Escherichia coli fatty acid synthesis

Wang, H., Cronan, J.E. 2004. Functional replacement of the FabA and FabB proteins of Escherichia coli fatty acid synthesis by Enterococcus faecalis FabZ and FabF homologues. J. Biot. Chem. 279 34489-34495. 

The second type of arrangement observed for sequentially related enzymes is exemplified by the Escherichia coli fatty acid synthase (see chapter 18). This synthase is a complex of most of the enzymes involved in fatty acid synthesis. The intermediates in this case are bound to the enzyme complex until synthesis is complete. 

All the reactions in the synthetic process are catalyzed by a multienzyme complex, fatty acid synthase. Although the details of enzyme structure differ in prokaryotes such as Escherichia coli and in eukaryotes, the four-step process of fatty acid synthesis is the same in all organisms. We first describe the process as it occurs in A1, coli, then consider differences in enzyme structure in other organisms. 

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in long-chain fatty acid biosynthesis (see Chapter 7.11). The overall reaction is catalyzed in two sequential reactions (Scheme 3). First, the biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin (which is attached to a carrier protein) using bicarbonate as a CO2 donor. In the second reaction, the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In mammals, both reactions are catalyzed by a single protein, but in Escherichia coli and other bacteria, the activity is catalyzed by two separate proteins, a biotin carboxylase and a carboxytransferase. Due to its role in fatty acid synthesis, inhibitors of the overall ACC reaction are proposed to be useful as antiobesity drugs in mammals as well as novel antibiotics against bacteria. 

The utility of this methodology is illustrated by the stereoselective synthesis of ( + )-cer-ulenin (759), an antifungal antibiotic first isolated from the culture filtrate of Cephalosporium caerulens. Its ability to inhibit lipid biosynthesis in Escherichia coli by irreversibly binding P-keto-acyl-carrier protein synthetase, the enzyme responsible for the chain lengthening reaction in fatty acid synthesis, has attracted interest in its mechanism of action. D-Tartaric acid... 

Naphthomycin-related compounds, named diastrovaricins I (63) and II (64), were isolated from the culture broth of Streptomyces diastochromogenes subsp. variabilicolor n. subsp. as new inducers of Friend mouse erythroleukemia cells [102]. In addition, naphthoquinomycins A (65), B (66) and C (67) were isolated as inhibitors of fatty acid synthesis in Escherichia coli [103]. Naphthoquinomycin C (67) was found to be identical with the antibiotic given the name naphthomycin H [104,105]. 

JanCen, H.J. and Steinbuchel, A. (2014) Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol. Biofuels, 7, 7. 

Fatty acid synthesis has been engineered as well. Two pathways to very long chain polyunsaturated fatty acids were realized in P. pastoris to demonstrate their feasibility for future reengineering in oilseed crops [163]. Fatty acids and their esters are also interesting potential biofuels. Fatty acid esters with branched chain alcohols are potential low-viscosity biodiesels, and were successfully synthesized in Escherichia coli and P. pastoris by metabolic engineering [164]. 

The 3-keto reductase step is equivalent to that found in fatty acid synthesis, although it occurs late (and only once) in this process. Indeed, thiol-bound acetoacetate proved inactive as a substrate for the aromatic complex, whereas it was reduced by fatty acid synthetase (Dimrothe/a/., 1972). Thus, the carbonyl group adjacent to the terminal methyl position is not susceptible to reduction by the aromatic synthetase, despite the apparent presence of a suitable reductase, possibly because it is held on the enzyme surface in an inappropriate enolic configuration (Packter, 1973). If so, it may not prove acceptable, since the 3-ketoacyl-acyl carrier protein (ACP) reductase from Escherichia coli only accepts keto substrates and does not react with or bind to the enol form of 3-ketoacyl derivatives (Schulz and Wakil, 1971). 

