Synthetic biology to address complex bottleneck challenges in high-value bioprocesses

Almost all materials and chemical products enjoyed today, from plastics to vitamins, are directly derived from fossil fuels. Microbial cell factories can produce many of these products via biological, enzymatic conversion of sugar to the value-added product of interest. Cell factory production has inherent benefits compared to petrochemical synthesis in its sustainability, safety, and potential profitability. Efficient cell factories require the coaxing of living organisms, in this thesis, bacteria, into fundamentally changing their metabolism. Even the relatively basic metabolism of bacteria has evolved sophisticated interactions and
regulation over billions of years, systems which we are not yet close to understanding in entirety. This thesis focuses on the use of synthetic biology, metabolic engineering, and bioinformatics for specifically improving the production of two high-value products, biotin, and lipoic acid, in Escherichia coli cell factories. Both biotin and lipoic acid cell factories are characterized by utilizing biosynthetic pathways, which require complex and toxic enzymes that act as bottlenecks for productivity. We use various tools and approaches to tackle some of these bottlenecks.

We first build a free lipoic acid cell factory from the ground up. Through several iterations of development, we design, test, and discover ways to improve the productivity of the cell factory. In the end, this allows us to produce many-fold more lipoic acid than previously reported in a cell factory. Next, we focus on the main enzymatic bottleneck of the lipoic acid cell factory, Lipoyl Synthase (LipA). While not ultimately improving this step, we make some fundamental discoveries related to this bottleneck enzyme and the diversity of lipoic acid biosynthetic pathways in nature. The biotin pathway is limited in production by the
enzyme Biotin Synthase (BioB). Using various bioinformatic tools coupled with biochemical analysis, we discover a new type of BioB. This new BioB type likely uses a never-before-seen mechanism, which has great potential for overcoming the critical bottleneck in biotin cell factory production. Finally, we engineer and discover novel electron-transfer strategies to BioB. These works present several improvements to both the productivity of lipoic acid and biotin cell factories, but also the fundamental understanding of these biosynthetic pathways and their bottleneck enzymes LipA and BioB. These can hopefully play a part in the goal of commercializing these cell factories, a small step in the right direction in transitioning from a fossil fuel-dependent society.