Primary Sequence of the \u3ci\u3eEscherichia coli fadBA\u3c/i\u3e Operon, Encoding the Fatty Acid-Oxidizing Multienzyme Complex, Indicates a High Degree of Homology to Eucaryotic Enzymes

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in α2β2 conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon. The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length. The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively. Recently, the sequence of thefadA gene was published in a separate report (Yang et al., J. Biol. Chem. 265:10424-10429, 1990). In this work, most of the DNA sequence for fad4 was confirmed, and 10 errors were corrected. Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide. By comparison to other peptide sequences, the a subunit encoded withinfadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides. In agreement with the work of Yang et al., the β subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide

Primary Sequence of the \u3ci\u3eEscherichia coli fadBA\u3c/i\u3e Operon, Encoding the Fatty Acid-Oxidizing Multienzyme Complex, Indicates a High Degree of Homology to Eucaryotic Enzymes

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in α2β2 conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon. The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length. The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively. Recently, the sequence of thefadA gene was published in a separate report (Yang et al., J. Biol. Chem. 265:10424-10429, 1990). In this work, most of the DNA sequence for fad4 was confirmed, and 10 errors were corrected. Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide. By comparison to other peptide sequences, the a subunit encoded withinfadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides. In agreement with the work of Yang et al., the β subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide

Nucleotide sequence of the \u3ci\u3efadR\u3c/i\u3e gene, a multifunctional regulator of fatty acid metabolism in \u3ci\u3eEscherichia coli\u3c/i\u3e

The Escherichia coli fadR gene is a multifunctional regulator of fatty acid and acetate metabolism. In the present work the nucleotide sequence of the 1.3 kb DNA fragment which encodes FadR has been determined. The coding sequence of the fadR gene is 714 nucleotides long and is preceded by a typical E. coli ribosome binding site and is followed by a sequence predicted to be sufficient for factor-independent chain termination. Primer extension experiments demonstrated that the transcription of the fadR gene initiates with an adenine nucleotide 33 nucleotides upstream from the predicted start of translation. The derived fadR peptide has a calculated molecular weight of 26,972. This is in reasonable agreement with the apparent molecular weight of 29,000 previously estimated on the basis of maxi-cell analysis of plasmid encoded proteins. There is a segment of twenty amino acids within the predicted peptide which resembles the DNA recognition and binding site of many transcriptional regulatory proteins

COMPOUNDS FOR INCREASING LIPID SYNTHESIS AND STORAGE

This invention relates to methods for increasing lipid production in cells. Methods of producing biofuel from cells and preparing mutraceuticals comprising lipids produced according to a method provided herein are also provided

Effects of Lipid Activating Chemical Compounds on the Growth and Production of Fatty Acids and Metabolites in Green Algae

This study examines the effects of small molecule lipid activating chemical compound, discovered from the high throughput screening (HTS) (2) methods of previous FATTT Lab studies on the growth and production of lipids and metabolites in microalgae. For this study, the effects of two lipid inducing chemical compounds from the HTS method were implemented with the microalgae strain Chlamydomonas reinhardtii CC-125. This experiment focused on scaling up to large amounts of culture, approximately 1L. These cultures were grown in specially designed large bioreactors that were able to accommodate for such large volume of algal culture. Algal cells were treated with 10 μM concentration of the chemical compounds at initial time of inoculation. A control set was also implemented to be compared against the treatment conditions. Daily samples of algae cultures were taken in order to analyze growth on a time course-based method in addition. After five days, algae cultures were harvested completely, spun down into pellet form to be freeze dried vacuum by lyophilizer machine. The dried biomass was then recorded and used to carry out analytical techniques to quantify total lipids and metabolites of the algal cells

Cloning and Characterization of a Gene (fadR) Involved in Regulation of Fatty Acid Metabolism in \u3ci\u3eEscherichia coli\u3c/i\u3e

The regulatory gene fadR has been previously characterized by classical genetic means as a diffusible protein which exerts negative control over fatty acid degradation and acetate metabolism. fadR has also been implicated in the regulation of unsaturated fatty acid biosynthesis. To facilitate the identification of the product of the fadR gene and to study the mechanism by which this multifunctional regulatory gene exerts its control, we cloned a segment of DNA containing the fadR gene in the phage vector λL47. Subsequent subcloning of a segment of the chromosomal DNA from the λfadR+ phage into various plasmid vectors resulted in the isolation of the fadR gene on a 1.3-kilobase-pair HindIII-EcoRV fragment. fadR strains harboring the clonedfadR\u27 gene showed inducible levels of fatty acid oxidation and crotonase (enoyl-coenzyme A-hydratase, fadB) activity. The cloned gene exerted transcriptional control over 13-galactosidase synthesis in an fadR strain that had a λΦ(fadE-lacZ+) operon fusion. An fadR mutation in fabA(Ts) strains prevents growth at permissive temperatures without unsaturated fatty acid supplementation (Nunn et al., J. Bacteriol. 154:554-560, 1983). Plasmids carrying the fadR+ gene suppress this unsaturated fatty acid auxotrophy in fadR fabA(Ts) strains at the permissive condition. Maxiceli analysis identified a 29,000-dalton protein encoded by the 1.3-kilobase fragment which appeared to be associated with functional fadR gene activity

