Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity

Abstract

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

“…To the best of our knowledge, cis -3-hydroxy-L-proline has only been found as one of the building blocks of telomycin, a peptide antibiotic produced by Streptomyces canus C15978, and these bacteria may also hydroxylate free L-proline to cis -3-hydroxy-L-proline enzymatically6. Since the amount of cis -3-hydroxy-L-proline in nature is markedly less than that of trans -4-hydroxy-L-proline, the possibility of catabolism by (micro)organisms had not previously been considered.…”

“…Overall, the L-hydroxyproline gene cluster from microorganisms is often related not only to the metabolism of trans -4-hydroxy-L-proline, but also to that of cis -3-hydroxy-L-proline and trans -3-hydroxy-L-proline, suggesting that the origin of these free L-hydroxyproline compounds in nature is the degradation of several peptide antibiotics, such as telomycin (containing cis -3-hydroxy-L-proline and trans -3-hydroxy-L-proline)78, microcolin A ( cis -4-hydroxy-L-proline)9, pneumocandins ( trans -4-hydroxy-L-proline and trans -3-hydroxy-L-proline)10, and etamycin ( cis -4-hydroxy-D-proline)11, rather than collagen.…”

“…In mammalian systems, the L-proline residue is post-translationally hydroxylated to trans -4-hydroxy-L-proline [(2 S ,4 R )-4-hydroxyproline] or trans -3-hydroxy-L-proline [(2 S ,3 S )-3-hydroxyproline] by a diverse set of prolyl 4-hydroxylase (EC 1.14.11.2) and prolyl 3-hydroxylase (EC 1.14.11.7) with different protein substrates, respectively1. A number of bacterial and fungal enzymes are known to directly hydroxylate free L-proline to trans -4-hydroxy-L-proline2, trans -3-hydroxy-L-proline34, cis -4-hydroxy-L-proline [(2 S ,4 S )-4-hydroxyproline]5, or cis -3-hydroxy-L-proline [(2 S ,3 R )-3-hydroxyproline]6, which are also found as components of several peptide antibiotics produced by these microorganisms7891011.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…[234][235][236] A further example of this amino acid hydroxylation strategy has been identied in telomycin biosynthesis, in which the P450 enzyme Tem23 hydroxylates the Leu-10 residue during NRPS-mediated biosynthesis of the peptide. 237 A related strategy to this has also been invoked in hydroxylation of a PCP-bound threonine residue during actinomycin G biosynthesis, although in this case it appears as though there is a relationship between the P450 (AcmG8) and a halogenase that modies the same position (AcmG9) that needs further clarication.…”

Characterization of a Novel cis -3-Hydroxy- l -Proline Dehydratase and a trans -3-Hydroxy- l -Proline Dehydratase from Bacteria

“…The telomycin biosynthesis is dependent on the tem1-tem34 gene cluster, in which both L-proline cis-3-hydroxylase (tem32) and L-proline trans-3-hydroxylase genes (tem29) are contained (35) (Fig. 8A).…”