Carbohydrates play an important role in many cellular functions and diseases; however, carbohydrate-based drug discovery research is lagging compared to other biologically important molecules because of the significant challenges in creating and obtaining carbohydrate-based molecules. This difficulty is exacerbated by the lack of easily available of starting materials created with rare sugars. One such sugar is L-ribose. L-ribose is the core of several nucleoside- based pharmaceuticals presently on the market or undergoing clinical trials. As the need for these drugs increases, demand for L-ribose is going to increase significantly. We propose a fermentative route to the production of L-ribose and other rare sugars using a mannitol-1- dehydrogenase (MDH). The MDH has a broad substrate specificity profile that allows the production of many different rare sugars from readily available and inexpensive polyols. Phase I of this research dealt with the recombinant expression of the MDH gene within Escherichia coli and demonstrated the synthetic utility of the system with L-ribose being our initial target. This phase II portion will expand upon the successful phase I research to study the scale-up of these conversions into a commercially viable process as well as the engineering of the MDH to expand the synthetic utility of the enzyme beyond the substrate specificity of the wild-type MDH. Specifically, we intend to 1) improve the expression and activity the mannitol dehydrogenase, 2) scale-up the L-ribose production, 3) test and scale-up in vitro bioconversion and/or fermentations of other rare sugars, 4) improve the NADH oxidase for cofactor recycling, and 5) modify the substrate specificity of the mannitol dehydrogenase. This technology will create a diversity of low-cost rare sugars to expand the portfolio of carbohydrate starting materials available to biochemists and medicinal chemists. Rare sugars are becoming increasingly important in the development of therapeutics, whether for attachment to natural products, or as the building blocks for nucleoside-based antivirals. Many of these rare sugars, such as L-ribose or D-mannose, are simply not available in large quantities for biochemical and pharmaceutical studies. Our work will create a portfolio of low- cost sugars that will enable the production of a wide variety of carbohydrate-based therapeutics for drug discovery. ? ? ?
| Woodyer, Ryan D; Christ, Trevor N; Deweese, Kara A (2010) Single-step bioconversion for the preparation of L-gulose and L-galactose. Carbohydr Res 345:363-8 |
