Unsaturated Fatty Acids

Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.

Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid was discovered 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway, in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin, were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase, followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and thus there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate protein.

This chapter focuses on the structure and regulation of the pathways utilized by various microbes (bacteria, algae, and yeasts) for the production of fatty acids and triacylglycerols (TAGs). An important observation with regard to the possibility of microbial biodiesel production was made when fatty acyl-ACP thioesterase (FAT) enzymes were overproduced in Escherichia coli. Thus far it represents the most efficient way to uncouple fatty acid formation from phospholipid and membrane biosynthesis in E. coli. Phosphatidic acid (PtdOH) is a key branching point in de novo lipid metabolism, and it is converted either to CDP-diacylglycerol (DAG) or DAG depending on the organism. CDP-DAG and DAG serve as intermediates in membrane phospholipid biosynthesis and, in addition, DAG is converted to TAG. Phosphatidate phosphatases (PAPs) in coordination with phospholipid-producing enzymes are key regulators of the flux of carbon towards TAGs. PAPs catalyze the conversion of PtdOH to DAG; the primary destination of DAG is the synthesis of membrane phospholipids, whereas excess DAG is directed towards TAG. The emerging theme from genome comparisons underlines the evolution of distinct regulatory mechanisms in various phylogenetic groups. All free-living organisms have the machinery to synthesize fatty acids, and conceptually, they could be exploited for biodiesel production. However, the photosynthetic organisms provide the unique opportunity to couple CO2 sequestration to lipid accumulation and subsequent biodiesel production.

The first portion of this chapter, entitled “Catabolic Pathways,” deals with the catabolism of amino acids and lipids, as they are the principal carbon and energy sources derived from prey bacteria. The second portion of the chapter, entitled “Anabolic Pathways,” highlights the synthesis of lipids because of their unusual chemical structures in myxobacteria, and also the spore-specific components trehalose and ether lipids. Most myxobacteria, including Myxococcus xanthus, can catabolize prey microorganisms. M. xanthus utilizes amino acids and lipids as carbon and energy sources, incorporates purines and pyrimidines via salvage pathways, and fails to utilize sugars. In most cases there is excellent agreement between the presence of a particular amino acid catabolic pathway and the ability of that amino acid to stimulate growth in defined and minimal media. Lipid oxidation has been demonstrated by 14C-labeling experiments in Myxococcus virescens and methyloleate feeding in M. xanthus. Fatty acids are usually degraded by β oxidation, where two carbon acetate units are sequentially removed from the carboxyl end of the fatty acid, also known as the Δ terminus, as opposed to the methyl end or ω terminus. Monosaccharides are used for exopolysaccharide, peptidoglycan, and lipopolysaccharide biosynthesis. In Escherichia coli, trehalose is degraded to glucose by a periplasmic trehalase; no homolog exists in M. xanthus.

This chapter focuses on the remarkable similarity of cells, whether the cell is a one-celled organism, such as a bacterium, or a highly specialized cell in a multicellular organism. All cells share certain basic features: they have molecular machinery for duplicating DNA and for breaking down and synthesizing molecules, they reproduce by dividing in two, they use the same molecular building blocks, and they are enclosed by a hydrophobic membrane that separates the cell from its surroundings. All biological phenomena are based on chemical interactions, so understanding the cellular processes described throughout the chapter requires a basic understanding of cell chemistry. Biotechnology is based on the use of living cells and their component parts. Therefore, knowing something about the structure and function of cells and the molecules they contain is essential to understanding biotechnology’s scientific foundations, potential applications, and possible limitations. All living cells carry out a number of essential processes that are the defining traits of life. Cells grow and reproduce, maintain their internal environments, respond to the external environment, and communicate with each other. Cellular processes can be reduced to a series of chemical reactions, most of which are catalyzed by enzymes. To carry out all of these processes, cells require a constant supply of energy. Finally, cells also regulate their processes to ensure they are carried out in an orderly and efficient way.

This chapter talks about membrane adaptations, with emphasis on the adaptive changes occurring in prokaryotes; where relevant, the distinctive changes in eukaryotes will be compared. Genotypic adaptation refers to adaptive changes on an evolutionary time scale (usually longer than that for phenotypic adaptation), which involve an alteration in genetic structure, i.e., mutations occur and are positively selected if favorable to become established as part of the genome. Of particular relevance to the membrane adaptations of psychrophiles are the phenotypic and genotypic adaptations in lipid composition (the cellular “lipiome”), for which there is much information. Like the membranes of higher organisms, those of microorganisms are comprised mainly of proteins and lipids, together with a smaller amount of carbohydrate in the form of glycoproteins, glycolipids, or other molecules, organized as described originally in the Fluid-Mosaic Model of membrane structure. The presence of lipids that have a tendency to form non-bilayer phases gives a certain tension to the membrane and may be important in helping to drive processes such as sporulation and cell division that involve segregation of membranes. Microorganisms modify their membrane lipid fatty acyl composition in response to thermal changes by altering unsaturation, (methyl) branching, or chain length. The trans-unsaturated fatty acids are synthesized by direct and non-reversible isomerization of cisunsaturated fatty acyl chains without a saturated intermediate. The gene for the cis/trans isomerase enzyme has been cloned and the enzyme purified. Anteiso-branched fatty acids seem to be particularly associated with growth at low temperatures.

