Of the many proteins that exist inside our bodies, most are decorated with complex sugars called glycans through a process called glycosylation. Glycosylation is often necessary for these proteins to function correctly. For therapeutic use in humans, glycoproteins must have human-like glycans. Thus production is often limited to mammalian cell culture, which is time-consuming, expensive, and susceptible to viral contamination. A strain of Escherichia coli capable of producing human-like glycoproteins could overcome some of these hurdles. This project seeks to take bacterial production of glycosylated proteins to the next level by using state-of-the art techniques in RNA engineering to dynamically optimize the output of the glycosylation pathway.
In this project, the investigators will use the principles of RNA engineering to create genetic networks that dynamically tune the expression of glycosylation enzymes as they are needed in order to increase product output and purity. The proposed studies and research training activities are expected to have a broad impact on society, ranging from the science of glycobiology and the engineering science of RNA gene regulation, to the development of human glycotherapeutics. This project will also cultivate the next generation of highly trained graduate students who will be introduced to the broad, interdisciplinary nature of biotechnology research. Moreover, this program will actively and aggressively broaden participation in science and engineering. This will be accomplished by providing interdisciplinary research opportunities for undergraduate students, developing experiential glycoscience learning modules for undergraduate and high school students, and creating a quantitative graduate-level course for biomolecular engineering and synthetic biology. Finally, the development of bacterial glycosylation and RNA engineering for biotechnological applications will be brought to a larger research community through partnership with local biotechnology companies.
The long-term goal of this research project is to genetically engineer and optimize bacterial cells for the routine production of authentic human N-linked glycoproteins. To date, the investigators have recreated the earliest steps of this complicated process in Escherichia coli. The objective of this particular application is to maximize the productivity of this pathway by engineering synthetic RNA-based genetic circuitry that will dynamically control the expression of glycosylation enzymes using two distinct strategies. The first will eliminate competitive side reactions by creating distinct stages of glycan construction followed by glycan targeting, which we anticipate will dramatically increase the purity of the glycoproteins produced. In parallel, pathway productivity will be optimized by expressing pathway enzymes 'just-in-time' in the order they are needed as is done in several essential metabolic pathways used by cells. Since glycosylation consists of sequential enzyme steps that take place in different parts of the cell, controlling the dynamics of enzyme expression so that they are most active when needed is expected to significantly boost pathway production. Successful completion of these studies will lead to the development of a novel bacterial glycoprotein expression platform with the potential to overcome many of the limitations of existing eukaryotic platforms. Moreover, the proposed studies and research training activities will impact: (i) biotechnological synthesis of novel glycoconjugates and potential immunostimulating agents for research, industrial and therapeutic applications; (ii) the development of new, broadly applicable strategies that can be used to optimize a wide array of metabolic processes; and (iii) the development of new tools and design principles for engineering genetic circuitry to control cellular behavior with far-reaching potential.
Due to the interdisciplinary nature of the project, this award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.
