Functional Characterization of nigO and schU2: Unveiling the Enzymatic Mechanisms of Spiroketal Formation in Nigericin and Calcimycin Biosynthesis.

• Grant number: 24/22109-9
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: June 14, 2025
• End date: June 13, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Luiz Alfredo Torres Sales
• Supervisor: Prof Barrie Wilkinson
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: John Innes Centre, England
• Associated to the scholarship: 21/01581-3 - Structural studies of spirocyclases involved in the polyketide biosynthesis, BP.DD

Abstract

The discovery of new specialized metabolites is vital in addressing the challenge posed by emerging superbugs and drug resistance. Soil and freshwater environments have fostered the evolution of microorganisms with unique adaptations, leading to the production of bioactive compounds with significant pharmacological potential. The genus Streptomyces has a rich history in antibiotic discovery. Notable among these metabolites are polyketides, including polyether ionophores, which exhibit diverse structural and functional properties. Two key polyketides, nigericin and calcimycin, produced by Streptomyces violaceusniger and Streptomyces chartreusis, respectively, contain spiroketal moieties that should be essential for their biological activities. This project aims to elucidate the functional roles of the nigO and schU2 genes in the biosynthesis of nigericin and calcimycin which we propose are involved in spiroketal formation. We will perform gene deletions using plasmids that can be prepared through Gibson assembly, followed by bacterial conjugation with E. coli ET12567 to introduce mutations into the two Streptomyces strains. Subsequently, HPLC/MS analysis will quantify metabolite production and identifythe accumulation of pathway intermediates (putative spirocyclase substrates) in the mutant strains. Following scale up fermentation and isolation of the intermediates, enzymatic assays will be conducted to demonstrate the utlization of these substrates by the proteins NigO and SchU2. Our work will provide a comprehensive understanding of the genetic and enzymatic mechanisms underlying spiroketal formation during nigericin and calcimycin biosynthesis by functionally characterizing NigO and SchU2 to reveal their roles in spirocyclization processes contributing to the production of these bioactive compounds. Ultimately, this project aims to enhance our understanding of polyketide biosynthesis pathways, facilitating the development of novel therapeutic agents and strategies to combat drug-resistant pathogens.