Genome Sequencing Identified a Novel Lasso Peptide
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EurAsian Journal of BioSciences Eurasia J Biosci 14, 7933-7941 (2020) Genome sequencing identified a novel lasso peptide Sirilak Namwong1*, Masahiro Yuki2, Takuji. Kudo2, Moriya Ohkuma2 Takashi Itoh2 Somboon Tanasupawat3 1Department of Biotechnology, Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok 10300, Thailand. 2Japan Collection of Microorganisms, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan 3Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand *Corresponding author: [email protected] Abstract Background: One of the biggest problems to global health is rising of antibiotic resistance, however, the novel bioactive compounds are less discovered. To overcome this situation, genome sequencing and annotating have been developed in order to predict the biosynthesis pathway of new drugs. Materials and Methods: Strain BKK1 was classified based on 16S rRNA gene sequencing, ANI- Blast (ANIb), ANI-MUMmer (ANIm) and digital DNA-DNA hybridization (dDDH). The antiSMASH was selected for prediction the secondary metabolism gene clusters. Results: Strain BKK1 was proposed as Actinopolyspora saharensis based on 16S rRNA gene sequence similarity (99.07%), ANIb (98.59%,), ANIm (99.04%) and dDDH (90.9%). The gene clusters of biosynthetic pathways predicted to synthesize a class IV lasso peptide. Its leader region, core sequences and N-terminal end were divergent form class IV lasso peptides. According to the results, an active peptide from A. saharensis BKK1 was assumed to be a new biosynthesis cluster of class IV of lasso peptide. Additionally, its supernatant exhibited inhibitory activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Conclusion: The genome sequence and antibacterial assessment of A. saharensis BKK1 provide the useful knowledge of the novel peptide antibiotic cluster for developing new medicines for further medical applications. Keywords: Actinobacteria, Actinopolyspora saharensis, Lasso peptide, Genome, Bioactive compound Namwong S, Yuki M, Kudo T, Ohkuma M, Itoh T, Tanasupawat S (2020) Genome sequencing identified a novel lasso peptide. Eurasia J Biosci 14: 7933-7941. © 2020 Namwong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License. INTRODUCTION YIM90600. It carried erythromycin biosynthetic gene cluster encoding two novel erythromycins; erythronolide The discovery of novel active compounds from the H (EH) and erythronolide I (Chen et al. 2014). Recently, potential Actinobacteria i.e., Saccharopolyspora eight draft genome sequence of Actinopolyspora erythraea (erythromycins) and Streptomyces species were published, notwithstanding, there was no griseoflavus (aborycin) is screened by using the existing report of peptide antibiotics, a specific family of traditional methods. These procedures were costly and ribosomally synthesized and post-translationally time-comsuming (Fujimono and Sakai 2013, Chen et al. modified peptide (RiPP). The first lasso peptide named 2014). Despite the screening experiment, a few natural as anantin was proposed in 1986 and then microcin J25 products from the genus of Actinopolyspora were (MccJ25) in 1992 and aborycin in 1994 (De Lorenzo and investigated. The genus Actinopolyspora, which Neilands 1986, Salomon and Farias, 1992, Potterat et belonged to the class Actinobacteria, order al. 1994). Anantin and microcin belonged to class II Actinomycetales and family Actinopolysporaceae and lasso peptide and exhibited the atrial natriuretic factor twelve species have been described (Gochnauer et al. antagonist antimicrobial activity (Wyss et al. 1991, 1975, Yoshida et al. 1991, Guan et al. 2010, Tang et al. Maksimov and Link, 2014, Pati et al. 2018). Aborycin 2011, Meklat et al. 2012a, 2012b, Guan et al. 2013, (class I lasso peptide) consisted of a tricyclic 21-peptide Meklat et al. 2013a, 2013b, Saker et al. 2015, Duangmal antibiotic was proved as the HIV protease inhibitor to et al. 2016). prevent the assembly of HIV (Potterat et al. 1994, The genome analysis allowed us to discover a new Frechet et al. 1994, Shao et al. 2019). In 2017-2019, natural product form Actinopolyspora erythraea three novel antibacterial peptides (achromosin, 7933 EurAsian Journal of BioSciences 14: 7933-7941 (2020) Namwong et al. specialicin and citrocin) isolated from Actinobacteria W (Thompson et al. 1997). The phylogenetic tree was were published (Kaweewan et al. 2017, Kaweewan et al. constructed by the neighbor-joining algorithms using 2018, Cheung-Lee et al. 2019). Now, the active peptides MEGA 7.0 with bootstrap analysis (Saitou and Nei have been grained increasing attention for medical 1987). treatment due to their simply biosynthetic pathway Genome sequencing and functional gene annotation (Hegemann, 2019). Nevertheless, the novel biosynthetic Whole genome sequence of strain BKK1 was gene clusters (BGCs) of lasso peptide were rarely analyzed using an Illumina Miseq platform (Illumina, distributed (Tietz et al. 2017). Therefore, genome-mining Inc., San Diego, US-CA) using 2 × 250 bp paired-end approaches are considerably challenged for the reads. A de novo assembling of the reads to contigs discovery of new lasso peptides for the next years (Su using SPAdes 3.12 was performed (Bankevich et al. et al. 2019). 