Modular control of multiple pathways using engineered orthogonal T7 polymerases

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.United States. Office of Naval Research (Award number N00014-10-1-0245)National Science Foundation (U.S.). (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Graduate Research FellowshipAmerican Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipHertz Foundation. Graduate Fellowshi

Orthogonal Expression of Metabolic Pathways

Microbial metabolism can be tailored to meet human specifications, but the degree to which these living systems can be repurposed is still unknown. Artificial biological control strategies are being developed with the goal of enabling the predictable implementation of novel biological functions (e.g., engineered metabolism). This dissertation project contributes genetic tools useful for modulating gene expression levels (extending promoters with UP elements) and isolating transcription and translation of engineered DNA from the endogenous cellular network (expression by orthogonal cellular machinery), which have been demonstrated in Escherichia coli for the production of lycopene, a 40-carbon tetraterpene carotenoid with antioxidant activity and a number of other desirable properties

Generalizable approaches for gene expression regulation in Saccharomyces cerevisiae

There is a current surge of interest in using synthetic biology for biotechnology applications. Metabolic engineers, for example, are interested in synthetic biology for its modular and well characterized transcriptional “parts”, such as synthetic gene promoters and terminators, which enable fine tuning in metabolic pathway optimization. Likewise, emerging gene editing methods, such as CRISPR-Cas9, are enabling quicker and more precise genomic integrations. Using both of these advances, there is an increase in the throughput for which altered pathway conditions can be screened. While some advances are being made, there are still several technological gaps, especially for eukaryotic yeast hosts. Therefore, this dissertation work focused on developing engineering methodologies for the yeast Saccharomyces cerevisiae to expand capacity in each of these areas. There were three main areas explored in this work. First, we developed a method for synthetic promoter design which establishes de novo upstream activating sequences (UAS) capable of regulating gene expression by growth phase. These UAS elements, discovered through a transcriptome mining approach, show an over 30-fold activation of a core promoter with completely synthetic designs. Secondly, we improved synthetic terminator design, whereby both minimal synthetic terminators and larger native terminators were improved by adjusting nucleosome occupancy in adjacent sequence space. Using this methodology, de novo synthetic terminators were designed for increased termination efficiency. Lastly, we developed a method for guide RNA expression in yeast organisms using T7 RNA polymerase in vivo. This method allowed guide RNA expression to be exportable across yeast hosts and enabled more complex regulation designs, such as dCas9 logic gates. Together, these approaches improved synthetic promoter design, synthetic terminator design, and guide RNA expression regulation in ways that both complement current ongoing research in S. cerevisiae and enable a generalized approach to be established for other yeast organisms.Chemical Engineerin