Modification of Escherichia coli Lipid A by the Introduction of Myristoyltransferase Gene Cloned from Klebsiella pneumoniae
Keywords:
Lipid A, Fatty acid transferase, Mass spectrometry, IL-6, E. coliAbstract
Late stages of lipid A biosynthesis of Escherichia coli are transfer reactions of lauric acid (C12:0) and myristic acid (C14:0) to the hydroxyl group of 3-hydroxy-myristic acid (3-OH-C14:0). In the previous study we constructed the mutant strains with disrupted C12:0-transferase and C14:0-transferase genes and used those mutant strains for the modification of lipid A by the introduction of foreign acyltransferase genes. In the study reviewed here, the C14:0-transferase gene (lpxL2) of Klebsiella pneumoniae was cloned and introduced to the mutant strains by transformation to modify the lipid A structure. Lipopolysaccharide (LPS) preparations of the transformants were analyzed through chemical modification and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and were proved to have the lipid A with one C14:0, or two C14:0, one of which replaced C12:0 bound to 3-OH-C14:0 at the C2-position of the non-reducing end glucosamine. The IL-6-inducing activity of the LPS with C14:0 was measured, and compared with that of the original LPS with C12:0. The activity of LPS with C14:0 was found to be comparable with that of LPS with C12:0, suggesting that C14:0 can replace C12:0 without changing the immune-stimulating activity of lipid A.
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