The field of metabolic engineering focuses on using molecular biology tools to genetically modify the metabolic pathways of cells for the production of chemical compounds. The field of directed evolution can alter the native abilities of proteins by taking inspiration from natural evolution. Both fields bring novel solutions to current problems in energy, the environment, and medicine. However, there is still no general higher throughput screening method for both of these fields. In this dissertation, we apply our designed fluorescence polarization assay to fill this need in the fields of metabolic engineering and directed evolution. Chapter 0 gives background information related to metabolic engineering, directed evolution, tetracyclines, the Tetracycline Repressor protein (TetR), TAN-1612, and fluorescence polarization. Chapter 1 describes our development of a quantitive, sensitive, and fast fluorescence polarization assay which uses the TetR protein to detect the binding of the small molecule tetracycline TAN-1612. Chapter 2 demonstrates that the binding affinity of the TetR protein for TAN-1612 can be improved using directed evolution and by incorporating our assay to screen TetR mutants. Finally, in Chapter 3 we apply our fluorescence polarization assay to the screening of yeast strains biosynthesizing TAN-1612, without the need of time and labor intensive extraction and purification steps.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key A- and C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature’s biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Chapter 1 provides background on antibiotics, and the tetracycline class in particular, and the metabolic engineering and directed evolution techniques available to us for heterologous expression of enzymes in yeasts. In Chapter 2, we describe the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts. Further, in Chapter 3, we highlight our Tang Laboratory collaborators’ work, where they reported the heterologous biosynthesis of a non-antibiotic fungal anhydrotetracycline derivative, TAN-1612, in S. cerevisiae from Aspergillus niger. We have built upon this system, allowing for the high-titer production of TAN-1612 in yeasts. Finally, in Chapter 4, we outline our efforts to convert TAN-1612 into a high titer tetracycline- and analog-producer by modifying the 2-, 4-, and 6-positions, proven critical for antibiotic activity. By hijacking biosynthetic hydroxylating and reducing enzymes, we attempted to modify the 6α-position, dearomatizing the C-ring. We also expressed heterologous enzymes within the TAN-1612 pathway that could furnish the 2-position with a carboxamido group instead of its natural hydrogen groups. By taking advantage of yeast’s natural biosynthetic pathways, we will create inexpensive, single-dose antibiotics, setting the stage to pursue yeast as a novel therapeutic. These state-of-the-art synthetic biology technologies will create entirely new paradigms, leading the charge against infections and diseases.