Isolation of the bioactive metabolite

Supernatants of stationary cultures of S. elongatus inhibited the growth of Anabaena variabilis. The inhibitory activity could be extracted from lyophilized culture supernatants with the polar solvent methanol, but not with chloroform, acetone, or ethyl acetate as visualized by agar-diffusion plate assays (Fig. 1a). The producer strain was not affected by these extracts. Significant production of the inhibitor required CO 2 supplementation of liquid cultures and was dependent on the cell density of the producer strain. Inhibitor content apparently peaked after about 2 weeks of growth of S. elongatus cultivated in batch cultures in BG11 medium (Fig. 1b).

Fig. 1 Extracts of supernatant of Synechococcus elongatus inhibits growth of Anabaena variabilis. a Agar-diffusion plate assay with the effect of organic extracts (1, methanol; 2, chloroform; 3, acetone, and 4, ethyl acetate) of lyophilized supernatant of stationary-phase S. elongatus cultures on the growth of the producer strain and A. variabilis. Neomycin (Neo, 20 µg) served as positive control. b Optical density of producer strain S. elongatus (black) and zone of A. variabilis growth inhibition (diameter) of methanol extracts of S. elongatus supernatant on agar diffusion plates (turquoise). Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file Full size image

The chemical characterization of the bioactive compound indicated high polarity and absence of UV absorption. The low levels produced demanded an optimized bioactivity-guided isolation protocol with several enrichment and purification steps. A chromatographically pure compound was obtained via successive size-exclusion chromatography, medium-pressure liquid chromatography (MPLC) on normal phase, and ligand/ion-exchange high-performance liquid chromatography (HPLC) coupled to evaporative light-scattering detection (ELSD) (Supplementary Fig. 1). The molecular formula of the bioactive molecule was determined by electrospray ionization high-resolution mass spectrometry (ESI-HRMS) to be C 7 H 14 O 6 (M R = 194.18 Da from m/z = 217.0675 [M+Na]+) (Supplementary Fig. 2).

We elucidated the structure of the chromatographically pure compound using nuclear magnetic resonance (1H-NMR, 13C-NMR, and two-dimensional spectra; Supplementary Table 1, Supplementary Figs. 12–16). The signals were assigned to the constitution of a 7-deoxyheptulose and indicated the relative configuration mainly present in the furanose form (Fig. 2b, c). In marked contrast to six-membered sugar rings (pyranoses), the five-membered furanoses exhibit complex multiple ring conformations, and the coupling pattern only allows a suggested relative configuration27. The occurrence of pyranose and furanose forms of d-2-heptuloses are known for d-altro-2-heptulose, d-manno-2-heptulose, d-galacto-2-heptulose, and d-gluco-2-heptulose28. The only d-2-heptulose existing mainly in furanose form corresponds to the altro configuration, which rendered this configuration most probable for the inhibitor isolated from culture supernatants of S. elongatus.

Fig. 2 Structure and chemoenzymatic synthesis of 7-deoxy-sedoheptulose (7dSh, 1). a Chemoenzymatic synthesis of 7-deoxy-sedoheptulose (7dSh). Absolute configurations of stereo-centers are indicated. b Chemical structure of 7dSh in the furanose form with given assignments of coupling constants (gray). c (1–4) 1H NMR spectra of 7dSh (CD 3 OD, 600 MHz) chromatographically purified from supernatants of stationary phase cultures of S. elongatus (1, green), of the purified 7dSh from the supernatants of Streptomyces setonensis as control (2, red), and of enzymatically synthesized 7dSh (3, black). Predicted from assigned NMR-data (4, blue) of 7dSh in the 7-deoxy-d-altro-heptulofuranose form (Bruker, TopSpin software). Additional proton NMR signals in 1–3 give evidence for the dynamic forms of 7dSh in solution (open chain tautomers, ring conformers) Full size image

