Molecular approaches to activate secondary metabolite biosynthesis

Today, a big challenge is the identification and development of new antibiotics to combat antibiotic resistance, one of the most serious threats to global health. Secondary metabolites, characterized by their low molecular weight, possess complex chemical structures and exhibit a range of biological activities, including antimicrobial, antitumoral and immunomodulatory, etc. We aim to explore pristine environments (i.e., organic farming soil, hydrothermal vents, saltern ponds, activated sludge, and cultural artifacts) to search for bacteria-producing secondary metabolites. Besides, since most biosynthetic gene clusters remain unexpressed under standard laboratory conditions, we are pursuing two molecular strategies. On one hand, we aim to modulate cytosine methylation levels by introducing a DNA methyltransferase gene that was previously demonstrated to affect antibiotic production in the model strain Streptomyces coelicolor; on the other one, we inted to overexpress regulatory genes whose corresponding transcription factors could determine the expression of silent or cryptic biosynthetic gene clusters, leading to discovering new bioactive compounds and/or improve their production. We assembled a collection of 500 bacterial strains, encompassing Gram-positive and Gram-negative microorganisms. Within this collection, we specifically selected 108 Actinomycetes strains isolated from soil, as they exhibit remarkable productivity in synthesizing secondary bioactive metabolites. We conducted the metabolomic profiling of ten antibiotic-producing bacteria, unravelling metabolites active against Gram-positive multi-resistant strains. We cloned the SCO1731 gene, which encodes for a specific cytosine methyltransferase, into the png4+sp44 plasmid. This approach allows us to modulate epigenetic levels that hinder Streptomyces antibiotic production. Secondly, we are introducing plasmids encoding various classes of transcriptional regulators from Streptomyces into four different rare actinomycetes. The so far obtained results will be discussed. These efforts hold immense promise as we push the borders of antibiotic research and unravel the potential hidden within these unexplored pathways.