S. charteusis is a gram-positive bacterium that produces a divalent cation ionophore named calcimycin that inhibits growth of gram-positive bacteria and is a widely used biochemical tool to study calcium signalling. However, of the well-characterized calcimycin biosynthesis gene cluster a few genes, such as calU1 and calU2, remain catalytically uncharacterized. Moreover, in the light of calcimycin transporting manganese and iron and 4-Br-calcimycin transporting copper, we hypothesize that naturally occurring calcimycin derivatives offer altered divalent cation transport properties ^[1].

We aim to elucidate catalytic roles of the unknown genes and to generate calcimycin derivatives through mutasynthesis to explore divalent cation transport properties. To this end, knockout mutants of the corresponding genes were generated by Redirect technology ^[2].

In the calU1 deletion mutant an intermediate accumulated, suggesting CalU1 to be a putative novel enzyme to release the polyketide chain. Bioinformatics analysis and literature review indicate CalU2 is a putative spirocyclase ^[3]. A feeding experiment with 3-hydroxy anthranilic acid deletion mutant produced a new derivative (m/z: 470.2638 [M+H]+) identified by LC-MS.

The characterization of the unknown genes will provide a more thorough understanding of the biosynthesis of this class of polyether ionophores that consists of three ring systems, only one of which is synthesized by a PKS. The generation of calcimycin derivatives by modifications at the benzoxazole moiety might expand the capability of this ionophore class as biochemical tools.

Senges, Christoph HR, et al. "Effects of 4‐Br‐A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore." Proteomics 22.17 (2022): 2200061.GUST, B., et al. PCR-targeting system in Streptomyces coelicolor. John Innes Center, Norwich, United Kingdom, 2003.BILYK, Oksana, et al. Enzyme-catalyzed spiroacetal formation in polyketide antibiotic biosynthesis. Journal of the American Chemical Society, 2022, 144. Jg., Nr. 32, S. 14555-14563.