Discovery of actinomycin L, a new member of the actinomycin family of antibiotics

Abstract

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L₁ and L₂ produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L₁ and L₂ are diastereomers, and the structure of actinomycin L₂ was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X_2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X₂ producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X₂ and for anthranilamide. Actinomycins L₁ and L₂ showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.

Figure 1

Differential production of metabolites depending on the carbon source. (a) PCA score plot of Streptomyces sp. MBT27 metabolites produced in cultures with different carbon sources, namely, 1% arabinose, 1% fructose, 1% GlcNAc, 1% glucose, 2% glucose, 1% glycerol, 2% glycerol, 1% mannitol, 1% mannitol + 1% glycerol and 2% mannitol (%ages in w/v). (b) OPLS-DA score plot. Triangles and crosses represent samples of active and inactive groups respectively, circular areas represent the 95% confidence region of each group. (c) OPLS-DA loadings S-plot. Arrows indicate the most discriminative features that positively correlate with the active groups.

Figure 2

GNPS molecular network of the ions detected in the crude extract of Streptomyces sp. MBT27. Cultures were grown for seven days in MM with 1% glycerol. Orange nodes represent ions of the metabolites produced by Streptomyces sp. MBT27, while blue nodes represent those of the media components. The actinomycin spectral family is enlarged. Results of ANOVA statistical analysis were mapped onto the molecular network to illustrate the differential production of actinomycin cluster members under various growth conditions. Box plots represent relative intensities of actinomycins X₂, X_0β, and D after log transformation and pareto scaling; together with a compound with an m/z value of 1387.67, in cultures grown in MM with the following carbon sources: 1. 1% arabinose; 2. 1% fructose; 3. 1% GlcNAc; 4. 1% glucose; 5. 2% glucose; 6. 1% glycerol; 7. 2% glycerol; 8. 1% mannitol; 9. 1% mannitol + 1% glycerol; 10. 2% mannitol (%ages in w/v).

Figure 3

Chemical structures of the new actinomycins. Shown are actinomycin L₁ (10′S) (1) and L₂ (10′R) (2) (a) and the key COSY, HMBC and NOESY correlations for 1 (b).

Figure 4

X-ray ORTEP drawing of the crystal structure of compound 2.

Figure 5

Box plots showing the relative intensities of actinomycin L₁, L₂ and X₂ and anthranilamide after log transformation and pareto scaling. Cultures of Streptomyces sp. MBT27 were grown for 7 days in MM with the following carbon sources: 1. 1% arabinose; 2. 1% fructose; 3. 1% GlcNAc; 4. 1% glucose; 5. 2% glucose; 6. 1% glycerol; 7. 2% glycerol; 8. 1% mannitol; 9. 1% mannitol + 1% glycerol; 10. 2% mannitol (% ages in w/v). Red box indicates the abundance of actinomycin L₁, L₂ and X₂ and anthranilamide in the cultures grown with fructose (1% w/v). Note that Streptomyces sp. MBT27 produced actinomycin L and anthranilamide in very low amounts when fermented with fructose (1% w/v) as the sole carbon source.

Figure 6

Anthranilamide is required for the biosynthesis of actinomycins L₁ and L₂. Box plots show the relative intensities of actinomycin L₁ and L₂ after log transformation and pareto scaling in the cultures of Streptomyces sp. MBT27 (a) and S. antibioticus (b) fermented for 7 days in MM with fructose (1% w/v) (1), fed with 0.7 mM anthranilamide (2) and 0.7 mM anthranilic acid (3). Note that S. antibioticus produces actinomycin L exclusively in the presence of anthranilamide and not with anthranilic acid; conversely, Streptomyces sp. MBT27 is able to convert anthranilic acid into anthranilamide, enabling the production of actinomycin L.

Figure 7

Proposed biosynthetic pathway for actinomycin L. We propose that actinomycin L is formed through the reaction of anthranilamide (blue) with the 4-oxoproline moiety of actinomycin X₂ prior to the condensation of two 4-MHA PPLs into the actinomycin L.