Modificación genética de escherichia coli para la optimización de la producción de hidrógeno y etanol utilizando glicerina como fuente de carbono / genetic engineering in escherichia coli for the optimization of hydrogen and ethanol yields when glycerol is used as a carbon source

Resumen

The Earth's climate is warming as a result of anthropogenic emissions of greenhouse gases, particularly carbon dioxide from fossil fuel combustion. This fact, together with the low availability and abundance of fossil fuels, has led to an urgent need to develop clean and renewable energy sources. Bioenergy, a term including biodiesel, biohydrogen and bioethanol, has emerged as an alternative to fossil fuels. However, the biodiesel industry currently generates glycerol as a by-product in such large quantities that it has become an environmental problem in its own right. A feasible alternative to address this issue is to use waste glycerol as a carbon source for microbial transformation into hydrogen and ethanol because this process is less energy-intensive and is sustainable and eco-friendly than the use of fossil fuels. The use of Escherichia coli -a microorganism commonly used for metabolic engineering in industrial applications- for this purpose is a very promising possibility. E. coli is able to consume glycerol and to synthesise hydrogen and ethanol under anaerobic conditions, however the yields of these compounds are very low in the wild type strain. In this regard, the goal of this Thesis is to outline different strategies in order to enhance the hydrogen and/or ethanol yields and the consumption of glycerol by E. coli. To this aim the production of hydrogen and ethanol and the glycerol consumption were firstly analysed in the E. coli BW25113 wild type strain grown in a glycerol-based medium. Once the experimental conditions was established for a reliable analysis of these parameters in the wild type strain, the same procedure was used to carry out a high-throughput screening in triplicates of more than 150 single knockout strains purchased from the Keio and Yale Collections in order to find novel mutant phenotypes whose hydrogen and/or ethanol yields were improved with respect to that of the wild type strain. The genes affected in these mutants codify for enzymes related to the central carbon metabolism and other associated pathways in most of the cases, although several mutant affecting to amino acid and lipid metabolism were also analysed. In this screening several mutants, whose altered hydrogen and ethanol productions have been previously described, were also included to validate the experimental procedure. After testing all of these pre-selected E. coli mutant strains, a non-parametric test was applied in order to compare the wild type and mutant yields. Those strains with the highest hydrogen and/or ethanol and/or glycerol consumption were further analysed to apply a more robust statistical analysis. The results obtained in this analysis allowed the identification of novel E. coli genetic backgrounds suitable for the use of glycerol in the production of hydrogen and ethanol. The evaluation of the results obtained in this screening by GO database, also could indicate the most suitable metabolic pathways for genetic engineering in order to rewire the metabolism towards the synthesis of hydrogen and/or ethanol by using glycerol in a more efficient way. Among the biological processes identified in this study are: fermentation; generation of precursor metabolites and energy; anabolic pathways; oxidation-reduction process; NAD(P)H availability and flagella motility. Given the importance of the central carbon metabolism in hydrogen and ethanol synthesis, the tricarboxylic acid (TCA) cycle, which is split into two branches in the anaerobic mode, together with other related reactions were deeply studied. As a result of this study, it was found that several mutants of the reductive branch of the TCA cycle -fumarate reductase complex and fumA mutants- showed higher ethanol and hydrogen yields. From this analysis can also be concluded that malate plays a central role in the rewiring of C4 metabolism toward the production of the target products in the experimental conditions described in this work. On the other hand, the mutant for the putative C4 dicarboxylate transporter DcuD, a strain, not previously related to any phenotype, was found to improve all of the target product yields. As a continuation of the previous task, the synthesis of different enzymes by using inducible expression systems was used in order to improve the metabolic rewiring in the reductive branch toward the production of pyruvate and therefore into hydrogen and ethanol synthesis. The most promising results were obtained by the heterologous expression of the human GTP-dependent PEP carboxykinase (mitochondrial) (hPEPCK-M), which promote the conversion from oxaloacetate (OAA) to phosphoenolpyruvate (PEP). In this sense, the heterologous expression of this gene's ORF in several E. coli mutant backgrounds related to the reductive branch (fumarate reductase complex -frdA, frdB, frdC, frdD- and dcuD mutants) improved the hydrogen and ethanol yields. In conclusion, the results described in this Thesis establish the basis for a wider high-throughput screening methodology that can be employed to engineer an E. coli strain to rewire the metabolism in a more efficient way for the synthesis of hydrogen and/or ethanol using glycerol as carbon source.