A New Labyrinthulid Isolate That Produces Only Docosahexaenoic Acid

We show here that a new labyrinthulid strain, L72, isolated from a fallen leaf in the Seto Inland Sea of Japan, produced only docohexaenoic acid (DHA) among all the long-chain polyunsaturated fatty acids (LCPUFAs). The main fatty acid composition was 16:0 (28.9%), 18:0 (7.2%), 18:1 (5.7%), 18:2 (10.4%), and DHA (45.9%) without any other LCPUFA. The lipid content of the strain was 27.4%. The cells had many lipid bodies, which were densely located in all of the cells. On phylogenetic analysis using the 18S rDNA sequence, the strain was located in the labyrinthulids group, forming a monophyletic group with Labyrinthula sp. (strain s) and Labyrinthuila sp. (strain L59). We further tested the culture optimization of strain L72 to evaluate the ability of the strain to produce DHA. The optimum salt concentration and the temperature of the strain were 100% of artificial seawater and 20°C. Strain L72 could grow well on soybean oil (SBO) or soybean lecithin (SBL) as the carbon source. When 20 g/l of SBL was added to the medium, DHA production reached the maximum amount at 0.67 g/l for 14 d. The two important facts, that the strain can use SBL as the main nutrient and contains only DHA among the LCPUFAs, will be of great advantage for industry.     

真菌发酵生产EPA及DHA影响因素的研究进展

对真菌发酵生产EPA及DHA的影响因素进行综述 ,介绍了菌种、碳源、氮源、C/N比、pH值、温度、发酵时间、通气量、代谢途径的调控、种龄和接种量等因素对EPA及DHA产量的影响。     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Copy number variation of a fatty acid desaturase gene Fads2 associated with ecological divergence in freshwater stickleback populations

Fitness of aquatic animals can be limited by the scarcity of nutrients such as long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). DHA availability from diet varies among aquatic habitats, imposing different selective pressures on resident animals to optimize DHA acquisition and synthesis. For example, DHA is generally poor in freshwater ecosystems compared to marine ecosystems. Our previous work revealed that, relative to marine fishes, several freshwater fishes evolved higher copy numbers of the fatty acid desaturase2 (Fads2) gene, which encodes essential enzymes for DHA biosynthesis, likely compensating for the limited availability of DHA in freshwater. Here, we demonstrate that Fads2 copy number also varies between freshwater sticklebacks inhabiting lakes and streams with stream fish having higher Fads2 copy number. Additionally, populations with benthic-like morphology possessed higher Fads2 copy number than those with planktivore-like morphology. This may be because benthic-like fish mainly feed on DHA-deficient prey such as macroinvertebrates whereas planktivore-like fish forage more regularly on DHA-rich prey, like copepods. Our results suggest that Fads2 copy number variation arises from ecological divergence not only between organisms exploiting marine and freshwater habitats but also between freshwater organisms exploiting divergent resources.     

DHA高产菌Schizochytrium sp.FJU-512脂肪酸组成与18S rRNA基因序列分析

采用松花粉垂钓法分离到一株docosahexaenoic acid(DHA)高产菌FJU-512。该菌株DHA含量高(占总脂肪酸的56．24％),其它长链杂酸含量少(仅有Docosapentraenoic acid,二十二碳五烯酸,DPA),极具开发应用价值。高密度培养获得33g／L生物量。该菌株呈二分裂生长,没有分生孢子。对其18S rRNA基因进行了克隆测序并登录GenBank(AY758384)。依据18S rRNA基因建立的系统进化树表明：该菌与Schizochytrium limacinum具有紧密的亲缘关系。     

Anti-Angiogenic Effects of Conjugated Docosahexaenoic Acid in Vitro and in Vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

