Dinámica de la acetilación de "Escherichia coli" para mejorar sus capacidades biotecnológicas

Resumo

E. coli is one of the most widely used microorganisms for biotechnological purposes and as a research model, as it has been extensively studied and its complete genome, proteome and transcriptome are known. However, the use of E. coli as a host organism in bioprocesses (recombinant protein production) can lead to several drawbacks, such as metabolic burden, acetate overflow and limited knowledge of some of its posttranslational modifications, such as lysine acetylation. Nε-acetylation of lysines is a posttranslational modification that is related to acetate metabolism via acetyl-CoA and acetyl-P intermediates, which act as acetyl donors. This modification occurs in many enzymes of central metabolism and is involved in the regulation of metabolic processes, translation processes, cellular localisation and interactions of proteins with other molecules. The main objective of this PhD Thesis was to understand the factors that govern the dynamics of protein acetylation and its effect on the control of central metabolism in E. coli, in order to design strategies that guarantee cell optimisation to produce recombinant proteins. Thus, the implications of the use of different culture media on the production of recombinant proteins, and on the protein profile and acetylation level in E. coli were studied (Chapters 3 and 6). In addition, different expression systems and deficient strains in genes involved in acetate metabolism and lysine acetylation were also used to optimise the production of recombinant proteins and to determine their acetylation level (Chapters 3 and 4). Finally, the implications of acetylation in an important metabolic pathway, the de novo pyrimidine biosynthesis pathway, were studied (Chapter 5). The results obtained will allow the design of rational strategies, both by strain engineering and culture media selection, to increase the biotechnological capabilities of the bacteria and to understand the implications of acetylation in these processes. From the results obtained in this PhD Thesis, the main conclusions drawn are: 1) The production of recombinant proteins is favoured by using a complex medium and glycerol as a carbon source. 2) The use of the ΔackA strain results in a reduction in acetate overflow and a 5-fold increase in recombinant protein production when the optimal expression system is employed. 3) The metabolic burden produced by transcription of expression vectors leads to a decrease in recombinant protein production when using high copy number expression systems with strong promoters. 4) The de novo pyrimidine biosynthesis pathway is regulated by the acetylation of lysines in the OPRTase enzyme. Acetylation of OPRTase at lysines 26 and 103 results in a decrease in activity that can be reversed by CobB deacetylase. 5) The relative abundance of proteins in E. coli depends on culture conditions, and the identity and number of acetylated lysines depends on the relative abundance of proteins. 6) The level of acetylation in E. coli depends mainly on acetate overflow, being higher in complex medium. 7) Lysines with differences in their level of acetylation between culture conditions have been found in the glyoxylate cycle, the tricarboxylic acid cycle and nitrogen assimilation, so these pathways could be differentially regulated by acetylation. In conclusion, the results of this PhD Thesis show the interconnection of acetylation regulation of central metabolic pathways with culture conditions in E. coli. Thus, the use of different culture conditions determines the fluxes of central metabolism in general, and specifically of acetate overflow, which has an impact on the relative abundance of proteins, the level of lysine acetylation and, consequently, the production of recombinant proteins.