Cloning and Characterization of a Gene (fadR) Involved in Regulation of Fatty Acid Metabolism in \u3ci\u3eEscherichia coli\u3c/i\u3e

The regulatory gene fadR has been previously characterized by classical genetic means as a diffusible protein which exerts negative control over fatty acid degradation and acetate metabolism. fadR has also been implicated in the regulation of unsaturated fatty acid biosynthesis. To facilitate the identification of the product of the fadR gene and to study the mechanism by which this multifunctional regulatory gene exerts its control, we cloned a segment of DNA containing the fadR gene in the phage vector λL47. Subsequent subcloning of a segment of the chromosomal DNA from the λfadR+ phage into various plasmid vectors resulted in the isolation of the fadR gene on a 1.3-kilobase-pair HindIII-EcoRV fragment. fadR strains harboring the clonedfadR\u27 gene showed inducible levels of fatty acid oxidation and crotonase (enoyl-coenzyme A-hydratase, fadB) activity. The cloned gene exerted transcriptional control over 13-galactosidase synthesis in an fadR strain that had a λΦ(fadE-lacZ+) operon fusion. An fadR mutation in fabA(Ts) strains prevents growth at permissive temperatures without unsaturated fatty acid supplementation (Nunn et al., J. Bacteriol. 154:554-560, 1983). Plasmids carrying the fadR+ gene suppress this unsaturated fatty acid auxotrophy in fadR fabA(Ts) strains at the permissive condition. Maxiceli analysis identified a 29,000-dalton protein encoded by the 1.3-kilobase fragment which appeared to be associated with functional fadR gene activity

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga \u3ci\u3eCoccomyxa subellipsoidea\u3c/i\u3e C169

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions

Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae \u3ci\u3eCoccomyxa subellipsoidea\u3c/i\u3e

Triglyceride (TAG) synthesis during nitrogen starvation and recovery was addressed using Coccomyxa subellipsoidea by analyzing acylchain composition and redistribution using a bioreactor-controlled time course. Galactolipids, phospholipids and TAGs were profiled using liquid chromatography tandem mass spectroscopy (LC–MS/MS). TAG levels increased linearly through 10 days of N starvation to a final concentration of 12.6% dry weight (DW), while chloroplast membrane lipids decreased from 5% to 1.5% DW. The relative quantities of TAG molecular species, differing in acyl chain length and glycerol backbone position, remained unchanged from 3 to 10 days of N starvation. Six TAG species comprised approximately half the TAG pool. An average of 16.5% of the acyl chains had two or more double bonds consistent with their specific transfer from membrane lipids to TAGs during N starvation. The addition of nitrate following 10 days of N starvation resulted in a dramatic shift from chloroplast-derived to endoplasmic reticulum-derived galactolipids (from \u3c12% to \u3e40%). A model for TAG synthesis in C. subellipsoidea was developed based on the acquired data and known plant pathways and data presented

Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae \u3ci\u3eCoccomyxa subellipsoidea\u3c/i\u3e

Triglyceride (TAG) synthesis during nitrogen starvation and recovery was addressed using Coccomyxa subellipsoidea by analyzing acylchain composition and redistribution using a bioreactor-controlled time course. Galactolipids, phospholipids and TAGs were profiled using liquid chromatography tandem mass spectroscopy (LC–MS/MS). TAG levels increased linearly through 10 days of N starvation to a final concentration of 12.6% dry weight (DW), while chloroplast membrane lipids decreased from 5% to 1.5% DW. The relative quantities of TAG molecular species, differing in acyl chain length and glycerol backbone position, remained unchanged from 3 to 10 days of N starvation. Six TAG species comprised approximately half the TAG pool. An average of 16.5% of the acyl chains had two or more double bonds consistent with their specific transfer from membrane lipids to TAGs during N starvation. The addition of nitrate following 10 days of N starvation resulted in a dramatic shift from chloroplast-derived to endoplasmic reticulum-derived galactolipids (from \u3c12% to \u3e40%). A model for TAG synthesis in C. subellipsoidea was developed based on the acquired data and known plant pathways and data presented