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Identification of the negamycin biosynthetic genes in Kitasatospora purpeofusca ATCC21470

ABSTRACT: Negamycin is a ribosome-targeting antibiotic with activity against Gram-positive and Gram-negative ESKAPE pathogens and the ability to promote premature stop codon readthrough. Its therapeutic potential is limited by low natural production and synthetic complexity. To enable scalable biosynthesis, we identified and characterized its genetic basis in Kitasatospora purpeofusca. Two distant chromosomal regions, neg1 and neg2, were found to be essential. Deletion of neg1, involved in nitrite formation for N-N bond biosynthesis, reduced production to ~2%, while deletion of neg2, which directs β-lysine formation and scaffold assembly, abolished it completely. Isotope-labeling confirmed nitrite incorporation, and complementation restored production. Transcriptomic and proteomic analyses further supported the essential roles of both regions. Expression of neg1 along with the neg2 region in Streptomyces albus reconstituted negamycin biosynthesis. This study reveals an unusual split biosynthetic pathway and provides a foundation for the biotechnological production and further development of this promising antibiotic.

ORGANISM(S): Kitasatospora purpeofusca

PROVIDER: GSE304071 | GEO | 2026/09/08

REPOSITORIES: GEO

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Project description:Nitrite-oxidizing bacteria are vital players in the global nitrogen cycle that convert nitrite to nitrate during the 2nd step of nitrification. Within this functional guild, the genus Nitrospira is among the most widespread and phylogenetically and physiologically diverse nitrite oxidizers and its members drive nitrite oxidation in many natural and biotechnological ecosystems. Despite their ecological and biotechnological importance, our understanding of Nitrospira’s energy metabolism is still limited. The main bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing organisms and fastidious to culture. In this study, we cultured Nitrospira moscoviensis in a continuous stirred tank reactor system (CSTR) allowing constant biomass harvesting. Additionally, this cultivation setup enabled accurate control of physicochemical parameters and thus avoided fluctuating levels of nitrite and accumulation of nitrate. We performed transcriptome analysis and confirmed constant gene expression profiles in the chemostat culture over a period of two weeks. The transcriptomic data supports the predicted core metabolism of N. moscoviensis, including the reductive TCA cycle as a CO2 fixation pathway, the novel bd-like oxidase as terminal oxidase and the octaheme nitrite reductase involved in nitrogen assimilation. Additionally, the expression of multiple copies of respiratory complexes suggests functional differentiation of these copies within the respiratory chain. Transcriptome analysis also suggests a soluble and a membrane-bound gamma subunit as part of the nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation. Overall, the transcriptome data provided novel insights into the metabolism of Nitrospira supporting the genome-based prediction of key pathways. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of canonical and complete nitrifying microorganisms.

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Identification of the negamycin biosynthetic genes in Kitasatospora purpeofusca ATCC21470

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