Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica

Abstract

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.

Figure 1

Candidate DNA-targeting genotypes based on genome mining of Salinispora tropica CNB-440. A) Representative 10-membered enediyene, dynemicin A, and predicted enediyne precursor of sporolides A/B. Red –enediyne scaffold. B) ST_pks1 gene cluster with predicted 10-membered enediyne polyketide biosynthetic machinery. C) spo (sporolide A/B) gene cluster with a predicted 9-membered enediyne biosynthetic machinery. D) ST_pks2 gene cluster with a predicted glycosylated anthracycline product.

Figure 2

Identification of the ST_pks2 (lom) gene cluster product, lomaiviticin C, from extracts of wild type Salinispora tropica by LCMS-based comparative metabolic profiling. A) Comparative metabolic profiling identified a compound at 670.27 m/z with abolished production in the ST_pks2 mutant (shown by corresponding base peak chromatogram (BPC)). B) MS spectrum of the ST_pks2 product. C) MS/MS spectrum of the ST_pks2 product showed deoxysugar-specific fragments (red) indicating a glycosylated natural product. D) The identified ST_pks2 product is lomaiviticin C.