Azoxy bond formation in azodyrecins from Streptomyces mirabilis P8-A2

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Azodyrecins are one out of few azoxy compounds that have been discovered in Streptomyces spp. (1) They contain azoxy moiety (RN=N+(O-)R) which links iso-fatty acid derived molecules to amino acid. (2) These molecules show wide structure diversity and different biological activities, especially cytotoxic, nematicidal and antimicrobial activities. (1) The biosynthesis of only two Streptomyces spp. azoxy compounds have been investigated to greater depth, however the azodyrecin BGC is significantly different. To have better understanding of the biochemistry and enzymatic machinery synthesizing these compounds, we investigated their biosynthesis. Firstly, we have successfully confirmed that the protein machinery required for the production are contained in biosynthetic gene cluster (BGC). We applied a modified(3) method of CATCH (4) cloning which allowed us using in-vitro CRISPR-Cas9 and synthetic sgRNAs to precisely capture the targeted hypothetical azodyrecin BGC and clone it in to shuttle plasmid that we thereafter transferred in S. albidoflavus J1074 and S. coelicolor M1146. The metabolomics analysis by LC-ESI-MS showed production of azodyrecins in the mutant strains. Secondly, we further investigated the biosynthesis of the compound in the S. mirabilis P8-A2 azodyrecin WT producer strain through construction of knock-out (KO) mutants using CRISPR-BEST (5) base editing toolbox for Streptomyces spp. This method allows precise nucleotide level modifications which can be used to make changes in the translation of genes. We used the toolbox to introduce stop codons in the upstream region of the genes, which results in synthesis of immature protein and there by KO of the function. Using this method, we generated 14 KO mutants of which many resulted in no production of azodyrecins, in some we saw production of new intermediates, while few did not produce anticipated metabolomic response. Through analysis of the LC-ESI-MS data we could propose function of specific genes and the order of reactions. By having additional data on the biosynthesis of azoxy compounds in streptomyces, we wanted to estimate the spread of different known and proposed azoxy BGC in Streptomyces spp. as well as general abundance of BGCs containing conserved core genes and potentially produces undiscovered novel azoxy compounds. We did mapping onto medium/high quality genomes of Streptomyces spp. available in NCBI database.
PeriodSept 2023
Event titleInternational VAAM Symposium 2023: Biology of Bacterial Natural Product Producers
Event typeConference
LocationSaarbrücken, Germany, SaarlandShow on map
Degree of RecognitionInternational