Fermentation process development of recombinant Hansenula polymorpha for gamma-linolenic acid production

Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.     

Differential expression of desaturases and changes in fatty acid composition during sporangiospore germination and development in Mucor rouxii

Polyunsaturated fatty acids (PUFAs), namely, oleic (C18:1), linoleic (C18:2), and gamma-linolenic acid (C18:3), constituted the majority in the total fatty acid content (44%) of sporangiospores of Mucor rouxii. At 30 degrees C, the germination begins within 1h at which time spore swelling occurs, followed by germ tube emergence within 3-4h. Throughout germination, an increase in gamma-linolenic acid (GLA) was observed and its content was highest at germ tube emergence. It took longer for sporangiospores of M. rouxii to germinate at sub-optimal temperatures (15 and 35 degrees C). However, the content of GLA was higher at the germ tube initiation than at the mycelial stage at all temperatures, suggesting the association of GLA and germination of sporangiospores. This finding was substantially confirmed by differential expression of delta9-, delta12-, and delta6-desaturase genes measured during spore germination. The expression of three desaturase genes parallels the pattern of GLA synthesis. By using RT-PCR techniques to follow gene expression, we found that mRNA of delta12- and delta6-desaturase genes were translated as soon as the spores were introduced into a fresh medium while the mRNA of delta9-desaturase gene could not be detected until 2h after introduction. A sharp increase in mRNA of delta6-desaturase genes correlated well with an increase in GLA content at germ tube emergence (4h). These results demonstrated that changes in fatty acid composition of sporangiospore of M. rouxii and differential expression of desaturase genes occurred during germination, and that extensive changes in GLA synthesis associated with some events in germination process.     

Substrate specificity of acyl-Delta(6)-desaturases from Continental versus Macaronesian Echium species

Echium (Boraginaceae) species from the Macaronesian islands exhibit an unusually high level of gamma-linolenic acid (18:3n-6; GLA) and relatively low content of octadecatetraenoic acid (18:4n-3; OTA) in the seed, while the amounts of both fatty acids in their Continental (European) relatives are rather similar. We have tested the hypothesis of whether a different specificity of the acyl-Delta(6)-desaturases (D6DES) towards their respective usual substrates, linoleic acid (18:2n-6; LA) for GLA and alpha-linolenic acid (18:3n-3; ALA) for OTA, was partly responsible for this composition pattern. To this aim we have expressed in yeast the coding sequences of the D6DES genes for the Continental species Echium sabulicola, and the Macaronesian Echium gentianoides. When the yeast cultures are supplemented with the two fatty acid substrates (LA and ALA), a similar utilization of both compounds was found for the D6DES of E. sabulicola, while a preference for LA over ALA was observed for the enzyme of E. gentianoides. This substrate preference must contribute to the increased accumulation of GLA in the seeds of the Macaronesian Echium species. Comparison among the amino acid sequences of these desaturases and other related enzymes, allowed us the discussion about the possible involvement of some specific positions in the determination of substrate specificity.     

Sex differences in n-3 and n-6 fatty acid metabolism in EFA-depleted rats

We studied the effect of sex on the distribution of long-chain n-3 and n-6 fatty acids in essential fatty acid-deficient rats fed gamma-linolenate (GLA) concentrate and/or eicosapentaenoate and docosahexaenoate-rich fish oil (FO). Male and female weanling rats were rendered essential fatty acid deficient by maintaining them on a fat-free semisynthetic diet for 8 weeks. Thereafter, animals of each sex were separated into three groups (n = 6) and given, for 2 consecutive days by gastric intubation, 4 g/kg body wt per day of GLA concentrate (containing 84% 18:2n-6), n-3 fatty acid-rich FO (containing 18% 20:5n-3 and 52% 22:6n-3), or an equal mixture of the two oil preparations (GLA + FO). The fatty acid distributions in plasma and liver lipids were then examined. GLA treatment increased the levels of C-20 and C-22 n-6 fatty acids in all lipid fractions indicating that GLA was rapidly metabolized. However, the increases in 20:3n-6 were less in females than those in males, while those in 20:4n-6 were greater, suggesting that the conversion of 20:3n-6 to 20:4n-6 was more active in female than in male rats. FO treatment increased the levels of 20:5n-3 and 22:6n-3 and reduced those of 20:4n-6. The increase in n-3 fatty acids was greater in females than that in males and the reduction in 20:4n-6 was smaller. Consequently, the sum of total long-chain EFAs incorporated was greater in females than that in males. The administration of n-3 fatty acids also reduced the ratio of 20:4n-6 to 20:3n-6 in GLA + FO-treated rats indicating that n-3 fatty acids inhibited the activity of delta-5-desaturase. However, this effect was not affected by the sex difference.     