Amide bonds are found in many proteins. One is the acyl carrier protein of Escherichia coli (see 90), which contains the peptide backbone, and a 4 -phosphopantetheine unit (in violet in the illustration) is attached to a serine residue. Note the amine bonds in the pantothenic acid unit and also the 0-P=0 unit, which is a phosphate ester (an ester of phosphoric acid). An acyl carrier protein is involved in fatty acid synthesis, linking acetyl and malonyl groups from acetyl coenzyme A and malonyl coenzyme A to form P-keto acid acyl carrier protein (abbreviated as ACP). The widely utilized acetyl CoA is an ester (91) attached to coenzyme A. Acetyl CoA is a key intermediate in aerobic intermediary metabolism of carbohydrates, lipids, and some amino acids. 

Enoyl-ACP reductase(ER) catalyses the second reductive step during the cyclical reactions of fatty acid synthesis. Brassica napus NADH-dependent ER has been purified in milligram quantities from Escherichia coli harbouring an expression plasmid containing the plant cDNA. The availability of large quantities of protein has enabled successful crystallization[l] and elucidation of the complete structure of this enzyme[2]. In addition the supply of recombinant protein has allowed detailed kinetic investigations to be carried out. TTiis was not possible with the small quantities of protein available from rape seed material [3]. 

The two ketoacyl-synthetases present in the fatty-acid synthetase complexes of higher plants exhibit differential sensitivities against cerulenin. In plants the B-ketoacyl-synthetase I (de novo fatty acid synthesis) is affected by cerulenin, whereas the B-ketoacylsynthetase II, which catalyzes the elongation from 16 0 to 18 0 is relatively insensitive (Jaworski et al. 1974). The B-ketoacylsynthetase of Cephalosporium caerulens, which produces cerulenin, is much less sensitive than the yeast enzyme (Kawaguchi et al. 1979) and in Escherichia coli two sensitive and one insensitive synthetase were described (Jackowski and Rock 1987). 

Siggaard-Andersen, M. Role of Escherichia coli P-ketoacyl-ACP synthase I in unsaturated fatty acid synthesis. CarlsbergRes Commun 1988 53 371-379. 

Garwin JL, Cronan JE. Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis. J Bacteriol 1980 141 1457-1459. 

Marinari LA, Goldfine H, Panos C. Specificity of cyclopropane fatty acid synthesis in Escherichia coli. Utilization of isomers of monounsaturated fatty acids. Biochemistry 1974 13 1978-1983... 

Monson KD, Hayes JM (1980) Biosynthetic control of the natural abimdance of carbon 13 at specific positions within fatty acids in Escherichia coli. J Biol Chem 255 11435-11441 Monson KD, Hayes JM (1982a) Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes. Geochim Cosmochim Acta 46 139-149 Monson KD, Hayes JM (1982b) Biosynthetic control of carbon 13 at specific positions within fatty acids in Saccharomyces cerevisiae. Isotope fractionations in lipid synthesis as evidence for peroxisomal regulation. J Biol Chem 257 5568-5575... 

In Escherichia coli an acyl-acyl carrier protein synthetase that uses acyl carrier protein instead of CoA for fatty acid activation has been described (Ray and Cronan, 1976). The hydrocarbon utilizing yeast, Candida lipolyti-ca fabricates two distinct long chain acyl-CoA synthetases one of them activates fatty acids exclusively for lipid synthesis, while the other one does so for p-oxidation (Numa, 1981). Comparisons of the mitochondrial and microsomal long chain acyl-CoA synthetases of rat liver have shown, however, that the two enzymes are very similar (Philipp and Parsons, 1979 Tanaka et al., 1979). 

Rehm BHA, Mitsky TA, Steinbtichel A (2001) Role of fatty add de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by Pseudomonads establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli. Appl Environ Microbiol 67 3102-3109... 

Cell-free protein synthesis was performed using polydimethylsiloxane (PDMS)-based microreactor arrays [22]. The microreactor array chip comprised a temperature control chip made of glass and a disposable reaction chamber chip made of PDMS. To evaluate the performance of this microreactor array, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. 

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