Periodic Reporting for period 1 - StrepCryptPath (NEW METHOD TO ACTIVATE STREPTOMYCES SECONDARY METABOLISM CRYPTIC PATHWAYS)
Periodo di rendicontazione: 2019-06-01 al 2021-04-30
Streptomycetes are important biotechnological bacteria from which two thirds of the bioactive secondary metabolites used in clinic (mainly antibiotics, but also antitumorals, immunosupressors, etc.) were discovered. Drug discovery became challenging once the most common antibiotics were found. Genomic annotation of Streptomyces strains revealed the existence of several cryptic secondary metabolite pathways not expressed in the traditional screening programmes. These cryptic pathways are majority in streptomycetes and awakening them is one of our best chances to discover novel secondary metabolites and fight against microbial resistances. In our ERC-StG Strp-differentiation project (280304), we discovered an unexpected pleiotropic effect of cytosolic copper modulating differentiation and secondary metabolism. A S. coelicolor mutant lacking the SCO2730/31 copper chaperone/P-type ATPase export system shows a dramatic enhancement of secondary metabolism. This mutant, is in fact the first Streptomyces strain able to produce secondary metabolites during its whole developmental cycle, including germination, the exponential growth phase and the stationary stage. In addition, the S. coelicolor ΔSCO2730/31 mutant expresses half of their predicted secondary metabolite clusters, including several cryptic pathways. The aim of the Strepcryptpath project was to reproduce this amazing phenotype in other streptomycetes. If we can activate half of secondary metabolite pathways encoded in the genome, we can revolutionise the screening for new bioactive compounds (antibiotics and others). This was the aim of the Strepcryptpath project.
To knockout the SCO2730/31 orthologues for every streptomycete would be tedious and unfeasible in high throughput screening campaigns. In the Strepcryptpath project we opted by knockdown these genes creating an antisense mRNA for a consensus sequence of the SCO2730/31 orthologues. This antisense mRNA was successful to knockdown the SCO2730/31 genes and to enhance secondary metabolism in all the streptomycetes analysed. Although interesting, secondary metabolism activation was much lower than in the original Streptomyces coelicolor ΔSCO2730/31 knockout mutant, and was not enough to justify its application in high throughput screening campaigns for secondary metabolites.
Reproduce the cryptic pathway activation observed in the ΔSCO2730/31 mutant still one of our best chances to find new bioactive compound. The antisense mRNA created in the Strepcryptpath project constitutes the first step to reach this goal. A further analysis of the ΔSCO2730/31 mutant revealed that it has an increased mutation frequency and chromosomal instability. We identified 15 mutations in the ΔSCO2730/31 knockout that are not present in our SCO2730/31 knockdown mutant. In the early future, we will study which of these mutations contribute, together with the SCO2730/31 inactivation, to secondary metabolism enhancement. With this knowledge, we will test if the inactivation of these genes in our SCO2730/31 knockdown strains can activate cryptic pathways. If successful, we will evaluate the possibility of patenting this methodology and creating a spin-off company to scale-up this method in screening campaigns.
To knockout the SCO2730/31 orthologues for every streptomycete would be tedious and unfeasible in high throughput screening campaigns. In the Strepcryptpath project we opted by knockdown these genes creating an antisense mRNA for a consensus sequence of the SCO2730/31 orthologues. This antisense mRNA was successful to knockdown the SCO2730/31 genes and to enhance secondary metabolism in all the streptomycetes analysed. Although interesting, secondary metabolism activation was much lower than in the original Streptomyces coelicolor ΔSCO2730/31 knockout mutant, and was not enough to justify its application in high throughput screening campaigns for secondary metabolites.
Reproduce the cryptic pathway activation observed in the ΔSCO2730/31 mutant still one of our best chances to find new bioactive compound. The antisense mRNA created in the Strepcryptpath project constitutes the first step to reach this goal. A further analysis of the ΔSCO2730/31 mutant revealed that it has an increased mutation frequency and chromosomal instability. We identified 15 mutations in the ΔSCO2730/31 knockout that are not present in our SCO2730/31 knockdown mutant. In the early future, we will study which of these mutations contribute, together with the SCO2730/31 inactivation, to secondary metabolism enhancement. With this knowledge, we will test if the inactivation of these genes in our SCO2730/31 knockdown strains can activate cryptic pathways. If successful, we will evaluate the possibility of patenting this methodology and creating a spin-off company to scale-up this method in screening campaigns.