In vitro reconstitution of the escherichia coli lipid IVa biosynthetic pathway [electronic resource] : enzymatic synthesis of lipid A analogues

Lipid A is not only the hydrophobic moiety of lipopolysaccharide (LPS), a major constituent of the Gram-negative bacterial outer membrane, but it is also one of the most potent small molecule elicitors of the eukaryotic innate immune responses. As part of the outer most defense layer of a living cell, lipid A takes part in protecting the bacteria from harmful compounds and host environment adaptation. Pertinent to clinical implications, lipid A contributes to antibiotic resistance and colonization resistance in gut microbiota. To initiate innate immune responses, lipid A binds to myeloid differentiation factor 2 (MD2) and toll-like receptor 4 (TLR-4) complex with nanomolar affinity. This recognition event triggers signaling cascades that produce pro-inflammatory cytokines and interferons, the latter of which promote T-cell activation. In the past two decades, a limited number of lipid A analogues have been exploited for potential uses as new therapeutics. For example, monophosphoryl lipid A (MPLA) was approved by United States Food and Drug Administration as a vaccine adjuvant. Although lipid A constitutes the majority part of bacterial cell surface, it is challenging to perform structural-activity-relationship (SAR) studies on the biological activities of lipid A molecules for several reasons. First of all, not all Gram-negative bacteria can be cultivated in the laboratory. Furthermore, acquiring lipid A from bacterial hosts requires complex isolation and purification processes. Nevertheless, commercially available lipid A and its analogues are often contaminated with glycolipids, and had led to conflicting results and irreproducible data. Hence, the lack of appropriate laboratory tools limit the ability of scientific community to acquire a structurally defined library of lipid A compounds for SAR studies. To address to those challenges, this dissertation provides a novel approach to produce structurally defined lipid A analogues. Biosynthesis of lipid A requires a joint action of two cellular pathways --fatty acid biosynthesis and lipid A biosynthesis. In the first part of this dissertation, we report successful reconstitution of Escherichia coli acetyl-CoA carboxylases and fatty acid synthases in vitro. The reconstituted system is capable of synthesizing a variety of fatty acids using acetyl-CoA and sodium bicarbonate as substrates. We examine in detail the degree to which this system can produce unsaturated fatty acids at a maximum turnover rate of 1 s-1, and identify a determining factor that controls the unsaturated fatty acid biosynthesis. The second part of this dissertation is dedicated to reconstituting the Escherichia coli lipid A biosynthetic pathway. Thus far, we have functionally reconstituted two primary metabolic pathways that contain 19 enzymes, which itself is unprecedented. This reconstituted system synthesizes lipid A precursor, lipid IVA, which is also a biologically active immune modulator. Although earlier literature reported that lipid A isolated from E. coli contained only saturated primary fatty acyl moieties, we demonstrate the acyl-transferases lack specificity for unsaturated versus saturated acyl chains. This observation leads to the important question as to why and how cells control the exclusive incorporation of saturated fatty acyl moieties in vivo. Finally, by exposing mammalian macrophages with the lipid IVA analogues produced in vitro, we verify that our reconstituted system does indeed produce biologically active compounds.

Keywords: Chemistry.
Advisor: Khosla Chaitan
Chen James Kenneth advisor. Thesis advisor
Kool Eric T. advisor. Thesis advisor