Cloning and heterologous expression of six orphan gene clusters from Streptomyces sp. CNB091

The rise of microbial genome sequences available has revealed that their potential for natural product biosynthetic clusters is considerably higher than the number of known molecules. An example is the marine Streptomyces sp. CNB091 in which genome sequence analysis predicted 33 clusters, but only one series of compounds, Salinamides, have been detected so far. In order to gain a more comprehensive understanding regarding the limitations in detecting compounds from predicted clusters as well as to investigate the capability of CNB091 to produce new secondary metabolites we have embarked on interogating multiple gene clusters from CNB091 in heterologous hosts to identifiy the associated compounds and regulatory limitations. Our strategy consisted of first direct cloning six gene clusters from genomic DNA using yeast recombination which was adapted in this work to enable fast capture and expression testing of gene clusters. The six clusters included two NRPSs, two PKSI-NRPS hybrids, a phenazine-PKSI and an aminoglycoside ranging between 25-60 kb.  We further selected Streptomyces coelicolor M512 as heterologous host for expression of each of these clusters since this strain does not encode for any homologous clusters. Extracts from cultures of cells harboring the clusters were evaluated by LC-MS-MS and analyzed by molecular networking which enabled rapid comparison of the metabolic profile of each strain and allowed identification of new unique ions for some of the expressed clusters. New molecular biology tools are now been explored to increase expression of these compounds to facilitate their structural investigation.