l-Glutamic Acid Fermentation

Structure of a sugar lipid produced by an oleic acid-requiring mutant of Brevibacterium thiogenitalis was studied and established as (I).

Relation between biotin and oleic acid was studied using a biotin-requiring organism accumulating l-glutamic acid and its blocked mutants lacking the biosynthetic system of biotin or/and oleic acid. The results support the following considerations. Biotin is not formed from oleic acid and does not substantially affect the growth of l-glutamic acid-accumulating bacteria and their productivity of l-glutamic acid.

Consequently, biotin serves only for the synthesis of fatty acids in the present organisms. The essential factor for their growth and metabolism is an unsaturated fatty acid like oleic acid and not biotin. And also, saturated fatty acids have substantially no relation with their growth and metabolism like accumulation of l-glutamic acid.     

Fatty acid control of cyclic AMP levels inNeurospora crassa

Several saturated, monosaturated, and polyunsaturated fatty acids produce rapid increases in cyclic AMP levels in the fungusNeurospora crassa when added to the growth medium at 10–50 M. The time courses of cyclic AMP increase resembled those previously shown to be induced by other agents, reaching peak cyclic AMP levels at about 2 min after fatty acid addition. These fatty acids had little or no influence on adenylate cyclase fromNeurospora crassa in vitro. On the basis of previous evidence that uncouplers of oxidative phosphorylation increase cyclic AMP levels and that fatty acids can act as uncouplers, we suggest that the fatty acids in vivo may act to increase cyclic AMP levels by acting as uncouplers of oxidative phosphorylation. In agreement with this suggestion, two fatty acids were shown to produce decreased ATP-ADP ratios inNeurospora at concentrations producing cyclic AMP increases.     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Biogenesis of mitochondria 19 the effects of unsaturated fatty acid depletion on the lipid composition and energy metabolism of a fatty acid desaturase mutant of

1. The lipid composition of a mutant ofSaccharomyces cerevisiae which cannot synthesize unsaturated fatty acid (UFA) can be extensively manipulated by growing the organism in the presence of added fatty acids.
2. Growth of the mutant is supported by a wide range of unsaturated fatty acids including oleic, palmitoleic, petroselenic, 11-eicosaenoic, ricinoleic, arachidonic, clupanodonic, linoleic and linolenic acids; 9- and 10-hydroxystearic acids support growth less effectively, but erucic, nervonic, elaidic and saturated fatty acids (C_8∶0?C_20∶0)^* are ineffective. All the fatty acids which support growth are incorporated into cell lipids, apparently without further metabolism.
3. The effects of altered lipid composition on the energy metabolism of yeast cells were investigated. Cells containing less than approximately 20% of their fatty acids as UFA cannot grow on non-fermentable substrates, and their growth on glucose is restricted to that which can be supported by fermentation alone.
4. UFA-depleted cells contain mitochondria which are apparently normal in morphology, furthermore they have normal levels of cytochromesa+a ₃,b,c ₁ andc and respire at normal rates. This suggests that the lesion in energy metabolism produced by UFA-depletion may be the loss of the ability of the mitochondria to couple respiration to phosphorylation.
5. UFA-depleted cells incorporate added UFA into their cell lipids and subsequently regain the ability to grow on non-fermentable substrates, showing that the lesion in energy metabolism is fully reversible.

     

Influence of growth rate on pigment and lipid composition of the microalgaIsochrysis aff.galbana clone T.iso

A continuous culture ofIsochrysis aff.galbana clone T.iso, used to feedPecten maximus larvae at IFREMER (Brest, France), was carried out in a chemostat at its optimum temperature for growth (26 °C). Changes in pigments, lipid class (neutral, glyco- and phospholipids) and degree of fatty acid unsaturation were studied at three different growth rates (0.33, 0.5, 1 d^–1). As predicted by chemostat theory, a slow growth rate produced higher cell numbers and higher biomass per unit volume. These cells were low in chlorophylla and carotenoids, but rich in neutral lipids. In contrast, cultures with a fast growth rate yielded lower cell concentrations, buth higher chlorophylla, carotenoid and membrane lipid contents per cell. Changes in polyunsaturated fatty acid distribution were related to differences in algal growth rates. Neutral lipids contained mainly saturated and monounsaturated fatty acids (C18:19) at low growth rates whereas they were enriched in polyunsaturated fatty acids, especially C22:63, at high growth rates. Therefore, it is suggested that the growth rate in continuous cultures be controlled so as to adjust the relative proportions of polyunsaturated fatty acids in lipid classes of the diet meant for larval nutrition.Author for correspondence     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism

In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L^?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L^?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L^?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L^?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L^?1 during test tube cultivation.

