Unsaturated Fatty Acid Requirement Research Articles

Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis

Considering the requirement for unsaturated fatty acids (UFAs) to produce phospholipids, we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence, UASINO, found in many phospholipid biosynthetic genes, and is the target of regulation by Ino2p, Ino4p and Opi1p. We then examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in an opi1 mutant. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the opi1 mutant. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2 hybrid assay was used to examine potential interactions between Opi1p and Ole1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p. While no unexpected Opi1p interactions were detected, we observed that Ino4p interacts with Mga2p in this assay. This preliminary work indicates that phospholipid regulators also play a role in regulating OLE1 expression.Support or Funding InformationWe acknowledge the support of the Hope College Biology Department, and the Hope College Dean of Natural and Applied Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Strategies for further development of aquatic feeds

: To date, aquatic feeds have depended heavily on fishmeal and fish oil as their source of protein and lipid. However, the feed industry is encountering shortfalls in the availability of these ingredients because of a decline in the number of fish captured in the wild and the increased human demand for some of the species currently being used for fishmeal and oil production. Therefore, efforts are now being directed in different parts of the world to finding alternative quality ingredients, which ideally are less expensive and readily available for use in practical diets. The data accrued have shown that a large proportion of both fishmeal and fish oil can be replaced by other protein and lipid sources. However, it is emphasized that an optimal essential amino acid balance be maintained and that the n-3 highly unsaturated fatty acid requirement be satisfied when combining economical protein and lipid ingredients. Newly developed feeds should aim at being nutrient-dense in order to reduce the output of solid, P and N waste. This can be done through improving nutrient availability, optimizing the digestible protein to energy balance of the diet, and replacing dietary fishmeal with alternate ingredients. These diets should also be effective for maintaining good health and improving disease resistance in fish through enhancement of immune responses. A wholesome approach to culturing fish would be to use appropriate feeding standards that are aimed not only at improving economic returns but also at developing a lasting cohabitation of sustainable aquaculture and a cleaner environment. Furthermore, in the 2lst century aquaculture would still retain its place as a prominent source of food protein, signifying that fish feed research remains a forceful area in discovering better feeds for the industry.

Cloning, sequencing, and functional analysis of H-OLE1 gene encoding delta9-fatty acid desaturase in Hansenula polymorpha.

H-OLE1, a gene encoding delta9-fatty acid desaturase (FAD) in Hansenula polymorpha strain CBS 1976, was isolated by hybridization based upon its homology with the P-OLE1 gene cloned earlier from a related species, Pichia angusta IFO 1475. The sequence of the H-OLE1 gene revealed high structural conservation with delta9-FADs from various organisms. A putative 451-amino acid polypeptide encoded by the gene, like all other delta9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidine clusters, and a C-terminal cytochrome b5-like domain which has been suggested to be involved in electron transport in desaturation reactions. The whole H-OLE1 gene complemented a H. polymorpha fad1 mutation leading to a defect in delta9-FAD. However, the unsaturated fatty acid requirement that the Saccharomyces cerevisiae ole1 mutant displays was complemented by only the open reading frame of H-OLE1 driven by S. cerevisiae glyceroaldehyde-3-phosphate dehydrogenase promoter, but not by the intact H-OLE1, suggesting that the H. polymorpha delta9-FAD was compatible with the desaturation system of S. cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells. It was shown by Northern hybridization that transcription of the H-OLE1 gene in H. polymorpha was slightly repressed by exogenous delta9-unsaturated fatty acid. An H. polymorpha disruption mutant (deltaH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupted H-OLE1::S-LEU2 linear DNA. It was shown by genetic and molecular analyses that input DNA was integrated in several copies into the chromosomal target to replace the mutated fad1 allele. Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and deltaHOLE1 disruption mutants.

Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis.

A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus.

Caulobacter crescentus fatty acid-dependent cell cycle mutant.

A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.

Unsaturated fatty acid requirement inEscherichia coli: Mechanism of palmitate-induced inhibition of growth of strain WN1

The minimum requirement for unsaturated fatty acids was investigated in E. coli using a mutant impaired in the synthesis of vaccenic acid. Exogenously supplied palmitic acid was incorporated by this mutant which led to a reduction in the proportion of cellular unsaturated fatty acids. Growth was impaired as the level of saturated fatty acids approached 76% at 37 degree C and 60% at 30 degree C. The basis of this growth inhibition was investigated. Most transport systems and enzymes examined remained active in palmitate-grown cells although the specific activities of glutamate uptake and succinic dehydrogenase were depressed 50%. Fluorescent probes of membrane organization indicated that fluidity decreased with palmitate incorporation. Temperature scans with parinaric acid indicated that rigid lipid domains exist in palmitate-grown cells at their respective growth temperature. Freeze-fracture electron microscopy confirmed the presence of phase separations (particle-free areas) in palmitate-grown cells held at their growth temperature prior to quenching. The extent of this separation into particle-free and particle-enriched domains was equivalent to that induced by a shift to 0 degree C in control cells. The incorporation of palmitate increased nucleotide leakage over threefold. The cytoplasmic enzyme beta-galactosidase was released into the surrounding medium as the concentration of unsaturated fatty acid approached the minimum for a particular growth temperature. Lysis was observed as a decrease in turbidity when cells which had been grown with palmitate were shifted a lower growth temperature. From these results we propose that leakage and partial lysis are the major factors contributing to the apparent decrease in growth rate caused by the excessive incorporation of palmitate. Further, we propose that membrane integrity may determine the minimum requirement for unsaturated fatty acids in E. coli rather than a specific effect on membrane transport and/or membrane-bound enzymes.