Chemoenzymatic synthesis of 7-deoxy-sedoheptulose

To unambiguously prove the chemical structure of the 7-deoxyheptulose from S. elongatus culture supernatants, we established the chemoenzymatic synthesis of 7-deoxy-d-altro-2-heptulose (1) (7-deoxy-sedoheptulose, 7dSh). C 7 -carbohydrate intermediates occur in the pentose phosphate pathway and can be biosynthesized by the transfer of a C 2 -unit onto a C 5 -precursor using the enzyme transketolase. Transketolase (EC 2.2.1.1) stereospecifically adds the nucleophile to the re-face of the d-enantiomers of 2-hydroxyaldehydes (aldoses) and controls the stereochemistry of the reaction to result in (3S, 4R)-configured ketoses29. We cloned the gene encoding the S. elongatus transketolase (Synpcc7942_0538) in an Escherichia coli His-tag (pET15b) overexpression vector and purified the recombinant protein by affinity chromatography (see Methods). In the enzymatic synthesis of 7dSh, recombinant S. elongatus transketolase transfers the C1–C2 ketol unit of β-hydroxypyruvate (3) to 5-deoxy-d-ribose (2) in the presence of thiamine diphosphate and divalent cations (Mg2+)30 (Fig. 2a). Release of CO 2 from β-hydroxypyruvate during the transketolase reaction prevents the back-reaction and enables a one-way synthesis of 7-deoxy-d-altro-2-heptulose (1), which is the only product according to NMR and MS data.

Transketolases efficiently react with phosphorylated sugars, but reactions with dephosphorylated sugars result in low yields31. In agreement, the chemoenzymatic synthesis of 7dSh from 5-deoxy-d-ribose gave yields of about 20%. We purified chemoenzymatically synthesized 7dSh following the same protocol used for purifying 7dSh from culture supernatants (Supplementary Fig. 1), except that size-exclusion chromatography on Sephadex LH20 could be omitted.

The 1H-NMR spectrum of chemoenzymatically synthesized 7dSh (1) was identical to that of the compound isolated from S. elongatus culture supernatant. The chemical structure of 7dSh was reported in 1970 as the metabolite SF-666B from Streptomyces setonensis nav. sp. by Ezaki, Tsuruoka32. SF-666B was described to show exclusive activity against Gluconobacter oxydans subsp. suboxydans at low micromolar concentrations (0.8 µg mL−1)33. Therefore, we isolated SF-666B from culture supernatants of the Streptomyces setonensis production strain following our purification protocol (Supplementary Fig. 1). NMR spectroscopy revealed that SF-666B is indeed identical to 7dSh isolated from S. elongatus culture supernatants and to chemoenzymatically synthesized 7dSh (Fig. 2c).

Activity of 7dSh against cyanobacterial strains

With the assigned structure of 7dSh (1) and milligram amounts of pure compound at hand, we aimed for detailed biological profiling of the compound. In contrast to the previously reported activity of SF-666B, none of the 7dSh preparations (chemoenzymatically synthesized, purified from culture supernatants of S. elongatus or Streptomyces setonensis) showed any activity against Gluconobacter oxydans under the previously described assay conditions33 and under various other tested conditions (not shown). By contrast, all 7dSh preparations inhibited the growth of the filamentous cyanobacterium A. variabilis. To clarify the biological activity of 7dSh and its biological mode of action, we first analyzed the effects of 7dSh on cyanobacteria in more detail (Fig. 3).

Fig. 3 Effect of 7dSh (1) on the growth and photosynthetic oxygen evolution of A. variabilis cultures. a Growth of A. variabilis (OD 750 ) at different concentrations of 7dSh after 48 h of incubation. Cultures were inoculated to an OD 750 of 0.05, 0.2, or 0.5 (marked by dashed lines). 7dSh in aqueous solution was added at time 0. Significant differences between adjusted initial OD 750 and OD 750 after 48 h were analyzed in a one sample t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s., not significant). b Photosynthetic oxygen evolution by A. variabilis (initial OD 750 = 0.3) in the presence of 7dSh or neomycin (positive control) or without supplementation (BG11, negative control). 7dSh (ca. 50 µM) and neomycin (ca. 65 µM) were added as aqueous solution. Values in both graphs represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file Full size image

The effect of 7dSh (1) on A. variabilis depended on the ratio between the 7dSh concentration and the cell density of the cultures (determined as optical density at 750 nm, OD 750 ; Fig. 3a). Cultures with an initial OD 750 of 0.5 were hardly affected by 7dSh concentrations up to 5 µg mL−1 (ca. 25 µM). When the cell density of A. variabilis was lowered to an OD 750 of 0.2, 7dSh had a dose-dependent effect. At a concentration of 2.5 µg mL−1 (ca. 13 µM), 7dSh showed a cytostatic effect. A further increase of 7dSh to 5 µg mL−1 resulted in lysis of the cells. With even lower initial cell densities (initial OD 750 < 0.05), the effect of 7dSh was even more pronounced; already 2.5 µg mL−1 7dSh had a bactericidal effect. Therefore, the effect of 7dSh on A. variabilis can be either bacteriostatic or bactericidal, depending on the amount of 7dSh available per cell. This result indicated a cellular binding site for 7dSh that reduces the titer of the compound in solution or a metabolic alteration of 7dSh.