共轭亚油酸甘油酯对大鼠血清脂肪酸组成影响的研究

目的：研究共轭亚油酸甘油酯对高脂饮食大鼠血清脂肪酸组成变化的影响;方法：60 只SD 大鼠随机分为5 组：正常对照 组、高脂对照组,共轭亚油酸甘油酯低（2 g/kg·bw）、中（4 g/kg·bw）、高（6 g/kg·bw）剂量组,除基础对照组外其余各组均喂饲高脂 饲料,建立高脂模型,以灌胃方式给予受试物,6 周后取血清测定其脂肪酸组成。采用一步法直接对血清中脂肪酸进行甲酯化,气 相色谱毛细管柱分离,氢火焰离子化检测器（FID）定性定量分析;结果：共轭亚油酸甘油酯低、中、高剂量组大鼠血清中单不饱和 脂肪酸含量为25.66%,18.74%,17.72%,与高脂对照组相比显著性下降（P<0.01）,各剂量组饱和脂肪酸和多不饱和脂肪酸含量分 别为48.08%,48.52%,51.15%和27.03%,29.28%,31.13%,与高脂对照组相比显著性升高（P<0.01）。几种代表性脂肪酸如各剂量 组中的油酸、亚油酸与高脂对照组相比分别增加了26.48%,41.56%,51.26% 和9.18%,8.61%,8.73%,各剂量组中棕榈酸与高脂对 照组相比降低了5.28%, 8.80%, 10.92%。结论：共轭亚油酸甘油酯能够增加大鼠血清中饱和脂肪酸和多不饱和脂肪酸的含量,减 少单不饱和脂肪酸含量,改变血清脂肪酸组成。     

Grouping Newly Isolated Docosahexaenoic Acid-Producing Thraustochytrids Based on Their Polyunsaturated Fatty Acid Profiles and Comparative Analysis of 18S rRNA Genes

Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.     

Cultivation of Microorganisms in the Cultural Medium Made from Squid Internal Organs and Accumulation of Polyunsaturated Fatty Acids in the Cells

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.     

Chronic nutritional deprivation of n-3 α-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n-3) were markedly reduced in brains of third-generation (F3) rats nutritionally deprived of alpha-linolenic acid (18 : 3 n-3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n-6), an arachidonic acid (AA, 20 : 4 n-6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3-generation n-3-deficient rats, compared with control concentrations. Brain DPA-CoA could be measured in the deprived but not control rats, and AA-CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA- and DHA-selective enzymes, respectively. Competition among n-3 and n-6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n-3 deprivation), or conversion to bioactive eicosanoids and other metabolites.     

Order from disorder, corralling cholesterol with chaotic lipids. The role of polyunsaturated lipids in membrane raft formation

A myriad of health benefits including the prevention of cancer and heart disease accompanies consumption of polyunsaturated fatty acids (PUFA). Of special importance is the omega-3-PUFA docosahexaenoic acid (DHA), with 22 carbons and six double bonds that constitute the most highly unsaturated fatty acid naturally occurring. Our experiments target the membrane as a likely site of action and focus upon the interaction of cholesterol with PUFA-containing phospholipids. They support the idea that steric incompatibility of the rigid steroid moiety for highly disordered PUFA chains promotes lateral segregation of lipids into PUFA-rich/sterol-poor and PUFA-poor/sterol-rich regions. Solid state 2H NMR and X-ray diffraction demonstrate that the solubility of cholesterol is low in polyunsaturated bilayers. In mixed membranes of phosphatidylethanolamine (PE) with the lipid raft-forming molecules sphingomyelin (SM) and cholesterol, diminished affinity of the sterol for 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylethanolamine ([2H31]16:0-22:6PE) relative to 1-[2H31]palmitoyl-2-oleoylphosphatidylethanolamine ([2H31]16:0-18:1PE) is identified by 2H NMR order parameters. Here, lies the origin of a potential biological advantage of the relatively modest increase in PUFA content of plasma membranes that would be conferred by dietary supplementation. We hypothesize that the enhanced propensity to form SM-/cholesterol-rich rafts as well as PUFA-rich/cholesterol-poor microdomains would modify the function of proteins for which these respective regions provide a platform.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

