Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Abstract

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad-spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ-butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure-activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.

Keywords: antibiotics; biosynthesis; genomics; natural products; terpenoids.

Figure 1.

Genome mining of atypical bacterial type II DTSs. A) Representative SSN of type II DTSs displayed at an e value of 10⁻⁵⁰. Large nodes depict characterized type II DTSs in bacteria. Colors of nodes represent the source of the protein; see inset legend. B) SSN of the 66 type II DTS homologues found in the TnlT2 subfamily displayed at e values of 10⁻¹⁴² and 10⁻¹⁵⁸. Shapes and colors of nodes represent bacterial genera and associated biosynthetic pathway, respectively; see inset legend. C) Sequence logo of the consensus motif of (E/D)(T/S)xE.

Figure 2.

Discovery of the TNLs. A) Metabolite profiles of Streptomyces strains upon HPLC analysis: I) CB03234; inset shows a magnification of part of the chromatogram; II) CB03238; III) SB23001 (CB03234 ΔtnlT2); IV) SB23004 (CB03234 ΔtnlM1); and V) SB23005 (CB03234 ΔtnlP5). B) Representative metabolite profile of the diterpene-enriched fractions of large-scale fermentation of CB03234 upon LC-MS analysis (total ion current).

Scheme 1.

Structures and gene clusters of four terpenoids found within the 66 putative diterpenoid producers. A) The TNLs were discovered in this study; PLA, BRA, and LON (KS-505a) were known. The absolute stereochemistries of BRA and TNL were determined; only the relative stereochemistries of PLA and LON were reported. B) Genetic organizations of the tnl (CB03234), tnl’ (CB03238), pla, bra, and Ion biosynthetic gene clusters, highlighting the genetic and potential structural diversities found within this family natural products. Genes are colored based upon their annotated Pfam IDs (Tables S3–S6). GenBank accession numbers for tnl CB03234 (BK010469) and tnl CB03238 (BK010470).

Scheme 2.

Structural characterization of the TNLs. A) Structures of TNLs A–K (1–11, respectively). B) Structural and relative stereochemical determination of 1 by means of 2D NMR spectroscopy. C) Absolute stereochemical determination of 8 by means of ROESY correlations and the modified Mosher method.

Scheme 3.

Proposed biosynthetic pathway for TNL A (1) supported by the isolation of congeners or shunt metabolites 4–11, as indicated by the dashed arrows. A) The four core proteins, TnlT1–T4, construct the diterpenoid scaffold and elongate the side chain. Then, a series of oxidative reactions form the γ-butyrolactone moiety and hydroxylate the C-18 and C-12 positions before acylation of the chloroanthranilate moiety. Finally, the N- and O-methylations, catalyzed by TnlP5 and TnlM1, respectively, complete the biosynthesis, although their timing is still unknown. IPP: isopentenyl diphosphate, DMAPP: dimethylallyl diphosphate, GGPP: geranylgeranyl diphosphate. B) Biosynthesis of the chloroanthranilate moiety from l-tryptophan follows that of tryptophan degradation through the kynurenine pathway. Chemical transformations are colored red; for multiple transformations in one step, red and blue are used.