We subsequently used bactericidal concentrations of 7dSh (1) for bioprofiling to obtain unambiguous results. Since 7dSh was active against a cyanobacterium but not against Gluconobacter oxydans, we speculated that 7dSh might target the photosynthetic apparatus. Treatment of A. variabilis with 7dSh (ca. 50 µM) led to a slow decrease in photosynthetic oxygen formation over a period of 24 h (Fig. 3b). This effect is in contrast to that of specific inhibitors of photosynthesis, such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which act almost immediately. The slow decrease resembled the effect of the protein synthesis inhibitor neomycin, which slowly decreases photosynthetic oxygen evolution by inhibiting the PSII repair cycle. This similarity suggested an indirect effect of 7dSh on photosynthesis, ultimately mediated by the inability to maintain the PSII repair cycle.

To narrow down the cellular processes targeted by 7dSh (1), we made use of the unique properties of the recovery of nitrogen-starved chlorotic cells as an experimental system, where different metabolic activities are activated in a sequential order34. Here, long-term nitrogen-starved Synechocystis sp. cells were allowed to resuscitate from chlorosis by adding nitrate. In a typical experiment, the cells return to vegetative growth within 48 h in a highly coordinated process. Almost immediately after nitrate addition, dormant cells switch on metabolism and re-establish the basic enzymatic machinery. After approximately 16 h, photosynthesis and CO 2 fixation are turned on, and at the end of recovery, cells divide again. To reveal whether and at which stage resuscitation is blocked by 7dSh, chlorotic Synechocystis sp. cells were treated with 7dSh immediately before nitrate was added to initiate resuscitation. Following the addition of nitrate, control cultures showed the expected re-greening and return of photosynthetic activity34. The presence of 7dSh prevented resuscitation and re-greening in a dose-dependent manner (Fig. 4c). Measurement of oxygen exchange (Fig. 4b) and of PSII activity through pulse amplitude modulation (PAM) fluorometry35 (Fig. 4a) showed that 7dSh-treated cells initially started respiratory glycogen consumption but then were unable to proceed further in the recovery and to restore their photosynthetic machinery. This clearly indicated that 7dSh affected metabolism at an early stage of resuscitation that is mainly characterized by anabolic reactions such as de novo amino acid synthesis34.

Fig. 4 7dSh (1) prevents regeneration of resuscitating Synechocystis. a Optical density (black) and PSII quantum yield (turquoise) of chlorotic Synechocystis cultures (initial OD 750 = 0.5) regenerating in the absence or presence of 7dSh. NaNO 3 (17.3 mM) and 7dSh (ca. 260 µM) were added in aqueous solution at 0 h. Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. b Oxygen evolution of resuscitating Synechocystis cultures (initial OD 750 = 0.5) upon addition of nitrate (NaNO 3 , 17.3 mM) in the presence or absence of 7dSh (ca. 206 µM). Significant differences between 7dSh treatment and untreated control for each timepoint were analyzed in an unpaired t-test (*p-value < 0.05; **p-value < 0.01; *** p-value < 0.001; n.s., not significant). Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. c Cultures of chlorotic Synechocystis (initial OD 750 = 0.5) 48 h after addition of nitrate (NaNO 3 , 17.3 mM) and 7dSh. Numbers indicate concentration (µg mL−1) of 7dSh added to the culture. Source data are provided as a Source Data file Full size image

