Engineered aldolase for the fermentative production of L-fucose and other rare sugars.

DESCRIPTION provided by applicant The goal of this proposal is to develop a fermentative strategy for the large scale production of L fucose and other rare sugars L fucose deoxy L galactose is an important hexose deoxysugar found in a variety of organisms attached to an array of macromolecules These L fucose containing glycans exhibit a wide range of medicinal properties including supporting infant health L fucose containing human milk oligosaccharides anticoagulant and antithrombotic antivirus antitumor anticancer and immunomodulatory anti inflammatory blood lipids reducing antioxidant activitiy against hepatopathy uropathy and renalpathy gastric protective effects and therapeutic potential in surgery L fucose containing polymers The L fucose monomer has therapeutic properties such as inhibiting virulence factors and is also an invaluable synthetic starting material for a wide range of molecules including human milk oligosaccharides blood group antigens E and P selectin antagonists and functionalized L fucose derivatives In addition to pharmaceutical relevance L fucose also possesses topical properties attractive to the cosmetic industry including anti aging wrinkle reducing and is safe for sensitive skin Despite the impressive range of bioactivity discovered thus far L fucose remains prohibitively expensive and unavailable in the scale needed to support these applications We feel a fermentative approach is needed to meet these large scale requirements and to provide the glycoresearch community with this important building block needed to prepare scarce or unavailable glycans A key aspect of our strategy is the production of an engineered L fuculose phosphate aldolase that no longer requires the phosphorylated donor substrate dihydroxyacetone phosphate Instead the novel L fuculose phosphate aldolase FucA will catalyze the condensation between dihydroxyacetone and L lactaldehyde to form L fuculose thereby bypassing the typical sugar phosphate intermediate Through metabolic engineering we envision an E coli system capable of integrating this engineered L fuculose phosphate for the specific synthesis of L fucose In Phase I we will demonstrate the feasibility of a fermentative L fucose system by identifying and engineering a mutant FucA enzyme capable of condensing dihydroxyacetone and L lactaldehyde to form L fuculose for the ultimate production of L fucose In Phase II we will focus on the genetic and metabolic engineering of E coli for the production and accumulation of the substrates needed for the engineered L fuculose phospahte aldolase We propose a yield of g L production of L fucose after integration of the engineered L fuculose phosphate aldolase and subsequent optimizations In Phase III we will commercialize L fucose as well as other rare sugars produced by the engineered L fuculose phosphate aldolase using various acceptor aldehyde substrates PUBLIC HEALTH RELEVANCE This project is aimed toward developing a fermentative process for the large scale production of L fucose and other rare sugars L fucose is used as a synthetic building block for a wide range of pharmaceutically relevant biomolecules Additional rare sugars produced in this program will include D psicose L tagatose L fructose D ribulose L fuculose and deoxy D psicose which have applications as glucosidase inhibitors used in chemotherapy and serve as building blocks precursors to various therapeutics