Abstract

Synthetic biologists design and engineer organisms for a better and more sustainable future based on a currently transitioning global economy grounded in biomanufacturing. While the prospects are encouraging, concerns about uncertainty and risks associated to genome editing, including uncontrolled proliferation of transgenic microorganisms, need to be considered in safe-by-design bioengineering strategies. This dissertation delves into different types of genetic safeguards to conditionally restrict cell host viability to defined environments using the popular metabolic engineering host Pseudomonas putida KT2440. Specifically, this thesis encompasses the generation of an industrially appealing synthetic phosphite auxotroph attained via metabolic rewiring, and a CRISPR-Cas9-based kill switch based on different genetic circuits that react to external signals for the activation of a highly genotoxic response. To also prevent horizontal gene transfer, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-ScCas9-mediated counterselection that was used to generate a minimally recoded P. putida strain of essential metabolic genes. To further improve our genome editing capabilities, we employed a serial enrichment workflow followed by a next-generation sequencing analysis to screen in a high-throughput manner a library of potential recombineering systems in different Pseudomonas species, eventually finding new variants for enhanced mutagenesis. While all these genetic safeguards and tools are attractive in theory, their implementation in industrial biotechnology and real-world applications is rarely exercised in practice, despite technological advances. In this dissertation we question why and what could be done about it and claim that an explicit strategy of contextualization, that is, an early emphasis on potential applications, can assist the development of genetic safeguards. Moreover, the conduction of a series of interviews and analysis inspired by social science methodologies resulted in different explorations of research and ethical questions. These included the learning of safety and safe-by-design approaches in the life sciences programmes at Wageningen University, the impact of tensions in stakeholder norms on designing for biosafety in the industrial biotechnology context, and the perceptions of industry and academia on production platforms and opportunities in the biotechnology field. Lastly, this thesis ends providing a snapshot of the state of biomanufacturing and proposing the idea of meta-workflows. These are unifying ecosystems amongst research infrastructures that emphasize the importance of interoperability, harmonization, and sustainability in making a successful transition to global manufacturing that utilizes bio-based and safe production platforms.