ELUCIDATION OF ACETIL-COA METABOLISM IN Streptomyces coelicolor THROUGH STABLE ISOTOPE-ASSISTED METABOLOMICS

LIVIERI AL ; DEL CARRATORE F; GRAMAJO H; RODRIGUES E

Congreso; SAIB 2020; 2020

MI-P53-259ELUCIDATION OF ACETIL-COA METABOLISM IN Streptomyces coelicolor THROUGH STABLE ISOTOPE-ASSISTED METABOLOMICSLivieri AL1, Del Carratore F2, Takano E2, Gramajo H1, Rodrigues E11Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) and Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR). 2Manchester Institute of Biotechnology, University of Manchester.E-mail: livieri@ibr-conicet.gov.arThe genus Streptomyces plays key roles in soil ecology based on their ability to scavenge nutrients and recycling deth insect and plants by hydrolysing polysaccharides like cellulose, chitin, xylan, and agar. However, the most interesting characteristics of Streptomyces spp. rely on their ability to produce bioactive secondary metabolites –such as antifungals, antivirals, antitumorals, and antibiotics– and their capability of accumulate triacylglycerols (TAGs). TAGs and some secondary metabolites (polyketides) are produced mainly from a key central precursor: acetyl-CoA. This acetyl-CoA participates in other important pathways, like Krebs cycle, and it is a precursor of several macromolecules. Nevertheless, how these bacteria assimilate acetyl-CoA precursor to produce building blocks and the influence of this assimilation on the production of secondary metabolites and TAGs in actinobacteria is poorly understood. With the purpose of understanding the metabolism of acetyl-CoA and its role in TAGs and secondary metabolite biosynthesis, a study about the assimilation of acetyl-CoA in Streptomyces coelicolor was started. Genomic analysis showed that in S. coelicolor there are two putative pathways for acetyl-CoA assimilation, the glyoxylate cycle (sco0982-0983) and the ethylmalonyl-CoA pathway (sco6469-6473). By constructing single (Δsco0982 or sco6473::Tn5066 (HygR)) and double mutants deficient in these pathways (Δsco0982 and sco6473::Tn5066 (HygR)), it was found that S. coelicolor still grow on minimal medium with acetate as the only carbon source, which suggests that there is a novel unknown pathway for acetyl-CoA assimilation. With the aim of identifying the acetyl-CoA metabolic intermediates in both, wild type and the double mutant strains of S. coelicolor, a stable isotope-assisted metabolomics with C13-Acetate was carried out. For this purpose, both strains were grown until exponential phase in minimal medium with acetate as only carbon source and then labelled acetate was added. The incorporation of the labelling was allowed for 0, 5, 10, 30 and 60 min. After this, the cytoplasmic metabolites were extracted, and the samples were run in a Q-exactive mass spectrometer. Metabolic analysis showed no differences between both strains, suggesting that the double mutant strain uses an acetyl-CoA assimilation pathway similar to the parental strain. Analysis of the sample also showed that central metabolites like glutamate, succinate, citrate, among others were labelled at early times. These are consistent with the intermediaries of some acetyl-CoA assimilation pathways of archaea, like the methylaspartate cycle. Currently, genetic studies are being performed with the aim to identify homologous genes of this pathway that could be involved in acetyl-CoA