# Lipopolysaccharide: Recent Advances in Its Biosynthesis and Controlling Cell Envelope Homeostasis
###### Abstract  
Typical Gram-negative bacteria, such as _Escherichia coli_, are diderms with two membrane bilayers separated by a periplasmic space containing a thin layer of peptidoglycan (PGN). Lipopolysaccharide (LPS) is the major component of the outer membrane (OM), whereas phospholipids (PL) are the main lipid component of the inner membrane (IM). The most defining feature of Gram-negative bacteria is the asymmetric nature of their OM, as they contain PL facing the inner leaflet and LPS located in the outer leaflet. LPS constitutes the major amphiphilic component of the OM. Bacteria, such as _E. coli_ and _Salmonella_, contain approximately 2-3 \(\times\) 10\({}^{6}\) molecules of LPS that cover more than 75% of the OM [1]. The OM is essential for the viability of Gram-negative bacteria, with few exceptions. The maintenance of this asymmetry is crucial for bacterial cell envelope integrity, as it imparts a permeability barrier. Structurally, LPS can be divided into three elements: the most conserved part is lipid A, to which a core polysaccharide is attached, which is further extended in smooth bacteria by the \(O\)-antigen. The biosynthesis of LPS begins with the acylation of UDP-GlcNAc with \(R\)-3-hydroxynistoyl-ACP by LpxA and proceeds in a discontinuous manner. The second reaction of lipid A biosynthesis is catalyzed by Lpxc [UDP-3-O-(\(R\)-3-hydroxynistoyl)-\(N\)-acetylglucosamine deacetylase], constituting the first committed step in the LPS synthesis, as the equilibrium constant for the first reaction catalyzed by LpxA is unfavorable [2]. Following the deacetylation step, four additional enzymes, LpxD, LpxH, LpxB, and LpxK, act successively to generate the lipid IV\({}_{\text{A}}\) intermediate [3]. In _E. coli_, all six enzymes are essential and, being unique to bacteria, are targets for new antimicrobials. Next, two Kdo residues are added to the lipid IV\({}_{\text{A}}\) precursor, which enables the addition of secondary lauroyl and myristoyl chains, resulting in the synthesis of Kdo2-lipid A, which constitutes the hydrophobic anchor of LPS [4]. The hexaacylated lipid A part of LPS constitutes the endotoxin principal, as it is highly immunogenic and is recognized by the innate immune cell receptor TLR4/MD2-CD14 complex [5]. LPS can also be recognized in a TLR4/MD2-independent manner, involving recognition by caspases 4/5 in humans and 11 in mice [6]. The biosynthesis of the three main essential cell envelope components, PGN, LPS, and PL, is intricately linked. Thus, the enzymatic activities of glucosamine-6-phosphate synthase GimS and deacetylase LpxC control the biosynthetic branch points using two key precursor molecules, UDP-\(N\)-acetyl-D-glucosamine (UDP-GlcNAcAc) and \(R\)-3-hydroxynistoyl acyl carrier protein (\(R\)-3-hydroxynistoyl-ACP), respectively [4]. UDP-GlcNAcNc is used as a substrate to initiate LPS synthesis via the LpxA/LpxC pathway and PGN biosynthesis by recruiting MurA/MurB enzymes. UDP-GlcNAcNc is at the same time used as a key substrate for enterobacterial common antigen (ECA) biosynthesis. Bacteria synthesize fatty acids using the FASII system [7]. Fatty acid biosynthesis is highly regulated at this initial,rate-limiting step. The first committed step of fatty acid synthesis is the carboxylation of acetyl-CoA by the acetyl-CoA carboxylase enzyme complex (comprising AccA, B, C, and D components) to produce malonyl-CoA [8]. Next, malonyl-CoA is converted to malonyl-ACP by FabD. FabH initiates fatty acid synthesis by catalyzing the Claisen condensation of acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP [9]. From here, the acyl-ACP is elongated by two carbons per cycle by elongation enzymes. Acyl-ACP of sufficient length is used by the acyltransferase system to produce phosphatidic acid from glycerol-3-phosphate. In _E. coli_, the acyltransferases PlsB and PlsC utilize long-chain acyl-ACPs to transfer acyl chains to the 1 st and 2 nd carbons of a glycerol phosphate moiety to generate phosphatidic acid [10].