二十碳五烯酸和二十二碳六烯酸的生物合成途径研究进展

以二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)为代表的长链多不饱和脂肪酸,对人体心血管系统、神经系统、抗炎免疫系统等有着理想的功效,因而倍受研究者关注。我们从结构、生物来源和合成、生理功能及生物工程途径的研究现状等方面阐述了EPA和DHA的相关信息,并对其生成途径中涉及的酶进行了简要概述。     

Metabolism of Hydrocarbons in Microorganisms

The fatty acid composition of the lipid of yellowfin tuna (Thunnus albacares) caught in two different localities, Philippine Sea (a tropical zone) and the Pacific coast area of Japan (a temperate zone) is described. The total lipids of various organs (dorsal ordinary muscle, ventral ordinary muscle, dark muscle, liver, heart, pyloric cecum, and orbital region) and of the stomach contents were extracted, and the fatty acid comosition was analyzed by gas-liquid chromatography (GLC).

Docosahexaenoic acid (DHA; 22:6n-3) was the major unsaturated fatty acid in the lipid of all organs in the specimens examined from both localities, the mean DHA content accounting for more than 25% (mean ± S.D. of 26.9 ±5.7%) of the total fatty acids. This value is markedly different from the fatty acid profile of other fish species, because, in general, the fatty acid composition of other species is variable and the DHA content is less than 20% of total fatty acids.

Although the mean DHA content of the total fatty acids in the lipid of yellowfin tuna caught in the tropical and temperate zones was markedly higher than that in other fish species, there was a small difference between that in the northern samples (temperate waters, 30.5 ±6.1%) and the southern samples (tropical waters, 25.9 ± 5.2%). It is suggested that this difference may be due to environmental effects, e.g., the fatty acid composition of the lipids of prey organisms, because there is also a small difference between the mean DHA content of northern prey fish (22.7 ±6.1%) and that of southern prey fish (19.2 ±4.0%).     

二十二碳六烯酸对大鼠脂肪细胞增殖分化的影响

体外培养大鼠脂肪细胞,分别以0 μmol/L（对照组）、40 μmol/L（低剂量组）和160 μmol/L（高剂量组）的二十二碳六烯酸（DHA）处理细胞。采用台盼蓝排斥试验和MTT比色法检测细胞活性及增殖状况;油红O染色化学比色法定量分析细胞内脂肪生成及细胞分化程度;逆转录聚合酶链反应 (RT-PCR) 分析过氧化物酶增殖物激活受体γ2（PPARγ2）mRNA表达情况,探讨DHA对前体脂肪细胞增殖分化的影响及其可能机制。结果显示,各组细胞活力及MTT测得的光密度值(OD值)均低于对照组,160μmol/L组在60～72h作用显著（P<0.05）;脂肪细胞经DHA处理后, 160μmol/L组细胞油红O染色的OD值及PPARγ2 mRNA表达量均显著下降(P<0.01)。以上结果说明,DHA对脂肪细胞增殖分化均有一定抑制作用,高剂量DHA（160μmol/L）可显著减少细胞内脂肪的合成、抑制脂肪细胞分化,PPARγ2 mRNA表达量的下降可能是DHA抑制细胞分化的部分原因。     

Fatty acid production characteristics of fungi with particular emphasis on gamma linolenic acid production

The fatty acid production characteristics of fungi are described. These characteristics are the relationship between the oil content of the cell and the fatty acid content of the oil. For example, for polyunsaturated fatty acid (PUFA) production by Mucor hiemalis IPD 51, the oil content of the cell and the GLA content of the oil are coupled. For fungal production of some PUFA, synthesized after the rate-limiting step in the fatty acid anabolic chain, a maximum fatty acid production model was developed to link the fatty acid content of the oil and the oil content of the cell. Maximum volumetric productivity of gamma linolenic acid (GLA) by molds was found to occur at a specific GLA content of the oil. For example, for M. hiemalis IPD 51, a maximum volumetric of 4.7 mg GLA/L . h was produced at a GLA content of the oil of 8% to 10%. Similarly for Mucor circinelloides v. Tieghem IPD 155 a maximum volumetric productivity of 4.8 mg GLA/L . h was produced at a GLA content of the oil of 14% to 16%. These results imply that, when screening microorganisms for GLA or other fatty acid production, a number of medium compositions need to be evaluated to determine the tradeoff between oil content of the cell and fatty acid content of the oil. (c) 1993 John Wiley & Sons, Inc.     

