Engineering Saccharomyces cerevisiae for the production of early benzylisoquinoline alkaloids [electronic resource] : strategies and tools for strain improvement and pathway construction

Natural products or compounds derived from natural products comprise a majority of successful drug molecules. However, the discovery, synthesis, and scalable manufacture of these molecules present complex challenges that have limited the exploration and testing of new natural product drug candidates. For example, the benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites that exhibit diverse pharmacological activities, including antiviral, antiprotozoan, and analgesic activities. However, these molecules cannot be economically produced by traditional chemical synthesis strategies; current commercial production relies on extraction and isolation of select BIAs from the native plant hosts. As such, a microbial platform for the production of BIAs has the potential to reduce the cost and increase the scale of current production strategies, as well as expand the diversity of molecules accessible beyond those that accumulate in plants. We have engineered strains of the yeast Saccharomyces cerevisiae for the production of BIAs from non-BIA precursors. First, we developed methods for culturing wild-type yeast strains to produce the BIA norcoclaurine from fed tyrosine and dopamine. Next, we modified yeast native metabolism to re-route carbon flux towards aromatic amino acid biosynthesis, enabling robust production of norcoclaurine in the absence of extracellular tyrosine. Finally, we extended our engineered pathway to include two additional BIA molecules, coclaurine and N-methyl-coclaurine. This work demonstrates the first microbial synthesis of these three BIA molecules. These yeast strains optimized for early BIA production will provide a platform for the introduction of plant enzymes for the synthesis of more complex and diverse natural products. Additionally, in constructing our BIA-producing yeast strains, we encountered limitations in previously described methods for chromosomal integration in S. cerevisiae. These methods relied on time- and labor-intensive protocols for cloning and selection marker rescue. To address these limitations, we developed a new design for yeast integrating plasmids (YIPs). These "directed pop-out" YIPs enable a simple, two-step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids exhibit three novel features: a dedicated "YIPout" fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multi-fragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation-free marker rescue event. We constructed and tested directed pop-out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation-free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways.

Keywords: Chemical Engineering.
Advisor: Smolke Christina D. primary advisor. Thesis advisor
Swartz James R. primary advisor. Thesis advisor
Spormann Alfred M. advisor. Thesis advisor