Biosynthesis of Lyngbyastatins 1 and 3, Cytotoxic Depsipeptides from an Okeania sp. Marine Cyanobacterium

Abstract

Lyngbyastatins (Lbns) 1 (1) and 3 (2) belong to a group of cyclic depsipeptides that inhibit cancer cell proliferation. These compounds have been isolated from different marine cyanobacterial collections, while further development of these compounds relies on their lengthy total synthesis. Biosynthetic studies of these compounds can provide viable strategies to access these compounds and develop new analogs. In this study, we report the identification and characterization of one Lbn biosynthetic gene cluster (BGC) from the marine cyanobacterium Okeania sp. VPG18-21. We initially identified 1 and 2 in the organic extract by mass spectrometry and performed the targeted isolation of these compounds, which feature a (2S,3R)-3-amino-2-methylpentanoic acid (MAP) and a (2S,3R)-3-amino-2-methylhexanoic acid (Amha) moiety, respectively. Parallel metagenomic sequencing of VPG18-21 led to the identification of a putative Lbn BGC that encodes six megaenzymes (LbnA-F), including one polyketide synthase (PKS, LbnE), four nonribosomal peptide synthetases (NRPSs, LbnB-D and -F), and one PKS-NRPS hybrid (LbnA). Bioinformatic analysis of these enzymes suggested that the BGC produces 1 and 2. Furthermore, our biochemical studies of three recombinant adenylation domains uncovered their substrate specificities, supporting the identity of the BGC. Finally, we identified near-complete Lbn-like BGCs in the genomes of two other marine cyanobacteria.

Figure 1.

Chemical structures of selected lyngbyastatin (Lbn) analogs belonging to the dolastatin 11/12 class. MAP: (2S,3R)-3-amino-2-methylpentanoic acid; Amha: (2S,3R)-3-amino-2-methylhexanoic acid; Ibu: 4-amino-2,2-dimethyl-3-oxopentanoic acid.

Figure 2.

MS² fragmentation of 1 and 2 isolated from the extract of VPG18-21.

Figure 3.

Proposed biosynthetic pathway of 1 and 2. A schematic representation of the Lbn BGC was shown at the top. PKS genes and domains are colored in orange, while those of NRPSs are in light blue. M-CoA: malonyl-CoA; KS: ketosynthase; AT: acyltransferase; T: thiolation; AmT: aminotransferase; MT: N-, C- or O-methyltransferase whereas *indicates an inactive domain; A: adenylation; KR: ketoreductase; C: condensation; TE: thioesterase.

Figure 4.

Relative activity of different substrates activated by recombinant A domains of LbnA (A), LbnD2 (B), and LbnD3 (C). The activity of the best substrate is set as 100% for normalizing other substrates. The data represent means ± s.d. of three independent experiments. 2-Hydroxy-3-methylvaleric acid: Hmva; Me-2-oxovaleric acid: Mova; l-alloisoleucine: l-alle.

Figure 5.

Organization of two mined Lbn-type BGCs and the predicted structures of their encoded compounds. (A). The Lbn-type BGC mined from the genome of Okeania sp. SIO1I7. The GenBank accession numbers of ORF1-8 are NET25238 to NET25245. The moiety incorporated by its LbnA homolog is colored in blue, and l-Phe is in red. (B). The Lbn-type BGC mined from Moorena sp. SIO1G6. The GenBank accession numbers of FAAL, DS, and ORF1-7 are NET65453 to NET65461. The moiety incorporated by its LbnA homolog is colored in blue, while the terminal alkyne is shadowed in green. In addition, d-Phe generated after epimerization is colored in red. GNAT: GCN5-related N-acetyltransferases; FAAL: fatty acid-AMP ligase; DS: fatty acid desaturase.