Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin

Abstract

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such "cryptic" or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin's MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.

Figure 1

Structure of new co-culture-dependent antibiotic keyicin (1).

Figure 2

LCMS-PCA metabolomics of Micromonospora sp. and Rhodococcus sp. co-culture. (A) PCA scores plot describing variance in metabolites in co-culture and monoculture extracts of the Micromonospora sp. and Rhodococcus sp. (B) PCA loadings plot displaying individual metabolites responsible for the variance observed between extracts; the high variance seen in co-culture extracts is attributable to metabolites highlighted by the yellow oval in plot 2B.

Figure 3

Cell-cell contact study between Micromonospora sp. WMMB-235 and Rhodococcus sp. WMMA-185. (A1 and A2) Each half of custom co-culture vessel enabling separation of two independent cultures with 0.2 μm filter. (B) Intact co-culture vessel with filter membrane separating cell types. (C) Aliquots of the Rhodococcus sp. and (D) aliquots of the Micromonospora sp. removed from culture, diluted, and streaked every 2 d.

Figure 4

Key HMBC (red) and ROESY (blue) correlations in core of (1) and for determination of glycosidic linkages. Carbon connectivities determined by ¹³C–¹³C COSY correlations (red). Detailed application of specific ROESY correlations were instrumental in determining S1–S7 configurations (Supporting Information).

Figure 5

Direct comparisons of the keyicin aglycone core to the intact structure of nogalamycin and the more classically arranged anthracycline aclacinomycin A.

Figure 6

Partial characterization of anthracycline core possibilities for keyicin via application of molecular modeling and DFT calculation approaches. See Supporting Information for experimental data for these and alternative variants.

Figure 7

Correlations of orfs (and resulting gene products) to key benzoxocin linkages for keyicin versus nogalamycin aglycones. Gene/enzyme similarities across the two NP systems are color coded with arrows indicating key linkages installed. Notably only kyc54 and snoN appear to be similar yet fail to carry out the same chemistry upon their respective substrates.