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Exceeding planetary boundaries, and in particular climate change, is quickly forcing the global economy to align with the principles of sustainability. In this context, there is a strong call from policy makers, scientists, and citizens to step out of the oil-based era and relentlessly pursue a biobased, sustainable economy. The industrial exploitation of microbes for the sustainable production of bio-based chemicals and fuels has contributed significantly to this effort. Nevertheless, it is becoming clear that industrial biotechnology requires a wider repertoire of more effective microbial hosts to expand and enhance its capabilities. At the same time, advancements in synthetic biology allow the rapid and efficient domestication of several unconventional microbes with appealing industrial characteristics. One bacterial host that has gained increasing attention as an efficient microbial cell factory is Pseudomonas putida KT2440. However, despite its many attractive inherent features, additional efforts must be made to elucidate and engineer its complex metabolic and regulatory architecture. Hence, this thesis aimed at contributing to the industrial domestication of P. putida by developing and applying cutting-edge synthetic biology technologies for engineering its cellular metabolism.
The first part of the research herein was focused on the development of genetic tools, namely: i) the SEVA 3.1 vector library, optimized for Gram-negative bacteria, built upon the principles of the SEVA, BioBricks, and Type IIS restriction enzyme standards; and ii) the first tunable CRISPRi system for gene repression in P. putida. The second part aimed at constructing three P. putida strains tailored to overproduce the key precursor metabolites pyruvate, malonyl-CoA, and chorismate. Each strain was approached from a different metabolic engineering perspective dealing with the concepts of dynamic growth control, high-throughput optimization, and directed adaptive laboratory evolution. The work performed follows the principles of the DBTL cycle using in combination innovative computational and genetic methods that can be readily applied to further expand the industrial potential of P. putida.
|Qualification||Doctor of Philosophy|
|Award date||28 Mar 2022|
|Place of Publication||Wageningen|
|Publication status||Published - 2022|
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