Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery

Abstract

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.

Keywords: Biosynthesis; Cyanobacteria; Genomics; Mass spectrometry; Natural products.

Fig. 1

Hydrocarbon producing pathways in cyanobacteria. a Organization of the Olefin Synthase (OLS) pathway—FAAL fatty acid-ACP ligase, ACP acyl carrier protein, KS ketosynthase, AT acyltransferase, KR ketoreductase, ST sulfotransferase, TE thioesterase. b Organization of the fatty acid ACP reductase (FAAR)/aldehyde deformylating oxygenase (ADO) pathway

Fig. 2

a Structure of curacin A, from Moorea producens 3L. b Representation of docking domains between successive PKS modules of curacin A pathway—DH dehydratase, ER enoyl reductase, MT methyltransferase

Fig. 3

a Structure of jamaicamide B. b Structure of carmabin A. c Enzymatic functions of JamABC: JamA acyl-ACP synthetase, JamB; membrane-bound desaturase, and JamC; acyl carrier protein

Fig. 4

Organization of the phormidolide pathway. The pathway, identified from the genome of a Leptolyngbya sp., has many of the reported hallmarks of a trans-AT PKS system, including (1) discrete AT enzymes upstream of the PKS megasynthase architecture; (2) tandem ACP domains; (3) split modules; (4) non-elongating KS⁰ domains; (5) embedded ECH pairs and (6) HCS cassette. ACPS acyl carrier protein synthase, oMT O-methyltransferase, FkbH FkbH-phosphatase, AT^d docking acyltransferase, KS⁰ non-elongating ketosynthase, PS pyran synthase, C NRPS-like condensation domain, Hal halogenase, Hydro hydrolase, 4′PP 4′-phosphopantetheine, FACL Fatty Acid CoA Ligase, HCS HMG-CoA synthase, PEP phosphoenolpyruvate

Fig. 5

Select molecular networks clusters derived from mass spectrometric analysis of extracts of M. producens 3L (red), M. producens JHB (blue), M. bouillonii PNG (green). In the nodes, squares indicate consensus MS/MS spectra to compounds in an in-house MS/MS-library of known molecules. The respective compound name of an identified molecule is given next to the square node. The node size is representative of the numbers of MS²-spectra obtained for that specific m/z, and is reflective of the relative abundance of the metabolite. Multiple clusters per compound derive from the fact that [M+H]⁺ and [M+Na]⁺ parent ions can fragment differently [60]

Fig. 6

Proposed biosynthetic pathway of the columbamides in M. bouillonii PNG–AS acyl-ACP synthetase, A adenylation domain, PCP peptidyl carrier protein, R reductase

Fig. 7

Comparison between PKS/NRPS clusters of 4 Moorea strains with 7 (out of 126) additional cyanobacteria reviewed by Calteau et al., 2014 [7]. The 7 genomes were selected to compare two low secondary metabolite producers (Synechococcus genomes), two average secondary metabolite producers producers (Microcoleus sp. and Gleocapsa sp.) and three high secondary metabolite producers producers (Nostoc punctiforme, C. stagnela and Fischerella sp.). With the exception of Fischerella sp. PCC9339, only final scaffolds were considered, as incomplete genomes tend to present fragmented biosynthetic gene

Fig. 8

a Comparison between hectochlorin and lyngbyabellin A biosynthetic genes versus the new gene cluster identified in M. producens PAL 15AUG08-1. The percentage values represent the amino acid identities of MAFFT alignments between PAL genes and their equivalents above and below in JHB and PNG pathways. b Comparison between structures and residues for the predicted new compound, hectochlorin and lyngbyabellin B. In red, post-translational modifications that are not clearly predicted by biosynthetic gene clusters. Cy heterocyclization domain, Ox oxidase

Fig. 9

Predicted biosynthesis of M. producens PAL 15AUG08-1 macrolide of structural similarity to hectochlorin and lyngbyabellin B. Red sections represent uncertain positions of P450 oxidation