Temporal dynamics of natural product biosynthesis in marine cyanobacteria

Abstract

Sessile marine organisms are prolific sources of biologically active natural products. However, these compounds are often found in highly variable amounts, with the abiotic and biotic factors governing their production remaining poorly understood. We present an approach that permits monitoring of in vivo natural product production and turnover using mass spectrometry and stable isotope ((15)N) feeding with small cultures of various marine strains of the natural product-rich cyanobacterial genus Lyngbya. This temporal comparison of the amount of in vivo (15)N labeling of nitrogen-containing metabolites represents a direct way to discover and evaluate factors influencing natural product biosynthesis, as well as the timing of specific steps in metabolite assembly, and is a strong complement to more traditional in vitro studies. Relative quantification of (15)N labeling allowed the concurrent measurement of turnover rates of multiple natural products from small amounts of biomass. This technique also afforded the production of the neurotoxic jamaicamides to be more carefully studied, including an assessment of how jamaicamide turnover compares with filament growth rate and primary metabolism and provided new insights into the biosynthetic timing of jamaicamide A bromination. This approach should be valuable in determining how environmental factors affect secondary metabolite production, ultimately yielding insight into the energetic balance among growth, primary production, and secondary metabolism, and thus aid in the development of methods to improve compound yields for biomedical or biotechnological applications.

Fig. 1.

¹⁵N labeling of nitrogen-containing metabolites from L. majuscula 3L, JHB and L. bouillonii over time. The culture conditions and time of inoculation were identical for the three species. (A) Isotope “heat map” view of m/z 300–950 MALDI spectra taken daily, with two technical and two experimental replicates per day, during the course of a 10-day feeding experiment (days 0–5, 7, and 10 shown). Shifting of the vertical spectral bands for each species indicates nitrogen-containing metabolites undergoing production and turnover. The red boxes and corresponding numbers highlight described, known metabolites; unknown metabolites can be also be seen undergoing nitrogen shifts (red asterisk). (B) Enlarged heat map showing region pertaining to carmabin A (5). The spectra for day 1 and day 10 are also provided, with the shift of 5 Da (pertaining to the 5 nitrogen atoms in carmabin A) appearing as heavier isotopic peaks that correspond to peak shifts in the heat map. (C) MALDI imaging of a L. majuscula JHB filament after six days of ¹⁵N incubation. From left to right: an epifluorescence image of the filament at ex. 590 nm, the distribution of the m/z 511 peak (red represents older jamaicamide B) the m/z 515 peak (green represents only newly biosynthesized jamaicamide B), the overlayed distribution of the 511 and 515 peaks (yellow) annotated with the calculated percentage of labeling in different areas. (D) Percent ¹⁵N labeling over the 10-day study for the compounds indicated by the red boxes: jamaicamide A, B, and hectochlorin from L. majuscula JHB, carmabin A and curazole from L. majuscula 3L, and apratoxin A from L. bouillonii. These calculations represent the percentage of fully ¹⁵N-labeled molecules for each compound (33). (N = 2, error bars are SEM, calculated from two experimental groups, each with two technical replicates. See Materials and Methods for calculation.) (E) Chemical structures as discussed in text.

Fig. 2.

Comparison of growth, percent ¹⁵N labeling of jamaicamide B and pheophytin a over 10 days from small cultures of L. majuscula JHB. (A) Optical images (8x) show the growth of a single filament over the course of 10 days. (B) MALDI-MS spectra from micro-extractions showing the relative changes in the pheophytin a (left) and jamaicamide B (right) isotope clusters over time. Top panels contain control spectra for each molecule. Because the ionization efficiency is the same for a labeled and unlabeled compound, the decrease in the unlabeled, monoisotopic peaks indicates the reduced abundance of the ¹⁴N containing species, while the increase in other peaks is indicative of newly synthesized, ¹⁵N labeled species. (C) Comparison of growth and percentage of ¹⁵N labeling of jamaicamide B and pheophytin a over 10 days. [Error bars are SEM. For ¹⁵N labeling N = 5, for growth N = 16 (day 2), 10 (day 4), 7 (day 6), 5 (day 8), 3 (day 10).]

Fig. 3.

Different ¹⁵N labeling states of jamaicamide B and A over 10 days in L. majuscula JHB. (A) Time course of single, double, total, and unlabeled jamaicamide B over 10 days, calculated from same data as in Fig. 2B. The arrows point to T₅₀ (time at which 50% of the molecules are ¹⁵N labeled), . (B) Time course of single, double, total, and unlabeled jamaicamide A over 10 days from the same dataset. (arrow). (C) Comparison of total ¹⁵N-labeled jamaicamide B and A. The initiation of ¹⁵N labeling in jamaicamide A is delayed by approximately 1.5 days when compared to jamaicamide B. (Errors are SEM, N = 5.)

Fig. 4.

Further investigation into jamaicamide A and B biosynthesis. (A) Total percent ¹⁵N labeling of jamaicamide A is enhanced by addition of NaBr to media. Bars indicate percent ¹⁵N labeling of pheophytin a, jamaicamide A, and jamaicamide B in small L. majuscula JHB cultures incubated for five days in media containing 100% [¹⁵N]NaNO₃ and either or NaBr (N = 6; error bars are SEM). (B) Total percentage ¹⁵N-labeled jamaicamide B (solid line) and A (dashed line) over 14 days in 16 h light/8 h dark conditions. (C) New production (total ¹⁵N labeling) is terminated for jamaicamide B (solid line) but continues for jamaicamide A (dashed line) when L. majuscula JHB is subjected to continuous dark for a five-day period from day 4 to day 9 (opaque box) (N = 8; error bars are SEM). (D) Ratio of jamaicamide A to jamaicamide B after six days in regular light conditions (16 h light/8 dark) and continuous dark (24 h dark) from larger scale experiments measured by LC–MS.