This Small Business Innovation Research (SBIR) Phase I project proposes to develop a metabolic engineering and synthetic biology toolkit for a scalable, industrial yeast host. Currently, the vast majority of synthetic biology tools are directed toward engineering E. coli or S. cerevisiae, model laboratory organisms that are often poorly suited for industrial fermentations. Furthermore, there is an absence of available synthetic biology tools for those hosts that are well suited for industrial fermentations. This research addresses this problem through the development of a foundational set of synthetic biology tools in an industrially tractable, but under researched yeast strain. The research objectives include construction and characterization of a series of expression vectors that facilitate transfer of genetic material into host cells, construction of a genetic library designed to perturb host metabolism and redirect carbon flux toward production of target small-molecules, and demonstration of an approach to reduce expression of competing metabolic pathways. Proof-of-principle application of the tools will be used to demonstrate improvements in malonic acid biosynthesis in engineered yeast.
The broader/commercial impacts of the proposed project, if successful, will be technology that enables genetic modification and engineering of a robust, industrial yeast host, removing significant technical barriers that have traditionally inhibited both commercial and academic research. In addition, the industrial yeast host genetic toolkit may accelerate research and development on, and improve the commercial economics of, a range bio-chemicals with over $30B in aggregate market value. The vast majority of these products are currently produced petrochemically, but there are potential cost and environmental advantages if they can be produced biologically. The technology will first be applied to commercialize malonic acid, a high-value specialty chemical currently derived petrochemically.
SBIR Phase I: Production of high-value chemicals in industrial yeast hosts The goal of this Small Business Innovation Research Phase I project was to develop a metabolic engineering and synthetic biology toolkit for a scalable industrial yeast host. Metabolic engineering and synthetic biology research is currently dominated by the model prokaryote and eukaryote hosts, E. coli and S. cerevisiae, respectively. Both of these hosts have the benefit of readily accessible genetic toolkits that facilitate rapid, efficient introduction of genetic modifications and hypothesis testing. Neither of these organisms, however, is ideal for broad-scale use in industrial fermentations (i.e., fermentative production of varied small-molecule fuels and chemicals). In fact, there exist very few metabolic engineering/synthetic biology tools for hosts most widely used in industry. Thus, this Phase I SBIR grant proposal was focused on providing a genetic toolkit – including vectors and genetic libraries – for use in a more industrially ideal yeast host. In brief, the Phase I work plan was to construct and characterize a set of foundational synthetic biology tools designed to accelerate metabolic engineering using the strain and subsequently demonstrate application of the toolkit for improving small-molecule biosynthesis. The objectives include construction and characterization of a series of expression vectors that facilitate transfer of genetic material into the strain, construction of a genetic library designed to perturb host metabolism and redirect carbon flux toward production of the target small-molecule, and demonstration of an approach to reduce expression of competing pathway proteins. All three objectives were successfully completed, and the metabolic engineering tools were used to improve target small-molecule biosynthesis.
