Engineering of the gram-negative Zymomonas mobilis as a future industrial chassis

Human activities have dramatically altered natural processes causing a significant impact on social-ecological systems. This led to the current environmental crisis, in which the industrialized world faces progressive depletion of its energetic resources, mainly based on non-renewable fuels. In this scenario, it is key to address the conversion of renewable carbon sources into value-added chemicals. In this thesis, it was studied whether the Gram-negative bacterium Zymomonas mobilis could serve as a biotechnological chassis to produce bioethanol, using cellulose as feedstock. Interestingly, Z. mobilis naturally produces a striking concentration of bioethanol, which can be employed as biofuel or fuel additive. However, the biodegradation of cellulose requires the activity of cellulolytic enzymes, that Z. mobilis does not produce. Therefore, to convert Z. mobilis into a cellulose to ethanol microbial cell factory, the efficient production and secretion of these enzymes is required. The present research has focused on the development of a Z. mobilis strain with improved secretion abilities. Previous studies have been proved that bacteria lacking surface proteins show a better secretion. Therefore, this research aimed at developing a surface-naked Z. mobilis strain, lacking the genes encoding for cell-surface structures, such as proteins involved in cell mobilization, interactions with surfaces or organisms, and biofilm formation. Therefore, this surface-naked strain may serve as a testing platform for the overexpression of secretion systems, essential to transport cellulolytic enzymes extracellularly. We attempted the deletion of these genes through the homologous recombination method. This included the construction of plasmids carrying the upstream and downstream regions of the genes of interest; however, this deletion strategy was unsuccessful. In parallel, we also attempted at expanding the molecular toolkit to regulate gene expression in Z. mobilis, by combining in silico research and in vivo experiments. Notably, we tested multiple Rho-independent transcription terminators through a synthetic circuit, that enabled the measurement of their strength. Overall, this allowed the construction of a terminator library, which had not been previously reported in Z. mobilis