A multiproducer microbiome generates chemical diversity in the marine sponge Mycale hentscheli

Abstract

Bacterial specialized metabolites are increasingly recognized as important factors in animal-microbiome interactions: for example, by providing the host with chemical defenses. Even in chemically rich animals, such compounds have been found to originate from individual members of more diverse microbiomes. Here, we identified a remarkable case of a moderately complex microbiome in the sponge host Mycale hentscheli in which multiple symbionts jointly generate chemical diversity. In addition to bacterial pathways for three distinct polyketide families comprising microtubule-inhibiting peloruside drug candidates, mycalamide-type contact poisons, and the eukaryotic translation-inhibiting pateamines, we identified extensive biosynthetic potential distributed among a broad phylogenetic range of bacteria. Biochemical data on one of the orphan pathways suggest a previously unknown member of the rare polytheonamide-type cytotoxin family as its product. Other than supporting a scenario of cooperative symbiosis based on bacterial metabolites, the data provide a rationale for the chemical variability of M. hentscheli and could pave the way toward biotechnological peloruside production. Most bacterial lineages in the compositionally unusual sponge microbiome were not known to synthesize bioactive metabolites, supporting the concept that microbial dark matter harbors diverse producer taxa with as yet unrecognized drug discovery potential.

Keywords: biosynthesis; microbiomes; natural products; sponges; symbiosis.

Fig. 1.

Representative natural products from the marine sponge M. hentscheli. Structures of selected congeners for the three polyketide families are shown. The methyl groups at numbered positions of pateamine A are thought to be introduced by a β-branching mechanism common for trans-AT PKSs.

Fig. 2.

Taxonomic classification of 22 Mycale bins and distribution of identified BGCs. Maximum likelihood placement of the 22 bins with >60% estimated genome completeness (values given in parentheses behind each bin number) in the GTDB-Tk reference tree (58) consisting of 17,435 leaves. Cyanobacteria were used as outgroup. Colored stars denote the placement of the two candidate phyla “Poribacteria” and Tectomicrobia known from previous sponge microbiome studies. Colored triangles denote positions of Mycale bins affiliated with phyla with few known genomes (Nitrospirae, Candidatus Dadabacteria, Oligoflexia). The three bins harboring the mycalamide, pateamine, and gananamide BGCs are shown in bold. The number of BGCs per bin is listed in the table and color coded according to the bin's assigned phylum. The symbol ≥ is used if multiple biosynthetic contigs were found that might belong to the same pathway. Contigs encoding NRPS–PKS hybrid systems were listed as PKSs. Arylp, arylpolyene; Phos, phosphonate. NRPS, nonribosomal peptide synthetase; RiPP, ribosomally synthesized and posttranslationally modified peptide; T1, type 1; T2, type 2.

Fig. 3.

Gene clusters and biosynthetic models for mycalamide, pateamine, and peloruside. Core PKS/NRPS genes are shown in red, tailoring biosynthetic genes in blue, and genes with unknown function in gray. Gaps between domains denote protein boundaries. Biosynthetic intermediates are shown tethered to the ACP/peptidyl carrier protein domains (small gray circles). Predicted substrates are shown above the KS and A domains (exomethyl/exoester refers to a clade of KSs that mainly accept intermediates containing β-branched methyl groups or a β-branched ester in case of bryostatin). Domains depicted in gray are predicted to be nonfunctional. (A) The myc gene cluster and biosynthetic model for mycalamide. The oxygenase thought to introduce oxygen into the growing polyketide chain is highlighted in orange. (B) The pam gene cluster and biosynthetic model for pateamine. Double slashes denote separate contigs. (C) The pel gene cluster and biosynthetic model for peloruside. (D) TransATor-based structure prediction for the putative pel BGC. The linearized peloruside structure is shown for comparison. AA, amino acid; AL, acyl-CoA/ACP ligase; DB, double bond; DUF, domain of unknown function 955; ECH, enoyl-CoA hydratase; GNAT, GCN5-related N-acetyl transferase superfamily; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase homolog; KR, ketoreductase; KS⁰, nonelongating KS; MT, C-methyltransferase; OMT, O-methyltransferase; OX, oxidoreductase; PS, pyran synthase. *Transposase gene.

Fig. 4.

Orphan PKS gene clusters. Core PKS/NRPS genes are shown in red, tailoring biosynthetic genes in blue, and genes with unknown function in gray. Gaps between domains denote protein boundaries. Small gray circles represent ACP/peptidyl carrier protein domains. Clusters 1 to 6 were identified in the pateamine producer Patea custodiens, clusters 7 and 8 were identified the mycalamide producer Entomycale ignis, cluster 9 was identified in the gananamide producer Caria hoplita, and clusters 14 to 18 were identified in the unbinned fraction. DUF, domain of unknown function 2156; FAAL, fatty acyl-AMP ligase; FkbH, hydroxylase; FkbM, methyltransferase; FT, formyltransferase; GT, glycosyltransferase; KR, ketoreductase; KS⁰, nonelongating KS; OX, oxidoreductase; PLP, pyridoxal phosphate-dependent enzyme; TD, terminal domain.

Fig. 5.

The gan BGC and functional characterization of modifying enzymes. (A) The gan cluster with genes color coded according to known enzymatic functions of the polytheonamide homologs. The core sequence of the precursor GanA (43 amino acids) containing the repetitive GANANA motif is shown; Ψ indicates rSAM C-methyltransferase pseudogene. (B) MS spectra of the GluC-digested precursor GanA produced in E. coli without (Upper) and with (Lower) the dehydratase GanF. (C) Tandem mass spectrometry (MS²) spectrum of the GluC-digested precursor GanA coproduced with GanF, localizing the dehydration to Thr1 at the N terminus of the core. (D) MS spectra of GluC-digested precursor GanA coproduced with the epimerase AerD in H₂O and D₂O. A mass shift corresponding to a total of 16 deuterations was detected. (E) Localization of epimerized amino acids (star symbols) in the core sequence of GanA introduced by the epimerases PoyD and AerD. RT, retention time.