In the past few decades, scientists have exploited the natural metabolic processes of microorganisms to achieve synthesis of many products such as pharmaceuticals, fuel and both bulk and fine chemicals. Metabolic engineering of microorganisms often exceeds the current ability of most organic synthetic chemist, 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. This proximity enzyme organization, also known as a metabolon, will facilitate in the passage of intermediates from one enzyme to another, thus limiting diffusion rates, enabling fast turnover of toxic intermediates and preventing crosstalk between competing metabolic pathways. Here we have developed a synthetic metabolon using DNA as a scaffold and nuclease-null Cas9 fusion proteins as binding partners. We have successfully bound three orthogonal dCas9 fusion proteins to a single DNA target. Then used this technology to increase the production of reducing sugar by as much as 4 fold via an endoglucanase and a carbohydrate binding module from <i>Clostridium thermocellum</i> fused to the aforementioned orthogonal dCas9 proteins. An added layer of conditional control can be appended by the use of aptazyme guide RNAs, which become functional upon the addition of the small molecule theophylline. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and possibility of fusion partners for a multitude of enzymatic pathways.<br/>

Designing modular synthetic metabolons via dCas9-guided assembly

2019 Spring National Meeting

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