Inhibition of 3-dehydroquinate synthase by 7dSh

To elucidate the mechanism of action, the effect of 7dSh (1) on the metabolic pattern of resuscitating Synechocystis sp. and exponentially growing A. variabilis was analyzed. Liquid cultures of the respective cyanobacteria were incubated in the absence or presence of 7dSh. At different time points, cells were collected and extracted with an acidic methanol/water solution (see Methods) for molecular analysis by LC-HRMS. Software-based subtraction (MetaboliteDetect 2.1, Bruker Daltonics) of the standardized MS chromatograms facilitated the detection of metabolic differences between cell samples of untreated and 7dSh-treated cultures. This analysis revealed a fast and massive accumulation of a metabolite with the sum formula of C 7 H 13 O 10 P (M R = 288.14 Da from m/z = 289.0325 [M+H]+ and 287.0171 [M−H]−) in 7dSh-treated cells. Within 1 h after 7dSh (1) addition to A. variabilis cultures, the concentration of the respective compound increased more than fifteen fold as compared to initial concentration of untreated control cultures (t = 0 h) (Supplementary Fig. 3a, b). The accumulation of the respective compound further increased over time, reaching the 72-fold concentration (about 1.1 µM) as compared to untreated control cultures (about 16 nM) after 4 h. The sum formula (C 7 H 13 O 10 P) and comparison of the MS/MS fragmentation pattern (Supplementary Fig. 3c) with MetFrag insilico fragmentation36 and data in the literature37 revealed that the accumulated compound was 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) (4, Supplementary Fig. 4). DAHP is the substrate of 3-dehydroquinate (DHQ) synthase, one of the first enzymes in the shikimate pathway, which converts DAHP to DHQ. This essential reaction in shikimate biosynthesis cannot be bypassed by alternative enzymes. The accumulation of DAHP is in accordance with DHQ synthase being the biological target of 7dSh38. Within the five-step reaction mechanism for conversion of DAHP to DHQ by DHQ synthase39, the second step represents the β-elimination of the phosphate group of DAHP (Supplementary Fig. 4). We propose that 7dSh (1) mimics DAHP (4), the natural substrate of DHQ synthase. The C-7-methyl group of 7dSh, which is absent in DAHP, would impede the β-elimination in step 2, thereby leading to an inhibition of DHQ synthase and consequently the accumulation of DAHP.

Inhibition of the shikimate pathway triggers a metabolic perturbation that leads to decreased pools of aromatic amino acids, and, as a result of perturbed protein synthesis, also to the accumulation of non-aromatic amino acids such as leucine, valine, and arginine40. Thus, to confirm our hypothesis, we analyzed the levels of aromatic and selected non-aromatic amino acids in 7dSh-treated and untreated A. variabilis cultures by LC-HRMS (Fig. 5). 7dSh (1) induced a significant accumulation of the non-aromatic amino acids leucine, isoleucine, valine and arginine. Within 4 h, the levels of isoleucine, arginine and valine increased almost threefold, and that of leucine about fivefold. By contrast, the levels of all aromatic amino acids significantly decreased in comparison to untreated control cultures (about 55% for tryptophan, 30% for phenylalanine and 20% for tyrosine).

Fig. 5 Effects of 7dSh (1) on amino acid levels in A. variabilis cells. Levels of selected amino acids in A. variabilis (initial OD 750 = 0.5) treated with 7dSh (260 µM) for 4 h and respective untreated control cultures. Significant differences between 7dSh treatment and untreated control were analyzed in an unpaired t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s., not significant). Values represent the mean values of five biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file Full size image

The significant accumulation of DAHP (4) and changes in amino acid levels were also detected in cultures of nitrogen-starved Synechocystis sp. that were resuscitating from chlorosis in the presence of 7dSh (1) (Supplementary Fig. 5). Because of the lower metabolic activity of chlorotic cultures, DAHP accumulation was delayed but comparable to 7dSh-treated A. variabilis cultures.

We obtained further evidence that 7dSh (1) is an inhibitor of the shikimate pathway in an amino acid feeding experiment. The uptake of aromatic metabolites should mitigate the effects induced by 7dSh. PAM fluorometry of A. variabilis cultures revealed a 7dSh-induced decrease in the PSII quantum yield to about 10% of that of untreated control cultures (Supplementary Fig. 6). This effect was alleviated by supplementation with a mixture of aromatic amino acids. By contrast, supplementation of untreated control cultures with the aromatic amino acid mixture did not affect their PSII quantum yield. Supplementation with aromatic amino acids similarly alleviates the effects of glyphosate on other cyanobacteria41.

