Construção de linhagens de Escherichia coli para produção heteróloga de fenazinas

Abstract

Phenazines are a class of secondary metabolites produced by some species of microorganisms, with great potential for biotechnological application. However, the production of these compounds has been performed only in naturally producing microorganisms, restricting the viability of its application, since some of these organisms are pathogenic and others present very limited production. Thus, the objective of this work is to produce these molecules in Escherichia coli cells, heterologously. To this end, genes involved in the biosynthesis of the selected phenazines were cloned and expressed using BioBricks-based vectors. Starting from the phzABCDEFG gene cluster from Pseudomonas aeruginosa, responsible for the production of phenazine-1-carboxylic acid (PCA), strategies were adopted for production of derived phenazines. For production of phenazine-1,6-dicarboxylic acid (PDC), it was necessary to remove the phzA gene from the mentioned cluster. However, when culture and subsequent mass spectrometry analysis were performed, it was not possible to identify the target molecule. Still on the PDC production, the homologous recombination technique was employed in order to replace the phzABG genes by the esmA1 and esmA2 genes from Streptomyces antibioticus Tü 2706, but no positive colonies were generated. For the production of 1-methoxyphenazine (PMO), the LaphzM gene from Lysobacter antibioticus was used, which, together with the phzS gene from P. aeruginosa, enabled the conversion of PCA into PMO. In this case, after culturing the recombinant strain, it was possible to identify the mass of the target molecule of interest, produced for the first time in a heterologous manner, to the best of our knowledge. In the production of PMO, different E. coli strains, concentrations of the inducer IPTG, and different carbon sources were evaluated, reaching a production of 421mg/L. It was also possible to successfully produce endofenazine A, using the ppzP gene from Streptomyces anulatus, which encodes a prenyltransferase, together with plasmids for the expression of genes from the S. cerevisiae mevalonate pathway, and the cluster for PCA production. The confirmation of the production of these molecules was performed by mass spectrometry and liquid chromatography, and in initial experiments it was possible to reach final concentrations around 200 mg/L of the product of interest.