There is a growing interest in developing methodologies for the robust, high-yield production of bioengineered natural polysaccharides that can be used as nutraceuticals or pharmaceuticals, such as chondroitin sulfate. Such methods, using recombinant microorganisms will allow, for the first time, the production of animal-free polysaccharides and will eliminate the risks associated with animal-derived products that include viral infections, product inconsistencies and possible adulteration as well as reliance on environmentally non-friendly processes. This project is a translational and multi-disciplinary research effort with the ultimate goal to facilitate the efficient and safe production of kilogram quantities of non-animal sourced bioengineered chondroitin sulfate. It seeks to unravel the role of precursor availability on the production of large molecules and to translate this knowledge to a highly efficient production platform of polysaccharides.
The project objective is to enhance the production levels of non-sulfated chondroitin through standard metabolic engineering methods, stoichiometry-based modeling and metabolic pathway balancing (specific aim 1). In specific aim 2, the investigators will demonstrate CRISPR interference (CRISPRi) as a metabolic engineering tool, enabling inducible knockdown and tunable repression of multiple genes simultaneously for controlling metabolic fluxes in E. coli. In specific aim 3, a scalable methodology will be developed for in vitro enzymatic production of USP chondroitin sulfate A (CSA, chondroitin-4-sulfate) using chondroitin substrate harvested from E. coli fermentation. The societal impact of the proposed project is expected to be broad, as the importance of having a non-animal-derived chondroitin cannot be overstated. Recombinant protein biotherapeutics have had a revolutionary impact on healthcare. A number of both basic and applied research areas are expected to be impacted as a result of this project, including: (i) identification of metabolic steps that play a key role in biosynthesis of chondroitin in E. coli; (ii) application of CRISPRi for metabolic engineering, further facilitating its application as a synthetic biology tool, where it could also be utilized for construction of complex gene circuits, logic gates, and biosensors (iii) the synthesis of bioengineered chondroitin sulfate will become reality and will pave the way for the animal-free synthesis of several other polysaccharides of pharmaceutical importance. The proposed project will be performed in a scientifically diverse environment with graduate students working together with industrial investigators to perform the experimental tasks with the aid of undergraduate students. In addition, a focused outreach effort by the PI and co-PI will include their continued participation in the Rensselaer Summer High School Research Program and the Scientific Research Program in Shaker High School as well as enrollment in new outreach opportunities such as the New Visions Math, Engineering, Technology and Science program. This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the GOALI Program of the Division of Industrial Innovation and Partnerships.