Antifungal and herbicidal effects of 7dSh

As the shikimate pathway occurs in other bacteria and in fungi and plants, we decided to investigate the effects of 7dSh (1) on organisms other than cyanobacteria. We chose the yeast model organism Saccharomyces cerevisiae as the fungal representative. When S. cerevisiae was cultivated in YPD complex medium, 7dSh did not affect growth. By contrast, when the yeast was grown in YNB minimal medium with defined carbon and nitrogen sources, 7dSh (10 µg mL−1, ca. 50 µM) inhibited growth (Supplementary Fig. 7), with a lower growth rate and a significantly lowered final optical density (OD 600 of about 0.5) compared to growth in the absence of 7dSh (final OD 600 of about 0.95). Glyphosate (100 µg mL−1, ca. 590 µM) had to be applied at more than tenfold higher concentration to achieve a similar effect. A similar decreased growth rate instead of complete growth inhibition of microbes has earlier been described for glyphosate42. If an antimetabolite binds reversibly to the targeted enzyme, it will be replaced by the accumulating natural substrate. Therefore, a high intracellular concentration of the antimetabolite and low abundance of target enzyme favor inhibition of the targeted reaction. The observed residual growth of S. cerevisiae is consistent with a putative reversible binding of 7dSh.

As the representative for testing the effects of 7dSh (1) on plants, we chose the model organism Arabidopsis thaliana. Seedlings of A. thaliana germinated in mineral salt medium were significantly affected by micromolar concentrations of 7dSh. After 7 days, seedlings of the untreated control formed distinct roots with numerous root hairs and green cotyledons (Fig. 6a). The control seedlings showed gravitropism, and the distance between the shoot and root apical meristem was about 6 mm (Fig. 6b). Even a low concentration of 7dSh (5 µg mL−1, ca. 25 µM) significantly affected the size of the seedlings. At concentrations of 25 or 50 µM, the growth inhibition effects of 7dSh were similar to those of glyphosate at the same concentrations. At concentrations of 130 and 260 µM, the inhibitory effect of 7dSh significantly surpassed that of glyphosate at the same concentrations, both in terms of seedling size (Fig. 6b) and morphological appearance (Fig. 6a). Impaired growth and aberrant morphology of the seedlings were particularly evident at higher concentrations of 7dSh (260 µM). In this case, the seedling growth was arrested within the first days. Only minor root and cotyledon formation was observed, and gravitropism was impaired (Fig. 6a and b). In comparison, A. thaliana seedlings that had been treated with higher concentration of glyphosate (260 µM) were less affected. They developed further, formed roots with small root hairs and bigger cotyledons. In the following days (day 7 to 14), no further plant growth or morphological change was observed in the presence of the inhibitors.

Fig. 6 7dSh (1) reduces the growth of A. thaliana seedlings. a Morphological appearance of autotrophically grown Arabidopsis thaliana seedlings 7 days after induction of germination. Seedlings were grown in constant light on agar plates without an antimetabolite (control) or in the presence of 7dSh or glyphosate. Plates were mounted vertically and illuminated from above. White arrows mark the root and shoot apical meristem. Scale bar (5 mm) applies to all images. b Measurement of the distance between root and shoot apical meristem. Significant differences between seedling sizes were analyzed in an unpaired t-test (*p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; n.s., not significant). Box-and-whisker plots represent the values of at least 16 seedlings. c Effect of 7dSh and glyphosate (each 260 µM) on the growth of A. thaliana on soil after 18 days in a day/night cycle. Statistical analysis was performed by using a one-way ANOVA. Tukey’s multiple comparison test was used as the post-hoc test. Means that were significantly different (p-value < 0.05) are marked with different capital letters in the diagram. Box-and-whisker plots represent the values of at least 58 A. thaliana seedlings. For b, c: Error bars indicate range, box bounds indicate second and third quartiles, center lines indicate median. Source data are provided as a Source Data file Full size image

LC-HRMS analysis of whole plant extracts of the A. thaliana seedlings revealed a 7dSh-induced accumulation of DAHP, which was not detectable for the control or glyphosate-treated seedlings (Supplementary Fig. 8). As a proof of principle the accumulation of shikimate 3-phosphate was detectable in glyphosate-treated seedlings but not detectable in control or 7dSh-treated plants.

