Synthetic Biology approaches for improved protein production in the bacterium Bacillus subtilis

Today’s society depends on oil-based resources for a long range of everyday products. The associated greenhouse gas emissions have well-documented adverse effects on the climate and the environment, which supports life on planet earth. Thus, transforming society from being dependant on oil-based resources into a sustainable bio-based alternative, is arguably the most important challenge of the 21st century. Lignocellulosic biorefineries are promising candidates as
alternatives to oil-based processes for producing fuels and chemicals. However, breaking down the polymers found in this type of biomass into fermentable sugars, constitutes a significant technical challenge. This challenge has yet to be truly solved, and hinders the broad implementation of lignocellulosic biorefineries. They key to solving this challenge, could lie within the family of enzymes known as Lytic Polysaccharide Monooxygenases. These enzymes oxidise the biomass
polymers, thereby increasing the effectiveness of already available enzyme cocktails. Unfortunately, our knowledge about these enzymes is still relatively limited, and more research is required for unlocking their true potential. The scientific progress of the field and the industrial utilisation of the enzymes, both depend on the generation of enzyme batches containing high concentrations of the desired enzymes. This can be achieved through heterologous protein production
in microbial hosts, one of the most popular being the bacterium Bacillus subtilis. However, bottlenecks in gene expression can be a limiting factor for the success of production efforts, ultimately resulting in failed projects in both academic and industrial settings. In order to avoid and remove such bottlenecks, engineering concepts from the field of Synthetic Biology can be applied to optimise production strains. This thesis aims to provide the reader with a general understanding of how Lytic Polysaccharide Monooxygenases can benefit the transition into a bio-based society, and how Synthetic Biology approaches can be employed to optimise production of these and other enzymes. The thesis is accompanied by publications, which are examples of methods that can improve workflows and production titers of heterologous proteins in B. subtilis.