     

Fatty acid cellular metabolism and lactone production by the yeast Pichia guilliermondii

Methyl ricinoleate conversion into γ-decalactone by fungi is already widely used by the aromatic industry. It offers an interesting alternative to chemical synthesis by permitting acquisition of a natural label. Peroxisomal β-oxidation has been described as the probable transformation mechanism. This paper provides information about this metabolism and shows the importance of the step catalysed by carnitine octanoyltransferase. After culture of the yeast Pichia guilliermondii on a medium containing methyl ricinoleate as sole carbon source, we confirmed that mitochondrial β-oxidation could not be responsible for the biotransformation. We also observed the effect of chlorpromazine, an inhibitor of carnitine octanoyltransferase, on peroxisomal β-oxidation and therefore on lactone production, and on lipid accumulation by the yeasts. The presence of chlorpromazine caused a reduction in aromatic specific production yield. This reduction was inversely proportional to the amount of chlorpromazine present in the medium. A considerable accumulation of methyl ricinoleate derivatives was also observed. We therefore concluded that the metabolism responsible for the bioconversion was peroxisomal β-oxidation. The effects of chlorpromazine suggested that the entry of fatty acids into the peroxisomes took place in a carnitine-dependent manner. This step might be a limiting step in the metabolism. Received: 26 June 1995/Received revision: 16 November 1995/Accepted: 4 December 1995     

Biochemical and physiological responses of <Emphasis Type="Italic">Selenastrum gracile</Emphasis> (Chlorophyceae) acclimated to different phosphorus concentrations

Algae are able to adjust their metabolism according to their environment, maximizing growth rate and production of biomolecules under adverse conditions such as pulses of excess of a contaminant or limitation of a nutrient. In order to evaluate the effects of phosphorus (P) availability on the biochemical composition of the freshwater microalga Selenastrum gracile, we acclimated the microalgae to different phosphorus concentrations. After acclimation, exponentially growing cells were inoculated and after 120 h, samples were processed for the determination of carbohydrate, lipid, fatty acid, chlorophyll, cell density, growth rate, and dry weight. Cell density, growth rate, and dry weight decreased with less P, while chlorophyll a, carbohydrates, lipids, and fatty acids per cell increased under P limitation. According to our lipid class and fatty acid results, algae alter their metabolism and membrane configuration to avoid more structural or metabolic damage under limitation, especially at 23 μmol P L^?1. The most sensitive parameters under P limitation were chlorophyll a, lipids, and poly- and monounsaturated fatty acids. The changes in fatty acids contributed to the fluorescence and photosynthesis changes under P limitation, and they occurred before changes were detected in other parameters, such as growth rate. Furthermore, we suggest that prior acclimation to different P affected microalgal physiology and metabolism.     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

Temperaure Compensation and Memberane Composition in Neurospora Crassa

The link between temperature compensation of the circadian rhythm and temperature-induced adjustment of membrane composition in Neurospora crassa is briefly reviewed. In common with most organisms, Neurospora responds to changes in growth temperature by adjusting its lipid composition, primarily by increasing the degree of unsaturation of its fatty acids at low temperature. This may result in maintenance of either membrane fluidity or phase transition behavior over a range of temperatures. In Neurospora, there are three mutations (frq, eel, and chol-1) that affect temperature compensation of the circadian rhythm; cel and chol-1 are defective in lipid synthesis, and frq interacts with the other two in double-mutant strains. This suggests that lipid metabolism may play a role in temperature compensation of the rhythm, and that the FRQ gene product may also be involved in membrane function, either in regulating lipid composition or as a sensor responding to changes in lipid composition. (Chronobiology International, 14(5), 445–454, 1997)     

The effect of temperature on growth and lipid and fatty acid composition on marine microalgae used for biodiesel production

