Functional nucleic acids for green biosynthesis

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Microbial-based production provides a powerful green alternative for producing fine chemicals and transport fuels while also limiting human exposure to hazardous synthetic chemicals and processing. Despite the recent success of yeast-based preparation of many high-value products, importing complex biosynthetic pathways with numerous synthetic steps into yeast requires balancing enzyme expression levels, minimizing pathway bottlenecks, and preventing self-toxic effects. Thus, current approaches require significant investment in time and resources for each individual target molecule, severely limiting the current generality and scope of fermentative production. My research program herein will be the first to harness nucleic acid tools for improving biosynthesis of chemicals and biofuels. This proposal outlines a research program for developing novel functional nucleic acids tools that (1) increase efficiency of complex enzymatic reactions; (2) reduce toxic effects to yeast, permitting increased titer; and (3) furnish pathways with molecular scaffolds for new chemical diversity and enhanced functionality. For example, we will design a new class of DNA-encoded biosensors to allow high-throughput fluorescent screening of millions of enzyme variants. We will also adapt a previously-described synthetic ribozymes to dynamically control key enzymes and metabolic pathways to reduce side-products. We will select functional nucleic acids that specifically bind to target molecules with high affinity (aptamers) to sequester or encapsulate toxic side products and intermediates. Finally, we will characterize the potential of replacing small functional groups with biorthogonal handles such as azide or alkyne, facilitating enzymatic incorporation into large molecular scaffolds. This would allow many different "click partners", including diverse functional groups with enhanced chemical properties, or larger biomolecules useful in drug-design (e.g., siRNA, aptamers) to be rapidly and specifically installed. The plans described in this proposal represent the groundwork needed for the development of a rigorous functional nucleic acids research program. Over the next five years, my group will aim to develop the tools and apply them towards a conceptually-new yeast-based biosynthesis platform that is both diversity and target oriented. In the future, we will apply the findings from our proof-of-principle experiments to biosynthetic pathways producing important targets, including antimicrobial and anticancer natural products, advanced fuels, and platform chemicals. This research program will contribute to the field by providing the conceptual framework and practical tools needed to realize the application of functional nucleic acids for energy efficient and safe green biosynthesis strategies. The strategy is innovative because functional nucleic acids have found applications in many fields of research, but are surprisingly lacking in biosynthesis and green chemistry.