Bioprospecting Portuguese Atlantic coast cyanobacteria for bioactive secondary metabolites reveals untapped chemodiversity

Abstract

Continental Portugal has an extensive Atlantic Ocean coastline; however little is known about the diversity of its marine cyanobacteria, with only a few reports published. Cyanobacteria are a prolific source of bioactive compounds with promising therapeutic applications. Previously, several cyanobacterial strains (Subsections I-IV) were isolated from the Portuguese Atlantic coast and characterized using a polyphasic approach. Preliminary screening indicated that these cyanobacteria lacked the genes for proteins involved in the production of conventional cyanotoxins. However, it was previously shown that extracts of marine Synechocystis and Synechococcus were toxic to invertebrates, with crude extracts causing stronger effects than partially purified ones. To further evaluate the potential of our temperate region Portuguese isolates to produce bioactive compounds, a PCR screening for the presence of genes encoding non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), targeting the adenylation (A) and ketosynthase (KS) domains respectively, was performed. DNA fragments were obtained for more than 80% of the strains tested, and the results revealed that PKS genes are more ubiquitous than NRPS genes. The sequences obtained were used in an in silico prediction of the PKS and NRPS systems. RT-PCR analyses revealed that these genes are transcribed under routine laboratory conditions in several selected strains. Furthermore, LC-MS analysis coupled with molecular networking, a mass spectrometric tool that clusters metabolites with similar MS/MS fragmentation patterns, was used to search for novel or otherwise interesting metabolites revealing an untapped chemodiversity.

Innovative omics-based approaches for prioritisation and targeted isolation of natural products – new strategies for drug discovery

“…8 In academic laboratories, the screening of collections of a few hundred extracts corresponding to a dened corpus is typical in focused studies. [9][10][11][12][13][14] These small collections are easier to handle. Their size is compatible with concomitant metabolite proling and mining and with the capacities of tools currently available for NP prioritisation studies as discussed below.…”

“…These studies have highlighted the strong potential of such a roadmap for NP discovery based on large-scale genomics and metabolomics used in microbial research. 41 Two recent studies 9,42 have combined a genomic method with an MN approach to explore the biosynthetic potential of cyanobacteria that serve as prolic NP producers. In the rst study, PCR screening was performed on 61 strains belonging to different genera to detect the presence of genes encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) to target their adenylation and ketosynthase domains.…”

“…In the rst study, PCR screening was performed on 61 strains belonging to different genera to detect the presence of genes encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) to target their adenylation and ketosynthase domains. 9 In the second study, three species of genus Moorea were subjected to genome sequencing to identify their biosynthetic genes (NRPS and PKS). 42 In both studies, LC-MS/MS-MN analyses allowed for the detection of novel NPs and showed that both PKS and NRPS genes were present in the majority of cyanobacterial strains and that the majority of NPs were strain-specic.…”

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

“…Since then, a good number of strains have been isolated and assessed in ecotoxicological studies or used for the discovery of biologically active compounds, the main research lines of the group. As a consequence, a considerable body of research (e.g., Vasconcelos et al 1995 ; Martins et al 2005 , 2013 ; Leão et al 2013b ; Brito et al 2015 ) emphasizes that several strains now deposited at the LEGE culture collection (CC) have the potential or actual capacity to produce a myriad of chemical compounds, including toxins or newly discovered bioactive molecules. Yet, most of the strains at BBE were kept independently by their isolators along these years, and were poorly characterized, named inconsistently or even unidentified.…”

Chassis optimization as a cornerstone for the application of synthetic biology based strategies in microbial secondary metabolism

“…Among the several bacteria used as natural product factories ( Gross and Loper, 2009 ; Weissman and Muller, 2010 ; Sansinenea and Ortiz, 2011 ; Mondol et al, 2013 ; Brito et al, 2015 ) the actinomycetes, in particular streptomycetes, stand out as the most prolific source of bioactive microbial metabolites ( Berdy, 2005 ). Moreover, the vast (meta)genomic data collected during the post-genomic era and the identification of multiple cryptic secondary metabolite biosynthetic gene clusters in the genomes, has identified actinobacteria as a renewed source of novel natural products ( Baltz, 2008 ).…”