Cultivation temperature is one of the major factors affecting the growth and lipid accumulation of microalgae. In this study, the effects of temperature on the growth, lipid content, fatty acid composition and biodiesel properties of the marine microalgae Chaetoceros sp. FIKU035, Tetraselmis suecica FIKU032 and Nannochloropsis sp. FIKU036 were investigated. These species were cultured at different temperatures (25, 30, 35 and 40 °C). The results showed that the specific growth rate, biomass and lipid content of all microalgae decreased with increasing temperature. With regards to fatty acids, the presence of saturated fatty acids (SFAs) in T. suecica FIKU032 and Nannochloropsis sp. FIKU036 decreased with increasing temperature, in contrast with polyunsaturated fatty acids (PUFAs). Moreover, Chaetoceros sp. FIKU035 was the only species that could grow at 40 °C. The highest lipid productivity was observed in Chaetoceros sp. FIKU035 when cultivated at 25 °C (66.73 ± 1.34 mg L^?1 day^?1) and 30 °C (61.35 ± 2.89 mg L^?1 day^?1). Moreover, the biodiesel properties (cetane number, cold filter plugging point, kinematic viscosity and density) of the lipids obtained from this species were in accordance with biodiesel standards. This study indicated that Chaetoceros sp. FIKU035 can be considered as a suitable species for biodiesel production in outdoor cultivation.     

Lipids associated with microconidial differentiation in a mutant ofNeurospora crassa

Thepeach-fluffy-cot mutant ofNeurospora crassa produces neither macroconidia nor ascospores but does differentiate microconidia after a defined length of time. Changes in the composition of sterols, sterol esters, triglycerides, free fatty acids, and phospholipids were followed during vegetative growth and differentiation of microconidia. The changes in free sterols before and during microconidial differentiation indicate a change in lipid metabolism associated with differentiation. Free sterols and sterol esters accumulated in the developing microconidia, but decreased rapidly during microconidial maturation. The fatty acid components remained relatively unchanged except for a significant increase in linoleic acid. The linoleic acid change might be associated with the development of microconidia or it might simply be a reflection of the NADP-deficiency common in many morphological mutants ofN. crassa.     

Studies on the ethanol-induced decrease of fatty acid oxidation in rat and human liver slices

In order to elucidate the mechanisms involved in the acute ethanol-induced liver triglyceride accumulation, the oxidation, esterification and β-keto acid formation have been studied in rat and human liver slices after incubation with albumin bound, long chain fatty acids (palmitic. oleic and linoleic acids).The addition of alcohol to rat and human liver slices depressed the formation of ¹⁴CO₂ from palmitic acid-1-¹⁴C, oleic acid-1-¹⁴C and linoleic acid-1-¹⁴C. The esterification to triglycerides and phospholipids was increased and the formation of β-keto acids was decreased by alcohol.Addition of 4-methylpyrazole, an inhibitor of liver alcohol dehydrogenase, almost prevented the alcohol effect on the lipid metabolism of the liver slices. The oxidation of alcohol is thus obligatory for the decreased β-oxidation of fatty acids, the increased esterification and for the decreased formation of β-keto acids. The results suggest that ethanol oxidation inhibits β-oxidation of fatty acids and that this primary effect leads to accumulation of liver triglycerides by increased esterification of plasma free fatty acids.     

Autophagy is required for mitochondrial function,lipid metabolism,growth, and fate of KRASG12D-driven lung tumors

Evidence suggests that the role of autophagy in tumorigenesis is context dependent. Using genetically engineered mouse models (GEMMs) for human non-small-cell lung cancer (NSCLC), we found that deletion of the essential autophagy gene, Atg7, in KRAS^G12D-driven NSCLC inhibits tumor growth and converts adenomas and adenocarcinomas to benign oncocytomas characterized by the accumulation of respiration-defective mitochondria. Atg7 is required to preserve mitochondrial fatty acid oxidation (FAO) to maintain lipid homeostasis upon additional loss of Trp53 in NSCLC. Furthermore, cell lines derived from autophagy-deficient tumors depend on glutamine to survive starvation. This suggests that autophagy is essential for the metabolism, growth, and fate of NSCLC.