Excess Linoleic Acid Increases Collagen I/III Ratio and “Stiffens” the Heart Muscle Following High Fat Diets

Controversy exists on the benefits versus harms of n-6 polyunsaturated fatty acids (n-6 PUFA). Although n-6 PUFA demonstrates anti-atherosclerotic properties, survival following cardiac remodeling may be compromised. We hypothesized that n-6 PUFA like linoleic acid (LA) or other downstream PUFAs like γ-linolenic acid or arachidonic acid alter the transforming growth factor-β (TGFβ)-collagen axis in the heart. Excess dietary LA increased the collagen I/III ratio in the mouse myocardium, leading to cardiac “stiffening” characterized by impaired transmitral flow indicative of early diastolic dysfunction within 5 weeks. In vitro, LA under TGFβ1 stimulation increased collagen I and lysyl oxidase (LOX), the enzyme that cross-links soluble collagen resulting in deposited collagen. Overexpression of fatty acid desaturase 2 (fads2), which metabolizes LA to downstream PUFAs, reduced collagen deposits, LOX maturation, and activity with LA, whereas overexpressing fads1, unrelated to LA desaturation, did not. Furthermore, fads2 knockdown by RNAi elevated LOX activity and collagen deposits in fibroblasts with LA but not oleic acid, implying a buildup of LA for aggravating such pro-fibrotic effects. As direct incubation with γ-linolenic acid or arachidonic acid also attenuated collagen deposits and LOX activity, we concluded that LA itself, independent of other downstream PUFAs, promotes the pro-fibrotic effects of n-6 PUFA. Overall, these results attempt to reconcile opposing views of n-6 PUFA on the cardiovascular system and present evidence supporting a cardiac muscle-specific effect of n-6 PUFAs. Therefore, aggravation of the collagen I/III ratio and cardiac stiffening by excess n-6 PUFA represent a novel pathway of cardiac lipotoxicity caused by high n-6 PUFA diets.     

被孢霉△~9脂肪酸脱饱和酶基因保守区的cDNA克隆及结构分析

被孢霉被广泛采用用于发酵生产γ-亚麻酸、花生四烯酸和EPA等多不饱和脂肪酸。为了解决发酵产率过低等诸多问题,我们拟采用基因工程技术改造生产菌株。通过对已克隆△~9脂肪酸脱饱和酶基因的分析,合成一组简并引物,PCR扩增了被饱霉△9脂肪酸脱饱和酶基因的保守区。结果表明被抱霉△9脂肪酸脱饱和酶基因保守区由537个核苷酸组成,共编码179个氨基酸。其与迄今为止发表的微生物面△9脂肪酸脱饱和酶基因有很高的同源性。这是被饱霉△~9脂肪酸脱饱和酶基因研究的次次报道。     

海洋真菌Thraustochytrium sp.FJN-10 Δ~4脱饱和酶基因的cDNA克隆及结构分析

海洋真菌Thraustochytriumsp.FJN-10是二十二碳六烯酸(Docosahexaenoic acid,DHA,C22:6n-3)的高产菌株。为阐明其DHA生物合成机制及采用基因工程技术提高DHA生物合成能力,通过RT-PCR扩增了DHA生物合成相关脱饱和酶基因的保守区,采用cDNA末端扩增技术(Rapid amplification of cDNA ends,RACE)获取全长基因,结果分析表明其克隆基因为Δ4脂肪酸脱饱和酶基因,开放阅读框由1560个核苷酸组成,共编码519个氨基酸,这是Thraus-tochytriumsp.FJN-10Δ4脂肪酸脱饱和酶基因研究的首次报道。