Defined enzymatic synthesis of lipid A analogs
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsXirui Xiao, Chaitan Khosla
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Abstract
Provided herein include methods and compositions for synthesis of Lipid IVA and derivatives thereof, using a defined set of pathway enzyme, which may be isolated and used to reconstitute all or part of the pathway in a cell-free reaction.
Claims13
A method of synthesizing a compound represented by one of the following structures:
- the method comprising:
- combining in a reaction mixture each of the following sets of enzymes:
- Module A:
- Acetyl-CoA carboxylases;
- Module B:
- chain initiation enzymes of fatty acid synthases;
- Module C:
- chain elongation enzymes of fatty acid synthases;
- Module D:
- Lipid A biosynthetic enzymes;
- in the presence of Acetyl-coenzyme A (CoA) or an analog thereof including, UDP-N-acetyl-D-glucosamine (GlcNAc), and cofactors and buffers required for synthesis.
A method of synthesizing a compound represented by one of the following structures:
- the method comprising:
- combining in a reaction mixture the following sets of enzymes:
- Module A, comprised of Acetyl-CoA carboxylase C (AccC), Acetyl-CoA carboxylase A (AccA), Acetyl-CoA carboxylase D (AccD), and holo-Acetyl-CoA carboxylase B (AccB);
- Module B comprised of Fatty Acid Biosynthesis H (FabH), Fatty Acid Biosynthesis D (FabD), Fatty Acid Biosynthesis G (FabG), Fatty Acid Biosynthesis A (FabA) or Fatty Acid Biosynthesis Z (FabZ), Fatty Acid Biosynthesis I (FabI), and holo-Acyl Carrier Protein (ACP);
- Module C, comprised of Fatty Acid Biosynthesis B (FabB) or Fatty Acid Biosynthesis F (FabF), FabD, FabG, FabA or FabZ, and FabI;
- and Module D, comprised of UDP-N-acetylglucosamine acyltransferase (LpxA), UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC), UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase UDP-2,3-diacylglucosamine hydrolase (LpxH), Lipid-A-disaccharide synthase (LpxB), and tetraacyldisaccharide 4′-kinase (LpxK), in the presence of Acetyl-CoA or an analog thereof including, UDP-GlcNAc, and cofactors and buffers required for synthesis.
A method of synthesizing a compound represented by one of the following structures:
- the method comprising:
- combining in a reaction mixture the following sets of enzymes:
- Module B comprised of FabH, FabD, FabG, FabA or FabZ, FabI, and holo-ACP;
- Module C, comprised of FabB or FabF, FabD, FabG, FabA or FabZ, and FabI;
- and Module D, comprised of LpxA, LpxC, LpxD, LpxH, LpxB, and LpxK, in the presence of malonyl-CoA, Acetyl-CoA or an analog thereof including, UDP-GlcNAc, and cofactors and buffers required for synthesis.
A method of synthesizing a compound represented by one of the following structures:
- the method comprising:
- combining in a reaction mixture the following sets of enzymes:
- Module C, comprised of FabB or FabF, FabD, FabG, FabA or FabZ, and FabI;
- and Module D, comprised of LpxA, LpxC, LpxD, LpxH, LpxB, and LpxK, in the presence of butyryl-ACP thioester, malonyl-CoA, UDP-GlcNAc, and cofactors and buffers required for synthesis.
A method of synthesizing a compound represented by one of the following structures:
- the method comprising:
- combining in a reaction mixture the following set of enzymes:
- Module D, comprised of LpxA, LpxC, LpxD, LpxH, LpxB, and LpxK, in the presence of 3-hydroxymyristoyl-ACP intermediate and UDP-N-acetylglucosamine (UDP-GlcNAc), and cofactors and buffers required for synthesis.
Dependencies are inferred from the claim text.