Metagenomics Unveils Posidonia oceanica "Banquettes" as a Potential Source of Novel Bioactive Compounds and Carbohydrate Active Enzymes (CAZymes)

Abstract

Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea. It produces large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." In recent years, interest in the potential uses of these P. oceanica banquettes has increased, and it was demonstrated that biomass extracts showed antioxidant, antifungal, and antiviral activities. The discovery of new compounds through the culture of microorganisms is limited, and to overcome this limitation, we performed a metagenomic study to investigate the microbial community associated with P. oceanica banquettes. Our results showed that the microbial community associated with P. oceanica banquettes was dominated by Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria. Pseudoalteromonas was the dominant genus, followed by Alteromonas, Labrenzia, and Aquimarina. The metagenome reads were binned and assembled into 23 nearly complete metagenome-assembled genomes (MAGs), which belonged to new families of Cyanobacteria, Myxococcota, and Granulosicoccaceae and also to the novel genus recently described as Gammaproteobacteria family UBA10353. A comparative analysis with 60 published metagenomes from different environments, including seawater, marine biofilms, soils, corals, sponges, and hydrothermal vents, indicated that banquettes have numbers of natural products and carbohydrate active enzymes (CAZymes) similar to those found for soils and were only surpassed by marine biofilms. New proteins assigned to cellulosome modules and lignocellulose-degrading enzymes were also found. These results unveiled the diverse microbial composition of P. oceanica banquettes and determined that banquettes are a potential source of bioactive compounds and novel enzymes. IMPORTANCE Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea that forms large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." These banquettes accumulate on the shore, where they can prevent erosion, although they also cause social concern due to their impact on beach use. Furthermore, Posidonia dry material has been considered a source of traditional remedies in several areas of the Mediterranean, and a few studies have been carried out to explore pharmacological activities of Posidonia extracts. The work presented here provides the first characterization of the microbiome associated with Posidonia banquettes. We carried out a metagenomic analysis together with an in-depth comparison of the banquette metagenome with 60 published metagenomes from different environments. This comparative analysis has unveiled the potential that Posidonia banquettes have for the synthesis of natural products, both in abundance (only surpassed by marine biofilms) and novelty. These products include mainly nonribosomal peptides and carbohydrate active enzymes. Thus, the interest of our work lies in the interest of Posidonia "waste" material as a source of new bioactive compounds and CAZymes.

Keywords: BGC; CAZyme; Myxococcota; NRPS; Posidonia oceanica; Pseudoalteromonas; banquettes; bioactive compound.

FIG 1

Taxonomic distribution of the microbial community recovered from Posidonia oceanica banquettes using different approaches (16S rRNA metabarcoding, 16S rRNA from metagenome shotgun sequencing, reads from metagenome shotgun sequencing, and contigs from metagenome shotgun sequencing) at the level of phylum (A), family (B), and genus (C).

FIG 2

Relative frequencies of the biosynthetic clusters detected in Posidonia oceanica banquettes and other environments using antiSMASH (in contigs of >5 kb). Black bars indicate the numbers of clusters detected among the bacterial contigs, and gray bars indicate those unclassified taxonomically. Pie charts above the bars indicate the relative contribution of each biosynthetic cluster category according to the legend. The number above each pie chart represents the number of different biosynthetic clusters. DCM, deep chlorophyll maximum; MES, mesopelagic; LAP, linear azol(in)e-containing peptide.

FIG 3

Ketoacyl synthase (KS) domains in polyketide synthases (PKSs) detected in Posidonia oceanica banquettes. (A) Classification of KS domains. (B) Box plot of similarities between the KS domains detected in P. oceanica and those found in the other environments analyzed in this work. (C) Box plot of similarities between the KS domains detected in each habitat used in this work against proteins in the nr database. (D) Taxonomic distribution of KS domains detected in P. oceanica banquettes. FAS, domains involved in fatty acid synthesis; hybrid-KS, biosynthetic assembly lines that include both PKS and NRPS components; iterative, PKS domains containing the characteristic domains of type I PKSs; modular, multidomain architecture consisting of multiple sets of modules; PUFA, polyunsaturated fatty acid; trans, modular PKS operons lacking cognate AT domains; DCM, deep chlorophyll maximum; MES, mesopelagic.

FIG 4

Condensation domains in nonribosomal peptide synthetases (NRPSs) detected in Posidonia oceanica banquette contigs (>5 kb). (A) Classification of C domains. (B) Box plot of similarities between the C domains from P. oceanica banquettes and those detected in other environments. (C) Box plot of similarities between the C domains from the different environments and proteins in the nr database. (D) Taxonomic distribution of C domains detected in P. oceanica banquettes. C, condensation domain; cyc, cyclization domains; DCL, domains that link an l-amino acid to a growing peptide ending with a d-amino acid; dual, dual domains catalyzing condensation and epimerization; epim, epimerization domains changing the chirality of the last amino acid in the chain from l- to d-amino acid; hybridC, hybrid PKS/NRPS secondary metabolite; LCL, domains that catalyze formation of a peptide bond between two l-amino acids; modAA, involved in the modification of the incorporated amino acid; start, acylate the first amino acid with a fatty acid, polyketide, or other molecule; DCM, deep chlorophyll maximum; MES, mesopelagic.

FIG 5

Classification of secondary metabolite biosynthetic gene clusters in the 23 metagenome-assembled genomes (MAGs) recovered from Posidonia oceanica banquettes.

FIG 6

Frequencies of the CAZymes detected using dbCAN in the different environments analyzed in this work. The bar graph shows the number of biosynthetic clusters per number of total ORFs per metagenome. Black indicates the number of CAZymes detected among the bacterial fraction, and gray indicates CAZymes found in the taxonomically unclassified sequences. Pie charts demonstrate the relative contribution of each CAZyme family to the total. The number above each pie chart represents the number of different CAZyme families. DCM, deep chlorophyll maximum; MES, mesopelagic.

FIG 7

CAZymes and auxiliary enzymes in Posidonia oceanica banquettes. (A) Similarities between the CAZyme sequences from the different environments and sequences in the nr database. (B) Similarities between the CAZymes detected in P. oceanica and those from the other environments. (C) Similarities between CAZymes from each family detected in P. oceanica and sequences in the nr database. DCM, deep chlorophyll maximum; MES, mesopelagic.

FIG 8

Classification of CAZyme families in the 23 MAGs recovered from Posidonia oceanica banquettes.