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Identification of the negamycin biosynthetic pathway in Kitasatospora purpeofusca genome

ABSTRACT: negamycin biosynthesis

PROVIDER: PRJEB89693 | ENA |

REPOSITORIES: ENA

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Identification of the negamycin biosynthetic genes in Kitasatospora purpeofusca ATCC21470

Project description: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.

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Genome analysis and elucidation of the biosynthetic pathway for the cRAS inhibitor rasfonin in Cephalotrichum gorgonifer

Project description:Background: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterization work we found that this strain is able to produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and shows anti-tumor activity in RAS-dependent cancer cells. Results: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is s indeed responsible for rasfonin biosynthesis. Conclusions: The results of this study lay the ground for molecular biology focused research in Cephalotrichum gorgonifer. Furthermore, strain engineering and heterologous expression of the rasfonin BGC is now possible which facilitates the construction of high producing strains or the synthesis of rasfonin derivates for diverse applications.

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