This proposed program is a competitive renewal of grant R01 GM085267 titled Biosynthesis of Polysaccharides, funded from 7/30/2009 - 7/29/2013. This program is our continuing efforts to use both chemical and biochemical tools to elucidate the mechanism of polysaccharide biosynthesis and to produce promising drug candidates for biomedical evaluation. Lipopolysaccharides (LPS) are characteristic components of cell walls of Gram-negative bacteria, localize in the outer leaflet of asymmetric outer membrane (OM) and expose on the cell surface. LPS typically consists of a hydrophobic domain known as Lipid A (or endotoxin), a nonrepeating core oligosaccharide (including both the inner core and outer core) and a distal polysaccharide (O-antigen or O-PS). The biosynthetic pathway to LPS consists of independent biosynthesis of O-antigen and Core-Lipid A, respectively, and combination of these two parts to make LPS, and then transport to OM. Thus, total biosynthesis of LPS in vitro includes three Milestone events: 1) assembly of O-PS; 2) assembly of Core-Lipid A; 3) finally assembly of LPS. The first Milestone has already been completed in the last funding period of this grant. Biosynthesis of O-antigen of LPS is a wzy-dependent pathway: the individual repeating oligosaccharide unit is synthesized in the cytoplasm by the sequential action of specific glycosyltransferases. The repeating unit is then transported to the periplasmic side of the membrane by Wzx where it is polymerized into a polysaccharide by the polymerase Wzy. The chain length of the polymer is regulated by an unknown mechanism that involves Wzz protein. We used purified enzyme Wzy and Wzz to reconstitute such polymerization process, and for the first time, achieved the synthesis of polysaccharides in a test tube! Moreover, the enzyme WaaL (which transfers the polysaccharide from its diphosphate-lipid precursor to core-lipid A) was found to accept almost any structures of sugar-diphosphate-lipid donors. These results lay out an excellent foundation for accomplishing the remaining two milestones. For Milestone 2 of assembly of Core-lipid A, we will chemically synthesize a number of Lipid A molecules. The glycosyltranferases involved in the biosynthesis of core oligosaccharide will be over-expressed and used for in vitro sequential assembly of Core-Lipid A. The synthesized chemically defined Lipid A and Core-Lipid A molecules are not only useful research tools for studying LPS biosynthesis, but also potential vaccine adjuvants. Finally, Milestone 3 will be achieved by transferring the O-PS to the Core-Lipid A by WaaL to produce full LPS. Based on the understanding of Lipid A-TLR4/MD2 complex, many Lipid A molecules and Lipid A analogs have been found to have strong immune activities and can be used as adjuvant either alone or with other adjuvants in a variety of vaccine formulations. Thus, we hypothesize that our chemo-enzymatically constructed E. coli Inner Core-Lipid A and Core-Lipid A conjugates would represent as a novel set of wide- spectrum and stand-alone vaccine candidates with defined structures for prevention of urinary tract infection (UTI). Specifically, the program includes the following aims: Milestone 1: Our efforts in the biosynthesis of O-PS will include the synthesis of O-PS from two most commonly used uropathogenic E. coli (UPEC) strain CFT073 (O6:K2:H1) and UTI89 (O18:K1), and X-ray structure determination of WaaL and Wzy. Milestone 2: Chemically synthesize both E. coli di- and mono-phosphorylated lipid A with either tetraacylated or hexaacylated lipids, or with fluorine-containing hexaacylated lipid (total of 9 lipid A compounds), then transfer either E. coli R3 or R1 core oligosaccharides to these lipid A structures by following the biosynthetic pathway using sequential glycosyltransferase-catalzyed reactions. Milestone 3: Total assembly of E. coli O86 LPS, and other natural and chimeric LPS structures by WaaL catalyzed transformation. Successful execution of this research program should provide two unmet biomedical needs. Although LPS is one of widely used biochemicals in immunology and other biomedical research, there is essentially no pure LPS available. All the commercial LPS coming from isolation and purification from natural resources are inevitably a mixture. This program will achieve the total chemo-enzymatic synthesis of LPS, and open the field to investigate the structure-activity relation of LPS with its TLR4-MD2 complex. Moreover, the reconstituted, synthetic core-lipid A structures are novel wide-spectrum and stand-alone vaccine candidates against urinary tract infection caused by uropathogenic E. coli.