RESUMEN
In response to Iron deprivation and in specific environmental conditions, the cyanobacteria Anabaena flos aquae produce siderophores, iron-chelating molecules that in virtue of their interesting environmental and clinical applications, are recently gaining the interest of the pharmaceutical industry. Yields of siderophore recovery from in vitro producing cyanobacterial cultures are, unfortunately, very low and reach most of the times only analytical quantities. We here propose a four-step experimental pipeline for a rapid and inexpensive identification and optimization of growth parameters influencing, at the transcriptional level, siderophore production in Anabaena flos aquae. The four-steps pipeline consists of: (1) identification of the promoter region of the operon of interest in the genome of Anabaena flos aquae; (2) cloning of the promoter in a recombinant DNA vector, upstream the cDNA coding for the Green Fluorescent Protein (GFP) followed by its stable transformation in Escherichia Coli; (3) identification of the environmental parameters affecting expression of the gene in Escherichia coli and their application to the cultivation of the Anabaena strain; (4) identification of siderophores by the combined use of high-resolution tandem mass spectrometry and molecular networking. This multidisciplinary, sustainable, and green pipeline is amenable to automation and is virtually applicable to any cyanobacteria, or more in general, to any microorganisms.
RESUMEN
In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores' family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.