Metabolism of linoleic and α-linolenic acids in cultured cardiomyocytes: Effect of different N-6 and N-3 fatty acid supplementation

The metabolites of linoleic (LA) and -linolenic (ALA) acids are involved in coronary heart disease. Both n-6 and n-3 essential fatty acids (EFAs) are likely to be important in prevention of atherosclerosis since the common risk factors are associated with their reduced 6-desaturation. We previously demonstrated the ability of heart tissue to desaturate LA. In this study we examined the ability of cultured cardiomyocytes to metabolize both LA and ALA in vivo, in the absence and in the presence of gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) alone or combined together. In control conditions, about 25% of LA and about 90% of ALA were converted in PUFAs. GLA supplementation had no influence on LA conversion to more unsaturated fatty acids, while the addition of n-3 fatty acids, alone or combined together, significantly decreased the formation of interconversion products from LA. Using the combination of n-6 and n-3 PUFAs, GLA seemed to counterbalance partially the inhibitory effect of EPA and DHA on LA desaturation/elongation. The conversion of ALA to more unsaturated metabolites was greatly affected by GLA supplementation. Each supplemented fatty acid was incorporated to a significant extent into cardiomyocyte lipids, as revealed by gas chromatographic analysis. The n-6/n-3 fatty acid ratio was greatly influenced by the different supplementations; the ratio in GLA+EPA+DHA supplemented cardiomyocytes was the most similar to that recorded in control cardiomyocytes. Since important risk factors for coronary disease may be associated with reduced 6-desaturation of the parent EFAs, administration of n-6 or n-3 EFA metabolites alone could cause undesirable effects. Since they appear to have different and synergistic roles, only combined treatment with both n-6 and n-3 metabolites is likely to achieve optimum results.     

Fatty acid changes in liver choline and ethanolamine glycerophospholipids in aspirin-treated rats fed linoleate, gamma-linolenate and fish oil

The effects of dietary linoleic acid, gamma-linolenic acid and marine fatty acids on the development of aspirin-induced gastric hemorrhage and the distribution of liver glycerophospholipid fatty acids in fat-deficient growing rats were studied. Aspirin (100 mg/day)-treated and nontreated rats were fed for 7 days, a mixed diet of 2.5% safflower oil and 7.5% hydrogenated coconut oil (SFO/HCO) or 7.5% fish oil (SFO/FO), or 2.5% gamma-linolenate concentrate and 7.5% fish oil (GLA/FO). Gastric hemorrhage was induced in animals by aspirin treatment to various extents. It was not affected by FO feeding, but was significantly alleviated by GLA feeding. Aspirin treatment reduced the proportions of 20:4n-6 in liver phosphatidylcholine. FO feeding (in SFO/FO and GLA/FO rats) further reduced the 20:4n-6 level and replaced it by n-3 fatty acids. GLA feeding, on the other hand, elevated the proportion of 20:4n-6. As a result, the reduction of 20:4n-6 by fish oil feeding, was less significant in GLA/FO rats than in SFO/FO rats. The degree of gastric hemorrhage appeared to relate negatively to the levels of 20:4n-6 in liver phosphatidylcholine, and to the sum of 20:4n-6 and 20:5n-3 when FO was included in the diet. It is suggested that long-chain polyunsaturated fatty acids (20:4n-6 and 20:5n-3) per se in addition to being precursors of prostaglandins, may also affect the development of gastric hemorrhage, possibly by modulating the permeability of cell membranes in the gastric mucosa.     

Isolation and functional characterization of a delta 6-desaturase gene from the pike eel (Muraenesox cinereus)

Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR methods. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.     

Dose-dependent effects of dietary gamma-linolenic acid on rat spleen lymphocyte functions.

