Princeton University Library Catalog

Applications of protein-level regulation and optogenetics in metabolic engineering of S. cerevisiae

Mehl, Justin [Browse]
Senior thesis
Avalos, Jose L. [Browse]
Princeton University. Department of Chemical and Biological Engineering [Browse]
Princeton University. Program in Environmental Studies [Browse]
Class year:
Restrictions note:
This content is embargoed until July 1, 2019. For more information contact the Mudd Manuscript Library.
Summary note:
In metabolic engineering, the requirement to balance production of desired metabolites and basic cellular function limits yields. However, some of these basic functions are required only at certain developmental times. Using temporal-control to optimize the magnitude and timing of the expression of these pathways, it is possible to redirect flux toward desired metabolites while ensuring cell viability. An example of such a pathway in Saccharomyces cerevisiae is ethanol fermentation, which consumes pyruvate, a valuable intermediate in the production of lactic acid, isobutanol, and other products. Cell growth on glucose substrate is contingent on ethanol fermentation, which in turn is contingent on the expression of pyruvate decarboxylase (Pdc), the first enzyme in the pathway. Knocking out all three Pdc isozymes renders cells unable to grow on glucose. We hypothesized that temporal control using optogenetics and nanobody-mediated protein-level inhibition could increase yields and serve as new tools in metabolic engineering. A light-sensitive transcription factor that is activated only under blue light, was shown to induce tunable transcriptional regulation. Anti-Pdc1p nanobodies, single domain recombinant antibody fragments, were shown to inhibit Pdc1p function, relative to control nanobodies. Optogenetic circuits, based on the galactose regulatory system, make it possible to invert the transcriptional response to light input. A lactate production circuit was designed and tested in which transcription of Pdc1p was stopped during fermentation. While yields from this strain were low (1.18 g/L of lactate), it serves as an important proof of principle for two-stage fermentation and it has potential for significant optimization, including the addition of induced nanobodies.