(56e) Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVa to Enable Structure-Activity Analysis
AIChE Annual Meeting
2021
2021 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Biomolecular Engineering and Analyses to Understand Cell Migration, Membrane Components, and Genomic Structure
Monday, November 8, 2021 - 9:12am to 9:30am
The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogs of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multi-enzymatic platform to access these structure-activity relation-ships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP-N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IVA, the first bioactive intermediate in the Lipid A pathway. Our reconstituted multi-enzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.
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