and many others (Liu et al., 2014a, b). The structural diversity of these metabolites is remarkably large, which is partly due to the incorporation of many unusual and modifed residues such as D- and β-amino acids, as well as a variety of hydroxyl and N-methylated acids (Liu et al., 2014a, b). Methylprolines are non-proteinogenic amino acids with a methyl group that is connected to the 3, 4, or 5 carbon or nitrogen atoms of proline (Fig. S1). In Cyanobacteria, 4-mPro was frst discovered in spumigin A and was produced by Nodularia spumigena AV1 strain (Fewer et al., 2009). It has been subsequently found in Cyanobacteria compounds such as nostopeptolide A1, thrombin inhibitor spumigin J, trypsin inhibitor spumigin E, protein kinase inhibitor bisebromoamide, and microcystin antitoxin nostocyclopeptide M1 (Liu et al., 2014a, b; Luesch et al., 2003). In addition, methylproline amino acids have also been found in bioactive secondary metabolites from plants, fungi and actinobacteria (Luesch et al., 2003). The synthesis of 4-mPro has been demonstrated by Luesch et al. (2003) at the genetic and enzymatic le- vels, and the coding genes of the enzymes were named nosE and nosF. Their homologues were also found in the nonribosomal biosynthetic pathways of nostocyclopeptide A and spumigin E. The diverse synthesis mechanisms reveal multiple strategies for the introduction of the rare amino acids in the natural products that are produced by Cyanobacteria and other organisms. https://doi.org/10.1016/j.phytol.2018.12.011 Received 6 September 2018; Received in revised form 1 December 2018; Accepted 4 December 2018 ⁎ Corresponding author. E-mail address: bahareh.nowruzi@srbiau.ac.ir (B. Nowruzi). 1 Current address: Laboratorio de diatomología y calidad de aguas. Instituto de Investigación de Medio Ambiente, Recursos Naturales y Biodiversidad. La Serna 58, 24007, León, España. Phytochemistry Letters 29 (2019) 199–211 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved. T