Development of synthetic biology tools for growth decoupled production and protein expression

With each passing day, the need for humankind to transition how our economy operates becomes clearer and clearer. One of the most important transitions is moving away from using non-renewable fossil fuels and towards renewable and sustainable technologies. While windmills and electric cars can go far in addressing fossil fuel consumption in the energy and transportation sectors, we also need to replace oil based chemicals and materials. Luckily, the solution may already be in our hands. Humans have used microorganisms for thousands of years to make fermented drinks and foods, long before discovering what microorganisms were. Technological breakthroughs in the last 50 years have enabled us to engineer and rewire microorganisms towards producing the compounds that are currently derived from fossil fuels, through the process of microbial fermentation. The key advantage of this bio-based production process is the ability to use renewable raw materials, such as sugars, agricultural and forestry waste, or even waste gasses from heavy industry.

However, there are still many challenges to overcome before bio-based production can become the predominant production process. Building and operating the production facilities is extremely costly. The microbial strains and production processes have to therefore be highly efficient. This thesis focuses on developing strains and tools towards improved two-stage fermentations in the bacterial platform organisms Escherichia coli and Bacillus subtilis. Two-stage fermentations divide the fermentation into dedicated growth and production phases, which allows for more efficient use of the feedstock, which is a major contributor. This division of phases can be mediated by synthetic biology tools that are used to dynamically control the metabolism of the cells. Lastly, this thesis touches on heterologous protein expression, which is an important part of microbial cell factory engineering.