Designing modular synthetic metabolons via dCas9-guided assembly and protein nanocages
Date
2020
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Publisher
University of Delaware
Abstract
In the past few decades, scientists have exploited the natural metabolic processes of microorganisms to achieve the synthesis of many products ranging from pharmaceuticals, and fuels to both bulk and fine chemicals. Metabolic engineering of microorganisms often exceeds the current ability of most organic synthetic chemists, but our existing techniques lack the ability to easily optimize cell productivity and product titer for expansion to a variety of enzymatic pathways. Our goal is to move away from traditional approaches of overexpressing rate limiting enzymes and competitive pathway deletion, towards a strategy in which a cell can sense the accumulation of an intermediate and elicit proximity control over enzyme organization. Throughout this work, several strategies were employed to mediate this enzyme organization, also known as a metabolon. ☐ Nuclease- null Cas9 (dCas9) fusion proteins were combined with engineered single-guide RNA (sgRNA) along with a DNA template for the formation of the synthetic metabolon. The dCas9 protein was chosen as our scaffolding partner due to its high binding affinity as well as its innate ability to be directed to any DNA target with high specificity. Using DNA as the scaffolding domain also offers its own unique advantages like the ease of synthesis, its ability to form secondary and tertiary structures and its flexibility. The ability to utilize three orthogonal dCas9 fusion proteins to bind to a single target was studied, as was the ability of our dCas9 fusion proteins to form a synthetic metabolon focused on cellulose hydrolysis. Furthermore, we employed an engineered toe-hold guide RNA (thgRNA) for conditional binding and subsequent metabolon formation. ☐ To optimize the modularity of our dCas9 mediated metabolon and reduce expression issues associated with larger engineered proteins, we employed a post translation ligation strategy. We fused a small peptide tag (Tag) to the dCas9 protein and fused its binding partner (Catcher) to the pathway enzymes of interest. This tag/catcher system has been shown to form a spontaneous isopeptide bond in a wide range of temperatures, pHs and buffering conditions. We sought to utilize two orthogonal tag/catcher systems in order to maintain the site specificity offered by the dCas9 proteins. ☐ Utilizing the post translational tag/catcher system not only optimized the dCas9 mediated scaffold but it opened up a pathway to study other scaffolding methods. Specifically using self- assembling protein nanocages for the spatio temporal control of pathway enzymes. We chose to use two protein nanocages, E2 and HBV, due to their differences in size and subunit composition. We decorated the exterior of the protein nanocages with different ratios of enzymes utilized in cellulose hydrolysis and studied the effects on reducing sugar titers. ☐ When it comes to enhancing product titers from a variety of metabolic pathways there isn’t a one option fits all. Here we have developed multiple ways to scaffold pathway enzymes, each offering their own advantages. We hypothesize, if enzyme location is important to a particular pathway, the dCas9 mediated platform is second to none due to the site specificity offered by the Cas9:gRNA complex. Other pathways show increased product titers from the co-localization of enzymes and order of enzyme is less important, here one may wish to employ the nanocage mediated scaffolding system since it has fewer components.
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Keywords
Cas9, Metabolon, Nanocage, Protein, Scaffold