Mutations resulting in an unsaturated fatty acid requirement in Neurospora. Evidence for delta9-desaturase defects.

Mutations resulting in an unsaturated fatty acid requirement in Neurospora. Evidence for delta9-desaturase defects.

Quantitative effects of unsaturated fatty acids in microbial mutants. VI. Selective growth responses of yeast and bacteria to cis-octadecenoate isomers.

The full series of positional isomers of cis-octadecenoate were tested for their suitability in meeting the nutritional requirement for unsaturated fatty acids by mutants of Escherichia coli and Saccharomyces cerevisiae that were unable to synthesize unsaturated fatty acids. Quantitative comparisons of the efficiencies of the various isomers showed a range from 0-48 cells per femtomole for the prokaryotic cells and 0-5 for eukaryotic cells. The delta 5 isomer was much more effective than the delta 6 isomer with the bacterial cells whereas the reverse was true with the yeast cells. In general, isomers containing a cis ethylenic bond between carbons 7 and 12 were able to support extensive growth of either type of mutant. Since all of the various isomers were incorporated into cellular lipids by both types of microorganism, the different efficiencies observed in supporting growth were not a simple reflection of the inability of an acid to be esterified. The differences may reflect the suitability of the resultant esterified product to function as a normal membrane lipid. The contents of various fatty acids in the cellular phospholipids when growth ceases may have a linearly cumulative relationship to the degree of expansion of the acyl chains.

The unsaturated fatty acid requirement in Escherichia coli Temperature dependence and total replacement by branched-chain fatty acids

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ( 12-Me-14:0) or cis-Δ 9- octadecenoate ( cis-δ 9-18:1 ). When this strain is grown for many generations on 12-Me-14:0, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of 12:0 (8%) and 14:0 (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.

Nutrition of coryneform bacteria from milk and dairy sources.

Summary. Growth requirements were determined for 112 cultures of coryneform bacteria including strains of Corynebacterium bovis, C. ulcerans and C. lacticum; in general they were quite distinctive. Of 42 C. bovis strains all had an unsaturated fatty acid requirement fulfilled in chemically defined media by Tween 80, and, but for an atypically nonureolytic strain, all utilized ammonia and urea nitrogen. Whereas vitamins were unessential for the majority of C. bovis. 18 strains failed to grow in the absence of nicotinic acid; however, for 11 of these Casamino Acids replaced nicotinic acid in supporting growth. Thirteen C. ulcerans strains from aseptically drawn milk, as well as two Type Culture strains (NCTC 7907 and 7908) from the human throat required amino acid nitrogen sources, nicotinic acid, pantothenate and biotin. Some nutritional heterogeneity was shown by 46 strains collectively described as C. lacticum obtained from market milk, milk products and dairy utensils; nevertheless the nutritional differences between the groups of these strains correlated broadly with other distinguishing properties. A group of 9 markedly caseolytic strains required amino acid nitrogen sources, thiamine and biotin, whereas a second group of 30 strains, some utilizing inorganic nitrogen sources, required thiamine and pantothenate, with or without biotin. The remaining C. lacticum strains together with a number of unidentified strains from similar sources were generally slow growing and had widely differing growth requirements.

Unsaturated fatty acid metabolism in Neurospora

Summary Five independent occurrences of Neurospora mutants requiring unsaturated fatty acids were found. Four of these were allelic and grew in the presence of either oleic, linoleic, or linolenic acid. The remaining mutant which was not allelic was partially blocked in the conversion of linoleic to linolenic acid. Biotin, aspartic acid, or pyridoxine did not affect the requirement for unsaturated fatty acids in these mutants. The mutants tested did not respond to arachidonic acid. The results were interpreted as indicating that the unsaturated fatty acids are synthesized through a route distinct from that of the saturated acids and that they are synthesized in the order of oleic to linoleic to linolenic acid.

THE USE OF UNSATURATED FATTY ACIDS IN THE TREATMENT OF ECZEMA

About two years ago, Hansen 1 conceived the idea of investigating the possible clinical bearing of a discovery made in 1929 by Burr regarding the requirement for unsaturated fatty acids in animal nutrition. Burr found that rats placed on completely fat free diets, but adequate in all other respects, soon developed a definite syndrome characterized in the early stages by scaliness of the skin and in the later stages by cessation of growth, necrosis of the tail and pathologic alterations in the kidneys. When, however, he added fats to the diet of these experimental animals, they again began to grow and the skin scaliness disappeared entirely. Hansen found that the iodine absorption value of the blood serum of eczematous babies is low and that it increases with clinical improvement. He could produce an increase in the serum iodine by feeding these babies linseed oil and he also reported a number