This chapter summarizes the knowledge about the genomes of psychrophilic bacteria, a subclass of the cold-adapted bacteria, with emphasis on the specific selective features relevant to cold adaptation. A detailed analysis of the general features of genomes and proteomes from psychrophilic bacteria is presented. All investigators involved in sequencing the genomes of psychrophilic Bacteria looked for common features which would account for cold-adaptation. The genomes of psychrophilic bacteria also have the counterpart of major chaperonins such as the essential GroES GroEL complex. A remarkable observation poses interesting questions about the role of this complex. In the presence of molecular oxygen (dioxygen), this has the consequence that reactive oxygen species (ROS) are more frequent and stable for a longer time. Membrane fluidity can be increased in two ways: either by incorporating unsaturated fatty acids or by including branched-chain fatty acids in the diglycerides. Photobacterium profundum SS9 was found to exhibit enhanced proportions of both monounsaturated and polyunsaturated fatty acids when grown at a decreased temperature or elevated pressure. Three main features can be observed in the genomes and proteomes of these organisms: a variety of means to cope with ROS, a multiplicity of nucleic acid folding and unfolding devices, and, finally, a bias in the amino acid composition of their proteome.

Antimicrobials are classified as traditional when they (i) have been used for many years, (ii) are approved by many countries for inclusion as antimicrobials in foods (e.g., lysozyme and lactoferrin, which are naturally occurring but regulatory-agency approved), or (iii) are produced by synthetic means (as opposed to natural extracts). Many organic acids are used as food additives, but not all have antimicrobial activity. Research suggests that the most active are acetic, lactic, propionic, sorbic, and benzoic acids. Acetic acid was the most effective antimicrobial in ground roasted beef slurries against Escherichia coli O157:H7 growth in comparison with citric or lactic acid. Sorbate is applied to foods by direct addition, dipping, spraying, dusting, or incorporation into packaging. The mechanism by which dimethyl dicarbonate (DMDC) acts is most likely related to inactivation of enzymes. A related compound, diethyl dicarbonate, reacts with imidazole groups, amines, or thiols of proteins. Lysozyme is most active against gram-positive bacteria, most likely because the peptidoglycan of the cell wall is more exposed. The primary use for sodium nitrite as an antimicrobial is to inhibit Clostridium botulinum growth and toxin production in cured meats. Sulfites may be used to inhibit acetic acid-producing bacteria, lactic acid bacteria, and spoilage bacteria in meat products. In the future, traditional food antimicrobials will continue to play an important role in food preservation.

The tricarboxylic acid (TCA) cycle plays two essential roles in metabolism. First, under aerobic conditions the cycle is responsible for the total oxidation of acetyl-CoA that is derived mainly from the pyruvate produced by glycolysis. Second, TCA cycle intermediates are required in the biosynthesis of several amino acids. Although the TCA cycle has long been considered a “housekeeping” pathway in Escherichia coli and Salmonella enterica, the pathway is highly regulated at the transcriptional level. Much of this control is exerted in response to respiratory conditions. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although a few loose ends remain. The realization that a “shadow” TCA cycle exists that proceeds through methylcitrate has cleared up prior ambiguities. The glyoxylate bypass has long been known to be essential for growth on carbon sources such as acetate or fatty acids because this pathway allowsnet conversion of acetyl-CoA to metabolic intermediates. Strains lacking this pathway fail to grow on these carbon sources, since acetate carbon entering the TCA cycle is quantitatively lost as CO2 resulting in the lack of a means to replenish the dicarboxylic acids consumed in amino acid biosynthesis. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although the identity of the small molecule ligand that modulates transcriptional control of the glyoxylate cycle genes by binding to the IclR repressor remains unknown. The activity of the cycle is also exerted at the enzyme level by the reversible phosphorylation of the TCA cycle enzyme isocitrate dehydrogenase catalyzed by a specific kinase/phosphatase to allow isocitratelyase to compete for isocitrate and cleave this intermediate to glyoxylate and succinate.

This review concerns the uptake and degradation of those molecules that are wholly or largely converted to acetyl-coenzyme A (CoA) in the first stage of metabolism in Escherichia coli and Salmonella enterica. These include acetate, acetoacetate, butyrate and longer fatty acids in wild type cells plus ethanol and some longer alcohols in certain mutant strains. Entering metabolism as acetyl-CoA has two important general consequences. First, generation of energy from acetyl-CoA requires operation of both the citric acid cycle and the respiratory chain to oxidize the NADH produced. Hence, acetyl-CoA serves as an energy source only during aerobic growth or during anaerobic respiration with such alternative electron acceptors as nitrate or trimethylamine oxide. In the absence of a suitable oxidant, acetyl-CoA is converted to a mixture of acetic acid and ethanol by the pathways of anaerobic fermentation. Catabolism of acetyl-CoA via the citric acid cycle releases both carbon atoms of the acetyl moiety as carbon dioxide and growth on these substrates as sole carbon source therefore requires the operation of the glyoxylate bypass to generate cell material. The pair of related two-carbon compounds, glycolate and glyoxylate are also discussed. However, despite having two carbons, these are metabolized via malate and glycerate, not via acetyl-CoA. In addition, mutants of E. coli capable of growth on ethylene glycol metabolize it via the glycolate pathway, rather than via acetyl- CoA. Propionate metabolism is also discussed because in many respects its pathway is analogous to that of acetate. The transcriptional regulation of these pathways is discussed in detail.