IMPLEMENTATION OF AN INNOVATIVE STRATEGY TO DISCOVER MOST NEEDED NOVEL ANTIBIOTICS FROM STREPTOMYCES BACTERIA – INNOVANTIBIO

Today, numerous pathogenic bacteria carry genes that make them resistant to most antibiotics. Moreover, the ability of these bacteria to form biofilms further strengthens their ability to resist antibiotics. These aspects constitute a serious threat to public health and the discovery of new antibiotics, of molecules capable of specifically inhibiting these resistance mechanisms or the formation of biofilm becomes an absolute necessity.
Streptomyces bacteria are already well known to produce more than 2/3 of all known antibiotics as well as molecules able to inhibit the enzymatic systems conferring antibiotic resistance. Each Streptomyces specie is known to produce 2 to 4 bioactive molecules but recently the sequencing of the genome of several Streptomyces species revealed that the latter contain between 20 and 40 biosynthetic pathways able to direct the synthesis of a much larger number of potentially bio-active molecules. This suggests that most of the pathways detected in silico are too weakly expressed (cryptic) under the usual laboratory conditions to allow the detection and thus characterization of the synthetized products. The design and implementation of innovative strategies is thus necessary to enhance the expression of these cryptic pathways in order to access this immense metabolic diversity and discover much needed novel antibiotics.
A new “decryptifying” tool has recently been developed by the group MES of I2BC. Indeed, this group demonstrated that the strong antibiotic production of the model strain S. coelicolor was correlated with an oxidative metabolism and a low content in storage lipid of the triacylglycerol (TAG) family, whereas the absence of the production of the same antibiotics in the phylogenetically closely related model strain, Streptomyces lividans, was correlated with a glycolytic metabolism as well as by TAG accumulation. This group demonstrated that it was possible to trigger a transition from a glycolytic metabolism to an oxidative metabolism via the generation of an artificial ATP depletion. To do so, the gene encoding the small synthetic protein DX exhibiting a very high affinity to ATP was cloned under the control of a strong promoter in a conjugative and integrative vector and introduced into S. lividans. The overexpression of this small protein induces an ATP deficiency which is correlated with a decrease in the TAG content and a sharp increase in the production of antibiotics. This tool was introduced in other Streptomyces strains and in all cases tested a strong increase in the production of antibiotics was observed. The proof of concept has therefore been made and a patent deposit is in preparation.
The continuation of this project requires the characterization of the novel bio-active molecules produced by the strains transformed by the "decryptifying" tool and requires the close collaboration between three groups of the University Paris Saclay (I2BC, ICSN, UPSud-EA7361-CNR) that have complementary expertises.
The proposed project involves six sequential steps 1 Introduction of the "decryptifying" tool in poorly studied Streptomyces strains characterized by low antibiotic production and high TAG content 2 Construction of a collection of E. coli strains carrying genes confering resistance to different classes of antibiotics 3 Evaluation of the antibiotic activity present in the culture media of the strains transformed by the "decryptifying" tool on model microorganisms, pathogenic or not and multi-resistant or not to most antibiotics in current use as well as on the collection of E. coli strains mentioned above . 4 Metabolome analysis by LC-MS2 and molecular networking visualization, extraction, isolation and characterization of the new bioactive molecules produced. 5 Evaluation of the antibiotic activity of the purified molecules (alone or in combination) on the microorganisms mentioned above. 6 Identification of gene clusters directing the synthesis of new bioactive molecules.