Feeding rodents a diet rich in evening primrose oil (EPO), which contains 5-10 g gamma-linolenic acid (GLA)/100 g total fatty acids, has been shown to decrease lymphocyte proliferation and natural killer cell activity. However, EPO contains a very high level of linoleic acid which itself can affect lymphocyte functions and it is not clear to what extent the effects of EPO can be attributed to GLA. The current study investigated the effect of two levels of GLA in the rat diet upon immune cell functions; the level of linoleic acid was maintained below 30 g/100 g total fatty acids. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g or 10 g GLA/100 g total fatty acids in place of a proportion of linoleic acid. The total polyunsaturated fatty acid content and the n-6 to n-3 polyunsaturated fatty acid ratio of the diet were maintained at 35 g/100 g total fatty acids and 7, respectively. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet, with an increase in the proportions of GLA and dihomo-gamma-linolenic acid when the diets containing GLA were fed; these diets also increased the proportion of arachidonic acid in spleen leukocytes. Spleen lymphocyte proliferation in response to concanavalin A was significantly reduced (by 60%) by feeding the diet containing the higher level of GLA, but not by the diet containing the lower level of GLA. Spleen natural killer cell activity and prostaglandin E (PGE) production by spleen leukocytes were not significantly affected by inclusion of GLA in the diet, although there was a tendency towards decreased natural killer cell activity by cells from rats fed the high GLA diet. Thus, this study shows that dietary GLA is capable of altering the fatty acid composition of cells of the immune system and of exerting some immunomodulatory effects, but that the level of GLA in the diet must exceed 4.4 g/100 g total fatty acids for these effects to become apparent.     

Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.     

Gamma-linolenic acid alters the composition of mitochondrial membrane subfractions,decreases outer mitochondrial membrane binding of hexokinase and alters carnitine palmitoyltransferase I properties in the Walker 256 rat tumour

Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n-6, 20:4 n-6, 22:4 n-6 and 22:5 n-6 in the TAG fraction as determined by gas chromatography-mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n-6 and decreases in 18:1 n-9, 18:2 n-6 and 22:6 n-3, when exposed to GLA diet. These changes were reflected in a large increase in the n-6/n-3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.     

Identification and functional characterization of polyunsaturated fatty acid elongase (McELOVL5) gene from pike eel (Muraenesox cinereus)

The cDNA coding for a polyunsaturated fatty acid elongase (McELOVL5) was isolated from the brain of the pike eel (Muraenesox cinereus) being based on available sequences in 23 types of fish. Four sequence variants were identified with different amino acid substitutions as compared with two clones of McELOVL5 gene (McELOVL5 11.7 and McELOVL5 12.4). When the two variants of McELOVL5 were expressed in Saccharomyces cerevisiae, the two recombinant yeasts elongated γ-linolenic acid (GLA, 18:3n-6) to di-homo-γ-linolenic acid (DGLA, 20:3n-6) but differed in the rate of GLA conversion to DGLA. Cells transformed with McELOVL5 12.4 also converted arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) to docosatetraenoic acid (22:4n-6) and docosapentaenoic acid (22:5n-3), respectively. However McELOVL5 11.7 lost its function for the elongation of C₂₀ fatty acids. The four sequence variants have changed substrate specificities. Three-dimensional models of the McELOVL5 proteins are suggested.     

卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

Isolation and functional characterization of fatty acid delta5-elongase gene from the liverwort Marchantia polymorpha L

Bryophyte Marchantia polymorpha L. produces C22 very-long-chain polyunsaturated fatty acid (VLCPUFA). Thus far, no enzyme that mediates elongation of C20 VLCPUFAs has been identified in land plants. Here, we report the isolation and characterization of the gene MpELO2, which encodes an ELO-like fatty acid elongase in M. polymorpha. Heterologous expression in yeast demonstrated that MpELO2 encodes delta5-elongase, which mediates elongation of arachidonic (20:4) and eicosapentaenoic acids (20:5). Phylogenetic and gene structural analysis indicated that the MpELO2 gene is closely related to bryophyte Delta6-elongase genes for C18 fatty acid elongation and diverged from them by local gene duplication.     

The influence of different combinations of gamma-linolenic, stearidonic and eicosapentaenoic acids on the fatty acid composition of blood lipids and mononuclear cells in human volunteers

