Study of acetate metabolism using different carbón and nitrogen sources in Escherichia coli

Resumen

Escherichiacoli, E.coli, is a model organism in biological and biotechnological processes. Thus, E. coli is used in many industries for the production of high-interest compounds, such as drugs, recombinant proteins or other bio-products (Blount, 2015; Glaser, 2005). Due to the large number of processes that are based on the employ of this bacteria, the study of its central metabolism and regulation is essential to optimize all these biotechnological processes. Furthermore, E. coli is capable to grow using diverse carbon and nitrogen sources, al-though shows a preference for glucose as a carbon source and inorganic ammonium as a nitrogen source (Commichau, Forchhammer, & Stülke, 2006; Reitzer, 2003). The carbon and nitrogen sources are important for the E. coli metabolism fluxes and the production of different compounds (Bren et al., 2016). Thereby acetate metabolism has been studied in E. coli, because of the relevance of acetate overflow has in biotechnological industrial pro-cesses, and the relation between this metabolism and protein lysine acetylation. Acetate is excreted and then reincorporated (acetate overflow) in the E. coli metabolism when glu-cose is employed as a carbon source (Figure 1) (Bernal, Castaño-Cerezo, & Cánovas, 2016). This phenomenon is a major drawback for industrial processes. Regarding acetylation, a relation between this and carbon fluxes in glucose supplemented cultures, has been repor-ted (Christensen, Orr, Rao, & Wolfe, 2017; Schilling et al., 2015).Our aim is to deepen in the knowledge of acetate metabolism in E. coli, employing di-verse carbon and nitrogen sources. Specify, we study the influence of carbon fluxes on lysine acetylation, through the study of acetyl donor intracellular concentrations (as acetyl donors play a great role in non-enzymatic acetylation). In this work, E. coli BW25113 (wild-type, wt) and five genes deletion strains involved in acetate metabolism and lysine acetylation, are grown in minimal medium MM9 and in com-plex medium TB7 (which are nitrogen sources based in inorganic ammonium and peptides, respectively), supplemented with glucose and glycerol as a carbon source. The deletion genes are acs (acetyl-CoA synthetase gene), ackA (acetate kinase gene), cobB (encodes the main E. coli sirtuin deacetylase), patZ (encodes the acetyl-CoA synthetase enzyme) and pta(phosphotransacetylase gene). Under these conditions, protein lysine acetylation and intracellular concentration of acetyl and succinyl donors such as acetyl-phospate, acetyl-Coenzyme A and succinyl-Coen-zyme A, have been evaluated. The concentration of acetyl donors, and protein acetylation are influenced by carbon and nitrogen sources.