Characterization of an Anthracene Intermediate in Dynemicin Biosynthesis

Abstract

Despite the identification of a β-hydroxyhexaene produced by the enediyne polyketide synthases (PKSs), the post-PKS biosynthetic steps to the individual members of this antitumor and antibiotic family remain largely unknown. The massive biosynthetic gene clusters (BGCs) that direct the formation of each product caution that many steps could be required. It was recently demonstrated that the enediyne PKS in the dynemicin A BGC from Micromonospora chersina gives rise to both the anthraquinone and enediyne halves of the molecule. We now present the first evidence for a mid-pathway intermediate in dynemicin A biosynthesis, an iodoanthracene bearing a fused thiolactone, which was shown to be incorporated selectively into the final product. This unusual precursor reflects just how little is understood about these biosynthetic pathways, yet constrains the mechanisms that can act to achieve the key heterodimerization to the anthraquinone-containing subclass of enediynes.

Streptomycin‐induced ribosome engineering complemented with fermentation optimization for enhanced production of 10‐membered enediynes tiancimycin‐A and tiancimycin‐D

“…The relevant medium optimization revealed the important effects of NaI and CuSO 4 on the biosynthesis of TNMs. The observed essentiality of NaI was coincident with the reported specific role of iodide, which is required for the production of crucial iodoanthracene intermediate in biosynthesis of another anthraquinone enediyne DYN‐A (Cohen & Townsend, ), while the significant influence of CuSO 4 may be related to activation of copper‐inducible promoter or regulators and resulted metal‐responsive transcriptional control (Waldron & Robinson, ; Zheng et al, ). The change of nitrogen source further stimulated the biosynthesis of TNMs, especially TNM‐D and its aromatized byproducts, in CB03234‐S (Figure b).…”

C−N‐Coupled Metabolites Yield Insights into Dynemicin A Biosynthesis

“…Owing to its higher yield, we first focused on characterization of 9 (Figure a). The 1 H NMR spectrum of 9 (Figure S2) immediately revealed a substructure corresponding to 6 , as the characteristic signals of the anthracene were readily apparent . Chemical shift changes for these peaks were all less than 0.2 ppm except for a hydrogen in the C‐ring, which was shifted dramatically upfield by about 1.5 ppm.…”

“…The force of the evidence brought by these findings dictates that the 16‐carbon β‐hydroxyhexaene 4 must partition into two distinct, but parallel pathways to prepare precursors of the enediyne and anthraquinone halves of DYN (each 14 carbons in the final natural product). Assuming that the synthetic flux through these two channels is not perfectly matched, the HPLC profiles of the wild‐type DYN producer, Micromonospora chersina, and a CRISPR‐Cas9 knockout strain of DynE8 were compared, which led to the identification of an accumulated iodoanthracene 6 bearing a γ‐thiolactone ring (Figure ) . This metabolite subsequently proved to be the first intermediate identified mid‐pathway to an enediyne natural product, which was a surprising discovery as iodine and sulfur occur rarely in natural products, especially aromatic thiols .…”

“…The fact that it accumulates in readily detectable amounts, however, suggests – but does not prove – that 6 could couple with an unknown X from the “enediyne” arm of the pathway (Figure ) to form a heterodimeric intermediate en route to DYN. Mechanisms to account for how such a process might occur have been discussed and point to a unimolecular radical process (S RN 1) rather than a nucleophilic aromatic substitution, where the former would involve single‐electron transfer followed by loss of iodide and C–N bond formation …”

The hidden enzymology of bacterial natural product biosynthesis

“…91 )), novel module compositions and domain organizations (for example, SxtA, saxitoxin, 16 (REF. 92 )) and single enzymes for multiple discrete scaffolds (for example, DynE8, dynemicin A, 17 (REFS 93,94 )). In addition, the first known polyketide from a strictly anaerobic bacterium, Clostridium beijerinckii, was also recently described 95,96 .…”