This study set out to identify whether stearidonic acid (18:4n-3; STA) can be used to increase the eicosapentaenoic acid (20:5n-3; EPA) content of plasma lipids and cells in humans and to understand more about the effects of increased consumption of gamma-linolenic acid (18:3n-3; GLA), STA and EPA in humans. Healthy young males were randomised to consume one of seven oil blends for a period of 12 weeks (9g oil/day) (n = 8-12 subjects/group). Palm oil, sunflower oil, an EPA-rich oil, borage oil (rich in GLA), and Echium oil (rich in STA) were blended in various combinations to generate a placebo oil and oils providing approximately 2g GLA + STA + EPA per day, but in different combinations. Blood was collected at 0, 4, 8 and 12 weeks and the fatty acid compositions of plasma triacylglycerols, cholesteryl esters and phospholipids and of peripheral blood mononuclear cells (PBMCs) determined. Significant effects were observed with each lipid fraction. Neither STA nor its derivative 20:4n-3 appeared in any of the lipid fractions studied when STA (up to 1g/day) was consumed. However, STA (1g/day), in combination with GLA (0.9 g/day), increased the proportion of EPA in some lipid fractions, suggesting that STA-rich plant oils may offer a novel means of increasing EPA status. Furthermore, this combination tended to increase the dihomo-gamma-linolenic acid (20:3n-6; DGLA) content of PBMCs, without an increase in arachidonic acid (AA) (20:4n-6) content. EPA consumption increased the EPA content of all lipid fractions studied. Consumption of GLA (2g/day), in the absence of STA or EPA, increased DGLA content with a tendency to increase AA content in some fractions. This effect was prevented by inclusion of EPA in combination with GLA. Thus, this study indicates that STA may be used as a precursor to increase the EPA content of human lipids and that combinations of GLA, STA and EPA can be used to manipulate the fatty acid compositions of lipid pools in subtle ways. Such effects may offer new strategies for manipulation of cell composition in order to influence cellular responses and functions in desirable ways.     

Gamma-linolenic acid dietary supplementation can reverse the aging influence on rat liver microsome delta 6-desaturase activity.

We have recently demonstrated that in rats the process of delta 6-desaturation of linoleic and alpha-linolenic acids slows with aging. One method of counteracting the effect of slowed desaturation of linoleic acid would be to provide the 6-desaturated metabolite, gamma-linolenic acid (18:3(n-6) GLA) directly. We have here investigated the 6-desaturation of both linoleic and alpha-linolenic acids in liver microsomes of young and old rats given GLA in the form of evening primrose oil (EPO) (B diet) in comparison to animals given soy bean oil alone (A diet), monitoring also the fatty acid composition of liver microsomes and relating this to the microviscosity of the membranes. In young rats the different experimental diets did not produce any difference in delta 6-desaturase (D6D) activity on either substrate suggesting that, when D6D activity is at or near its peak, the variations in diet tested are unable to influence it. In the old animals the rate of 6-desaturation of linoleic and particularly of alpha-linolenic acid was significantly greater in the B diet fed animals than in the A diet fed. The effects of the diets on the fatty acid composition of liver microsomes were consistent with the findings with regard to 6-desaturation. Administration of GLA partially corrected the abnormalities of n-6 essential fatty acid (EFA) metabolism by raising the concentration of 20:4(n-6) and other 6-desaturated EFAs. Furthermore, the GLA rich diet also increased the levels of dihomo-gamma-linolenic acid and of 6-desaturated n-3 EFAs in the liver microsomes. The microviscosity of microsomal membranes as indicated by DPH polarization was correlated with the unsaturation index of the same membranes. There was a very strong correlation between the two. In both young and old rats the B diet reduced the microviscosity and increased the unsaturation index. However, the effect was much greater in the old animals.     

n-3 and n-6 polyunsaturated fatty acids induce the expression of COX-2 via PPARgamma activation in human keratinocyte HaCaT cells

Polyunsaturated fatty acids (PUFA) n-3 inhibit inflammation, in vivo and in vitro in keratinocytes. We examined in HaCaT keratinocyte cell line whether eicosapentaenoic acid (EPA) a n-3 PUFA, gamma-linoleic acid (GLA) a n-6 PUFA, and arachidic acid a saturated fatty acid, modulate expression of cyclooxygenase-2 (COX-2), an enzyme pivotal to skin inflammation and reparation. We demonstrate that only treatment of HaCaT with GLA and EPA or a PPARgamma ligand (roziglitazone), induced COX-2 expression (protein and mRNA). Moreover stimulation of COX-2 promoter activity was increased by those PUFAs or rosiglitazone. The inhibitory effects of GW9662 and T0070907 (PPARgamma antagonists), on COX-2 expression and on stimulation of COX-2 promoter activity by EPA and GLA suggest that PPARgamma is implicated in COX-2 induction. Finally, PLA2 inhibitor methyl arachidonyl fluorophosphonate blocked the PUFA effects on COX-2 induction, promoter activity and arachidonic acid mobilization suggesting involvement of AA metabolites in PPAR activation. These findings demonstrate that n-3 and n-6 PUFA increased PPARgamma activity is necessary for the COX-2 induction in HaCaT human keratinocyte cells. Given the anti-inflammatory properties of EPA, we suggest that induction of COX-2 in keratinocytes may be important in the anti-inflammatory and protective mechanism of action of PUFAs n-3 or n-6.