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We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of\n                    <jats:italic>lptA<\/jats:italic>\n                    slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated\n                    <jats:italic>lpxL1<\/jats:italic>\n                    mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of\n                    <jats:italic>lpxP<\/jats:italic>\n                    , an enzyme known to add a secondary 9-hexadecenoic acid to the 2\u2019 acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.\n                  <\/jats:p>","DOI":"10.1038\/srep36575","type":"journal-article","created":{"date-parts":[[2016,11,14]],"date-time":"2016-11-14T05:14:48Z","timestamp":1479100488000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide"],"prefix":"10.1038","volume":"6","author":[{"given":"Afshin","family":"Zariri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elder","family":"Pupo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elly","family":"van Riet","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos P. 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