Engineering Atypical Tetracycline Formation in Amycolatopsis sulphurea for the Production of Modified Chelocardin Antibiotics

Abstract

To combat the increasing spread of antimicrobial resistance and the shortage of novel anti-infectives, one strategy for the development of new antibiotics is to optimize known chemical scaffolds. Here, we focus on the biosynthetic engineering of Amycolatopsis sulphurea for derivatization of the atypical tetracycline chelocardin and its potent broad-spectrum derivative 2-carboxamido-2-deacetyl-chelocardin. Heterologous biosynthetic genes were introduced into this chelocardin producer to modify functional groups and generate new derivatives. We demonstrate cooperation of chelocardin polyketide synthase with tailoring enzymes involved in biosynthesis of oxytetracycline from Streptomyces rimosus. An interesting feature of chelocardin, compared with oxytetracycline, is the opposite stereochemistry of the C4 amino group. Genes involved in C4 transamination and N,N-dimethylation of oxytetracycline were heterologously expressed in an A. sulphurea mutant lacking C4-aminotransferase. Chelocardin derivatives with opposite stereochemistry of the C4 amino group, as N,N-dimethyl-epi-chelocardin and N,N-dimethyl-2-carboxamido-2-deacetyl-epi-chelocardin, were produced only when the aminotransferase from oxytetracycline was coexpressed with the N-methyltransferase OxyT. Surprisingly, OxyT exclusively accepted intermediates carrying an S-configured amino group at C4 in chelocardin. Applying medicinal chemistry approaches, several 2-carboxamido-2-deacetyl-epi-chelocardin derivatives modified at C4 were produced. Analysis of the antimicrobial activities of the modified compounds demonstrated that the primary amine in the R configuration is a crucial structural feature for activity of chelocardin. Unexpectedly, C10 glycosylated chelocardin analogues were identified, thus revealing the glycosylation potential of A. sulphurea. However, efficient glycosylation of the chelocardin backbone occurred only after engineering of a dimethylated amino group at the C4 position in the opposite S configuration, which suggests some evolutionary remains of chelocardin glycosylation.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin

“…The chdAR cassette (encoding efflux pump ChdR, its regulator ChdA, and an operator/promoter region) was deleted from A. sulphurea Δ chdPKS mutant [ 46 ], which does not produce CHD. For that purpose, two 1 kb homology cassettes for deletion of chdAR via homologous recombination (primer pairs chdARLF/chdARLR and chdARRF/chdARRR listed in Table S9 ) were cloned via Sph I and Eco RI into plasmid pNV18 [ 78 ], which was transformed into A. sulphurea Δ chdPKS according to previously described protocols [ 26 , 46 ]. In a selected transformant, the deletion of chdAR was identified by colony PCR.…”

“…[ 19 ], these gaps were further closed by generating the more potent lead molecule CDCHD [ 15 ]. The chemical space of both compounds has been further extended in recent studies by biosynthetic engineering and medicinal chemistry approaches to explore the structure–activity relationships of these scaffolds [ 26 , 27 ].…”

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation

“…Chelocardin, which was investigated in a phase II clinical trial in 1977, is active against tetracycline-resistant pathogens 41 . The biosynthetic gene cluster of Amycolatopsis sulphurea was recently engineered for the synthesis of derivatives 58 . The dual mode of action of chelocardin was first described based on proteome profiling 22 .…”

Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis

“…In the recent years, we have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete A. sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CDCHD) [8]. Our work also provided an understanding of the structure-activity relationship of CHD [9]. Furthermore, CDCHD was semi-synthetically modified [9,10] with the aim to optimize the potent CHD derivative even further.…”

“…Our work also provided an understanding of the structure-activity relationship of CHD [9]. Furthermore, CDCHD was semi-synthetically modified [9,10] with the aim to optimize the potent CHD derivative even further.…”