Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of “inducer” microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.
Co-cultivation has been used as a
promising tool to turn on or
up-regulate cryptic biosynthetic pathways for microbial natural product
discovery. Recently, a modified culturing strategy similar to co-cultivation
was investigated, where heat-killed inducer cultures were supplemented
to the culture medium of producer fermentations to induce cryptic
pathways. In the present study, the repeatability and effectiveness
of both methods in turning on cryptic biosynthetic pathways were unbiasedly
assessed using UHPLC-HRESIMS-based metabolomics analysis. Both induction
methods had good repeatability, and they resulted in very different
induced metabolites from the tested producers. Co-cultivation generated
more induced mass features than the heat-killed inducer cultures,
while both methods resulted in the induction of mass features not
observed using the other induction method. As examples, pathways leading
to two new natural products, N-carbamoyl-2-hydroxy-3-methoxybenzamide
(1) and carbazoquinocin G (5), were induced
and up-regulated through co-culturing a producer Streptomyces sp. RKND-216 with inducers Alteromonas sp. RKMC-009
and M. smegmatis ATCC 120515, respectively.
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