In order to evaluate the herbicidal activity of 7dSh (1) in more natural conditions, growth of A. thaliana in presence of the inhibitor was investigated in soil in a day/night cycle (Fig. 6c). After 18 days, seedlings were harvested and weighted. The weight of the seedlings was significantly reduced in 7dSh and glyphosate treatment as compared to untreated control. Furthermore, the inhibitory effect of 7dSh significantly surpassed that of glyphosate as the weight of the 7dSh-treated seedlings was less than half as much as that of the glyphosate-treated seedlings.

Early germination events are characterized by the efficient reactivation of metabolic pathways43. Metabolites required for the induction of germination are stored in the seeds. Once these reserve materials are depleted, the proliferation of the seedlings relies on de novo synthesis of intermediates and growth factors. Glyphosate- or 7dSh-induced inhibition of the shikimate pathway therefore leads to an effective arrest of the seedling growth.

Cytotoxicity of 7dSh on mammalian cells

To determine whether 7dSh (1) affects mammalian metabolism, we tested various human cell lines (THP-1 macrophages, A549 human lung epithelial cells, HepG2 human liver epithelial-like cells, 293 human embryonal kidney cells) and primary human neutrophils in cytotoxicity assays. 7dSh did not show any cytotoxic effects on tested human cell lines and primary cells (Supplementary Fig. 9a), even at 5 mM, a concentration that is two orders of magnitude higher than that required for its herbicidal effect. Neither 7dSh nor glyphosate at 5 mM lysed cells, as measured by the release of lactate dehydrogenase. Further, the morphological appearance of 7dSh-treated macrophages did not differ from that of the untreated control (Supplementary Fig. 9b,c).

The biology of 7dSh for the producer strain

It is surprising to us that 7dSh (1), which has never before been described in cyanobacteria, was isolated from the unicellular cyanobacterium S. elongatus. S. elongatus is a common laboratory strain but has never been described as a producer of hydrophilic secondary metabolites. The role of 7dSh in metabolism and for the physiology of the producer strain yet remains enigmatic. Due to the streamlined genome, which lacks classical secondary metabolite gene clusters25, the biosynthesis of 7dSh is not yet clear. However, to the best of our knowledge, a specific biosynthetic gene cluster or pathway may not be necessary for the biosynthesis of 7dSh. It has been shown for another cyanobacterial strain with a small genome, that enzymatic promiscuity enables the production of a large variety of secondary metabolites without the need of specific enzymes23. One of the enzymes known for enzymatic promiscuity is the transketolase, the enzyme we used for chemoenzymatic synthesis of 7dSh. The transketolase plays a fundamental role in cyanobacterial metabolism, e.g., in the Calvin cycle, and exhibits a wide substrate specificity. To shed light on the biosynthesis of 7dSh, we screened S. elongatus cultures for the presence of a potential 7dSh precursor. The screening resulted in the isolation of the monosaccharide 5-deoxy-d-ribose (2), a metabolite never isolated from nature before (Supplementary Fig. 10a, b, Supplementary Fig. 17–21).

The effective in vitro conversion of 5-deoxy-d-ribose (2) to 7dSh (1), carried out by the S. elongatus transketolase in our chemoenzymatic synthesis, suggests this reaction is the final step in the biosynthesis of 7dSh. The affinity of the cyanobacterial transketolase to 5-deoxy-d-ribose was about 100-fold lower than the affinity to the natural transketolase substrate d-ribose 5-phosphate (Supplementary Fig. 10c, d), enough to explain the minute levels of 7dSh produced by S. elongatus in later stages of growth, where CO 2 fixation and consequently d-ribose 5-phosphate levels decrease. Nevertheless, the synthesis of the assumed precursor 5-deoxy-d-ribose remains enigmatic. Future studies shall clarify whether this compound is a side product of a primary metabolic pathway or whether yet unidentified enzymes are involved.

The biological role of 7dSh (1) for the producer strain remains obscure. Since 7dSh shows allelochemical characteristics and is excreted to the medium, its production could also be a strategy of S. elongatus to protect its niches against other competitors. Even though the low production level of 7dSh in laboratory test conditions questions this hypothesis, the 7dSh concentration could be higher under certain natural conditions, such as in biofilms. Although S. elongatus laboratory strains usually grow planktonically, the re-isolated wild-type grows in a biofilm44. When growing in a biofilm or a microbial mat, the concentration of 7dSh could increase to a level sufficient to provide physiological effects such as controlling the surrounding microbial community.