Enduracidin analogues with altered halogenation patterns produced by genetically engineered strains of Streptomyces fungicidicus

Abstract

Enduracidins (1, 2) and ramoplanin (3) are structurally and functionally closely related lipodepsipeptide antibiotics. They are active against multi-drug-resistant Gram-positive pathogens, including MRSA. Each peptide contains one chlorinated non-proteinogenic amino acid residue, Cl(2)-Hpg or Cl-Hpg. To investigate the timing of halogenation and the importance of chlorination on bioactivity and bioavailability of enduracidin, and to probe the substrate specificity and portability of the ramoplanin halogenase, we constructed the mutant strain SfDelta30 in which the enduracidin halogenase gene orf30 had been deleted and complemented it with the ramoplanin counterpart orf20. We also expressed orf20 in the enduracidin wild-type producer. Metabolite analysis revealed SfDelta30 produced the novel analogues dideschloroenduracidins A (4) and B (5), while the recombinant strains SfDelta30R20 and SfR20 produced monodeschloroenduracidins A (6) and B (7) and a trichlorinated enduracidin (8), respectively. In addition, orf30 self-complementation yielded the strain SfDelta30E30, which is capable of producing six peptides including 6 and 7. MS/MS analysis positioned the single chlorine atom in 6 at Hpg(13) and localized the third chlorine atom in 8 to Hpg(11). Biological evaluation of these enduracidin analogues indicated that all retained activity against Staphylococcus aureus. Our findings lay the foundation for further utilization of enduracidin and ramoplanin halogenases in combinatorial biosynthesis.

Figure 1

(A) Schematic presentation of the halogenase gene orf30 in-frame deletion mutation. (B) PCR analysis confirming chromosomal in-frame deletion of orf30 in the mutant strain SfΔ30 and BglII digestion of the gel-purified PCR products confirming the BglII site introduced into the chromosome of SfΔ30 (C). PCR products were separated on a 2% agarose gel. (B) Lane 1, molecular marker (Fermentas GeneRuler 1 kb DNA Ladder); Lane 2, PCR product from wild-type S. fungicidicus (1.5 kb); Lanes 3-6, PCR products from independent mutant colonies (0.13 kb). (C) Lane 1, gel-purified PCR product from SfΔ30; Lane 2, gel-purified PCR product digested with BglII; Lane 3, molecular marker (Fermentas GeneRuler 100 bp DNA Ladder).

Figure 2

LC-ESI-MS analysis of partially purified dideschloroenduracidins A (A) and B (B). MALDI-TOF MS analysis of the peaks corresponding to dideschloroenduracidins A (C) and B (D).

Figure 3

MALDI-TOF MS analysis of the metabolites produced by the fermentation of the recombinant strain SfΔ30E30.

Figure 4

HPLC analysis of the crude extract from the fermentation of the recombinant strain SfΔ30R20 (A) and MALDI-TOF MS analysis of purified monodeschloroenduracidins A and B from SfΔ30R20 (B).

Figure 5

MALDI-TOF MS analysis of metabolites produced by recombinant strain SfR20.

Figure 6

Localization of the chlorine atom on monodeschloroenduracidin A and chloroenduracidin A by MS/MS analysis.