Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Microbial lipid metabolism is an attractive route for producing aliphatic chemicals, commonly referred to as oleochemicals. The predominant metabolic engineering strategy centers on heterologous thioesterases capable of producing fatty acids of desired size. To convert acids to desired oleochemicals (e.g. fatty alcohols, ketones), metabolic engineers modify cells to block beta-oxidation, reactivate fatty acids as coenzyme-A thioesters, and redirect flux towards termination enzymes with broad substrate utilization ability. These genetic modifications narrow the substrate pool available for the termination enzyme but cost one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. In this work, we demonstrate an alternative acyl-transferase strategy for producing medium-chain oleochemicals. Through bioprospecting, mutagenesis, and metabolic engineering, we developed strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones using the transacylase strategy.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

Purification and properties of lysophospholipase isoenzymes from pig gastric mucosa

Two lysophospholipases, named gastric lysophospholipases I and II (enzymes I and II), were purified 3730- and 2680-fold from pig gastric mucosa. The preparations showed 22 and 23 kDa single protein bands on SDS/PAGE respectively. Both enzymes lacked transacylase activity and appeared to exist as monomers. Their activities were not affected by Ca2+, Mg2+ or EDTA. Enzyme I was most active at pH 8.5 and hydrolysed a variety of lysophospholipids including acidic lysophospholipids and the acyl analogue of platelet-activating factor, whereas enzyme II was most active at pH 8 and its activity was confined to lysophosphatidylcholine and lysophosphatidylethanolamine. When 1-palmitoylglycerophosphocholine was used as substrate, enzymes I and II showed half-maximal activities at 11 and 12 microM respectively. The enzymes exhibited no phospholipase B, lipase or general esterase activity. Enzyme II was significantly inhibited by lysophosphatidic acid whereas enzyme I was only moderately inhibited. Peptide mapping with V8 protease and papain revealed structural dissimilarity between the two enzymes. Antiserum raised against enzyme I did not recognize enzyme II, but did recognize the small-sized lysophospholipase purified from rat liver. Anti-(enzyme II) consistently did not cross-react with enzyme I or the liver enzyme. These antisera specifically recognized neither the 60 kDa lysophospholipase transacylase purified from liver nor any peritoneal macrophage protein. Thus gastric mucosa contains two different small-sized lysophospholipases: one is closely related to the small-sized lysophospholipase of liver, but the other appears to be a novel isoform.