2012). The draft genomes of strain BKK1 was publicly This work aims to characterize Actinopolyspora available on the GenBank with the accession number of strain, BKK1 from salted egg collected from Bangkapi JAANYZ000000000. The genome was annotated on market in Bangkok province, Thailand based on the Prokka software 1.12 in line with the NCBI prokaryotic polyphasic approach, to analyze the draft genome genome annotation pipeline (PGAP) (Seemann 2014). sequence, to annotate the secondary metabolite genes Average nucleotide identity (ANI) was calculated and to determine the antibacterial activity. amongst the genome of two Actinopolyspora strains and the closely related type strains standing on ANI-Blast MATERIALS AND METHODS (ANIb) and ANI-MUMmer (ANIm) algorithms within the JSpeciesWS web service (Ritcher et al. 2016). The Polyphasic characteristics of strain BKK1 genome-to-genome distance calculator (GGDC 2.1) with Twenty-eight salted eggs were collected from the BLAST+ method was used to evaluate the digital Bangkapi market in Bangkok province, Thailand and DNA-DNA hybridization (dDDH) (Meier-Kolthoff et al. stored in the plastic bags at 4 C. Strain BKK1 was 2013). The polyphasic and genotypic (phylogenetic tree, isolated by spread-plate technique on complete medium ANI and dDDH) results were summarized for (CM, 15%, w/v NaCl) incubated at 30 C for 2 weeks identification of strain BKK1. (Meklat et al. 2012b). Phenotypic properties were The gene clusters were analyzed using antiSMASH assessed as described by Gordon et al. (1974), i.e., to permit us to predict the secondary metabolite utilization of carbon sources, range of NaCl for growth biosynthesis gene clusters (Medema et al. 2011). and resistance of antibiotics. All media used for biochemical tests included 15% (w/v) NaCl i.e., CM Antibacterial Activity assay agar, nutrient agar (NA) and the international Antimicrobial activity was determined by using paper Streptomyces project (ISP 2 and ISP 4). Cultural disc diffusion assay (Chen et al. 2010). Briefly, 100 μL characteristics and the colors of substrate. For culture of Staphylococcus aureus ATCC 25923 and Escherichia were investigated (Shirling and Gottlieb 1966). The coli ATCC 25922 we swabbed on the surface of NA. The colors of the substrate and aerial mycelia and any test extract was introduced into 7 mm discs incubated at soluble pigments were determined by comparison with 37 °C for 24 h. Inhibition zones were measured in the ISCC-NBS centroid color chart (Kelly and Judd millimeter (mm) using a vernier's caliper and tests were 1976). In chemotaxonomic studies, menaquinones and carried out in triplicate. polar lipids were analyzed (Minnikin et al. 1984, Komagata and Suzuki 1987). The isomeric form of RESULTS AND DISCUSSION diaminopimelic acid and the presence (or not) of glycine Polyphasic characteristics of strain BKK1 in the cell wall were realized as described by Becker et Strain BKK1 exhibited good growth on CM agar, NA al. (1964). The composition of whole-cell sugars was and ISP 2 media and developed long chains of spores determined according to the method of Lechevalier et al. on white aerial mycelium and fragmentation of yellow (1970). The fatty acid profile was determined following substrate mycelium. It was sensitive to chloramphenicol the procedure of Sasser (1990), using the TSBA40 (30 µg/mL), but insensitive to lysozyme (0.005 % w/v). method on a Microbial Identification System (MIDI). Strain BKK1 used sugars for their growth i.e., cellibiose, 16S rRNA gene sequencing and phylogenetic galactose, glucose, lactose, maltose and sucrose. It tree analysis grew in a wide range of NaCl concentrations (10-30%, 16S rRNA genes were amplified and purified. The w/v). This strain consisted of meso-diaminopimelic acid purified PCR products were sequenced using the (but not glycine) in its cell wall and arabinose including universal primers as researched by Coenye et al. galactose in whole-cell hydrolyzates. Three fatty acids (1999). The 16S rRNA sequence has been deposited in (anteiso-C17:0, iso-C15:0, and C15:0) and two the GenBank data library (strain BKK1=LC493220). The menaquinones