Deciphering Chemical Mediators Regulating Specialized Metabolism in a Symbiotic Cyanobacterium

Abstract

Genomes of cyanobacteria feature a variety of cryptic biosynthetic pathways for complex natural products, but the peculiarities limiting the discovery and exploitation of the metabolic dark matter are not well understood. Here we describe the discovery of two cell density-dependent chemical mediators, nostoclide and nostovalerolactone, in the symbiotic model strain Nostoc punctiforme, and demonstrate their pronounced impact on the regulation of specialized metabolism. Through transcriptional, bioinformatic and labeling studies we assigned two adjacent biosynthetic gene clusters to the biosynthesis of the two polyketide mediators. Our findings provide insight into the orchestration of specialized metabolite production and give lessons for the genomic mining and high-titer production of cyanobacterial bioactive compounds.

Keywords: Biosynthesis; Cyanobacteria; Genomic Mining; Quorum Sensing; Specialized Metabolism.

Figure 1

Cell‐density dependent expression of the pks1 BGC, and establishment of an AraC_PKS1 overexpression mutant. A) Expression values of a representative biosynthetic core gene of the BGC pks1 (RS10485) from a 9 d time‐course experiment performed on filters grown on diazotrophic agar plates. The baseline level estimated for constitutively expressed BGCs is indicated by the dashed line. TPM: transcripts per million. B) Schematic representation of the pks1 BGC. C) Schematic overview of the overexpression plasmid containing the potential pathway‐specific regulatory gene RS10525 from the pks1 BGC, a strong promoter region from N. punctiforme (5′ region of RS16340) and a streptomycin resistance cassette for selection. D) Transcription level of the potential pathway‐specific regulator AraC_PKS1 (RS10525) in the AraC_PKS1 mutant strain compared to the transcription level in the WT strain. E) HPLC profiles of cell pellets and culture supernatants from AraC_PKS1 mutant strain and WT under conventional and HL/HC cultivation conditions. HPLC profiles are shown at a wavelength of 199 nm. Known metabolite peaks are labeled (Mvd: microviridin, Nos1052: nostopeptolide 1052, NosA: nostopeptolide A, Apt: nostamide A).

Figure 2

Identification of bioactive metabolites from AraC_PKS1 strain. A) HPLC profiles of the extracts of culture supernatant and cells from AraC_PKS1 grown under HL/HC cultivation condition for 20 d. Several yet uncharacterized metabolite peaks are visible in the mutant strain. Main supernatant peaks were fractionated and labelled with I–V as indicated below the chromatogram. HPLC profiles are shown at the wavelengths of 220 nm (black) and 350 nm (light blue). B) Fluorescence micrographs of ripp4 reporter strain treated with fractions I–V and of an untreated ripp4 reporter control culture. All cultures were grown conventionally for 24 h. The reporter strain shows a strongly increased expression upon treatment with fraction V. CFP fluorescence signal (blue) indicating promoter activity of the ripp4 BGC. Chlorophyll‐a autofluorescence (red) indicating living vegetative cells. Scale bar (yellow)=10 μm. C) Obtained structures of new compounds, nostoclide N1 (1), N2 (2), and nostovalerolactone (3) from fraction V, and a minor congener 9‐dehydronostovalerolactone (4). These structures were elucidated by a combination of MS and NMR analyses. Selected ¹H‐¹H COSY (black bold line), ^2,3 J‐HMBC (black arrow), ^4,5 J‐HMBC (red arrow), and NOESY (blue arrow) correlations are shown.

Figure 3

Production of nostoclides and nostovalerolactone in N. punctiforme WT and the AraC_PKS1 mutant strain. HPLC profiles are shown at a wavelength of 350 nm. Culture supernatant (3) and cell pellets (1 and 2) were used for HPLC analysis. The heatmaps visualize the TPMs (transcripts per million) for each gene of the pks1 BGC.

Figure 4

Assignment of nostovalerolactones and nostoclides to the pks1 BGC. A) The pks1 BGC can be split into the nostovalerolactone BGC (nvl) and the nostoclide subcluster (ncl) based on sequence homology to characterized BGCs of structurally related tetronate antibiotics, as well as the BGC of cyanobacterin. B) Model for the biosynthesis of 9‐dehydronostovalerolactone (4) and nostovalerolactone (3) considering the results of labeling studies with 1‐¹³C acetate and 1,2‐¹³C₂ acetate (Figure S46–S51, Table S4). The proposed biosynthesis shares many parallels with the biosynthesis of tetronate antibiotics, but also comprises a non‐canonical cyclization sequence together with an oxidative rearrangement reaction to yield nostovalerolactone's unprecedented bicyclic ring system as evidenced by the unconventional labeling pattern of carbon atoms C7 and C3 of 3 and the increased number of oxygen atoms in the first proposed cyclization product (O₇) compared to its linear precursors (O₃+O₃). FAAL: fatty acyl‐AMP ligase, ACAD: acyl‐CoA dehydrogenase, ACP: acyl carrier protein, KS: ketoacylsynthase, AT: acyl transferase, KR: ketoreductase, DH: dehydratase, MT: C‐methyltransferase.

Figure 5

Effect of nostoclide and nostovalerolactone addition on BGC expression. The heatmaps visualize the log₂ fold change between treated and untreated WT. A schematic representation of the corresponding BGC is given above the heatmap.

Figure 6

Potential signaling pathway of the pks1‐derived products N1, N2 and nostovalerolactone are involved in. The factor required to activate pks1 BGC expression is not yet identified. *The PKS BGCs pks3 and pks4 are upregulated only in presence of nostovalerolactone and both nostoclides.