Feel the burn: a collection of stories on hot’n’sharp DNA engineering

The global demand on chemicals and fuels is exponentially increasing. At the same time, the excessive exploitation of fossil-based resources for the coverage of this demand has a high environmental impact, motivating the production of green chemicals and biofuels from renewable resources. Nowadays, the microbial production of green chemicals and fuels gains increasing attention, especially due to the ease in the construction of metabolically engineered microorganisms with high production capacities. This ease was achieved via the development of efficient genome engineering tools. This thesis describes the development of novel genetic tools for mesophilic and thermophilic bacteria, for metabolic engineering purposes. More specifically, here is reported: 1) the development of the first CRISPR-SpCas9-based genome engineering tools for the mesophilic bacteria Rhodobacter sphaeroides and Pseudomonas putida, as well as for the moderate thermophilic bacterium Bacillus smithii, 2) the in vitro characterization of one of the first reported thermotolerant Cas9 homologs, denoted as ThermoCas9, as well the development of the first CRISPR-ThermoCas9-based genome engineering tools for strictly thermophilic bacteria, and 3) the use of the developed tools for the metabolic exploration and exploitation of the moderate thermophilic bacterium B. smithii, towards the characterization of its acetate production pathway and the enhancement of its dicarboxylic acids productivity.