Selection-aided Metabolic Engineering of Microbial Cell Factories for Vitamin Production in Escherichia coli

Microbial cell factories provide a promising alternative to the carbon-intensive chemical synthesis of a wide range of products. A key to the development of new, cost-competitive cell factories is the designing, building and testing of high-performing microorganisms and learning from this process. Success and throughput of this process is dependent on efficiently building microbial diversity and subsequent filtering it to identify the most effective design. In this thesis, metabolic engineering approaches are combined with selection strategies to establish and improve cell factories for vitamin production in Escherichia coli. Single mutations in the global transcriptional regulator IscR leading to improvements in a range of cell factories dependent on iron-sulfur (FeS) cluster enzymes are identified.
Characterization with proteomics and genetic engineering experiments revealed that the enhanced performance in biotin, thiamine and lipoic acid production strains was centered around FeS cluster formation and repair. Transport proteins are the gatekeepers between the chemical world and intracellular metabolism. Functional annotation and understanding of sequence-function relationship are important for metabolic engineering, while also increasing our fundamental scientific knowledge. Through auxotrophic selection of metagenomic libraries, transporters with affinity for biotin sulfoxide (BSX) were found. These may improve biotin purity
of future cell factories since import of BSX allows reduction of BSX to biotin by biotin sulfoxide reductase, BisC. Similarly, transporterknowledge was gained in a directed evolution study of the nicotinamide riboside transporter, PnuC. Using random and directed mutagenesis strategies combined with a riboswitch-based selection system, PnuC transporters were engineered to accept thiamine as substrate, with only one to four amino acid changes. Overall, the results of this thesis illustrate how selection systems can assist in removing production bottlenecks of cell factories, increasing the throughput, and enhancing the success rate of metabolic engineering, hopefully accelerating the development towards a more sustainable future.