Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: Identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal Delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).

Comparative Analysis of Fatty Acid Desaturases in Cyanobacterial Genomes

Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N-2-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Delta 9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution. Copyright (c) 2008 Xiaoyuan Chi et al.

Effect of dietary linolenic acid/linoleic acid ratio on growth performance, hepatic fatty acid profiles and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco

The present study aimed to evaluate the effect of dietary linolenic acid (LNA)linoleic acid (LA) ratio on growth performance, hepatic fatty acid profile and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco. Six isonitrogenous and isolipidic diets were formulated to contain incremental levels of LNA from 0 to 5% at the expense of corn oil (rich in LA), resulting in six dietary treatments with LNA to LA ratios ranging from 0.35 to 14.64. The experiment continued for 7 weeks. Best growth and feed intake were obtained in the fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). In contrast, feed conversion ratio was the lowest for fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). Dietary LNA to LA ratios significantly influenced viscerosomatic index and hepatosomatic index (P<0.05), but not condition factor (P>0.05). Body composition was also significantly influenced by dietary LNA to LA ratios (P<0.05). Generally, liver FA compositions reflected dietary FA profiles. Declining LA and increasing LNA contents in liver were observed with the increasing dietary LNA/LA ratios (P<0.05). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased with the increasing LNA to LA ratios, suggesting that yellow catfish could elongate and desaturate C18 polyunsaturated fatty acids into highly unsaturated fatty acids. As a consequence, the n-6 fatty acids (FA) declined, and total n-3 FA and n-3/n-6 ratios increased with the dietary ratios of LNA/LA (P<0.05). Dietary LNA to LA ratios significantly influenced several enzymatic activities involved in liver intermediary metabolism (P<0.05), such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate dehydrogenase, malic dehydrogenase and lactate dehydrogenase, suggesting that dietary LNA/LA ratios had significant effects on nutrient metabolism in the liver. To our knowledge this is the first demonstration of the effects of dietary LNA to LA ratios on the enzymatic activities of liver in fish, which provides information on diet quality and utilization, and can also be used as an indicator of the nutritional status of this fish. (C) 2009 Elsevier B.V. All rights reserved.

Discrimination of Type 2 diabetic patients from healthy controls by using metabonomics method based on their serum fatty acid profiles

Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields, including work in cardiovascular research and drug toxicology. In this study, metabonomics method was employed to the diagnosis of Type 2 diabetes mellitus (DM2) based on serum lipid metabolites. The results suggested that serum fatty acid profiles determined by capillary gas chromatography combined with pattern recognition analysis of the data might provide an effective approach to the discrimination of Type 2 diabetic patients from healthy controls. And the applications of pattern recognition methods have improved the sensitivity and specificity greatly. (C) 2004 Elsevier B.V. All rights reserved.

Study on the fatty acid composition of jellyfish Rhopilema esculentum

The fatty acids composition in different parts of full-grown Rhopilema esculentum jellyfish from Yellow Sea was investigated. The lipids, extracted from the umbrella and oral arms and gonads of R. eculentum jellyfish, respectively were analysed by combined capillary gas chromatography/mass spectrometry. The results show that there are more than thirty kinds of fatty acids in jellyfish, and the fatty acid compositions of three parts of R. esculentum are almost the same. In the three parts, percentages of polyunsaturated fatty acids (PUFA) are high, and range from 36.23% to 38.74%. Docosahexaenoic acid (DHA), eicosatetraenoic acid (AA) and eicosapentaenoic acid (EPA) are three major PUFA.

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentration on the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) was cultured in media with different nitrogen sources and concentrations. During the pre-logarithmic phase, the alga grew faster with ammonium as N source than with nitrate, but the reverse applied during the post-logarithmic phase. The alga grew poorly in N-free medium or medium with urea as the sole N source. In the same growth phase, ammonium medium resulted in higher yield of total lipid, but the EPA yield did not differ significantly different from that using nitrate medium. The maximum growth rate occurred in medium containing 1.28 mmol L-1 sodium nitrate, while maximum EPA and total lipid contents were reached at 1.92 mmol L-1, when EPA accounted for 27.9% total fatty acids. The growth rate kept stable when NH4Cl ranged from 0.64 to 2.56 mmol L-1, and the maximum content of total lipid and EPA occurred in the medium with 2.56 mmol L-1 NH4Cl. The EPA content was higher in the pre- than post-logarithmic phase, though the total lipid content was lower. The highest EPA content expressed as percent total fatty acid was 27.9% in nitrate medium and and 39.0% in ammonium medium.

H-OLE1基因的克隆与功能鉴定及维管组织分化的激素调节

脂肪酸是生物体内普遍存在、具重要生理功能的物质，亦是重要的化工原料。研究脂肪酸生物合成及其调控，既是揭示生命活动基本规律的需要，又具巨大的经济价值。多形汉逊氏酵母(Hansenula polymorpha)是一种甲基嗜热酵母，能合成多聚不饱和脂肪酸，是研究脂肪酸生物合成的理想材料之一。为阐明多形汉逊氏酵母细胞中脂肪酸生物合成途径、关键步骤、调节机理，并利用此系统生产有用脂肪酸，我们开展了不饱和脂肪酸生物合成关键酶基因--△9-脂肪酸去饱和酶基因研究。 以P. angusta IFO 1475的P-OLE1基因为探针，Southern杂交分析，发现在亲缘关系很近的不同种类的甲基嗜热酵母如H. pofymorpha、Pichia angusta、P. pastoris、P. methanolica和Candida boMinii中Δ9-脂肪酸去饱和酶基因的结构多形性。 构建了H. polymorpha CBS 1976染色体Δ9-脂肪酸去饱和酶基因座位的限制性酶切图谱，进而分离了3.4 kb BamHI-XhoI基因片段并进行全序列分析，结果表明这个片段含1个与已克隆的酵母Δ59-脂肪酸去饱和酶基因高度同源的、由1353 bp组成的ORF。推导的H-OLE1多肽具脂肪酸去饱和酶的一些基本特征，如含2个结构域：1个位于N一端、含3个保守的组氨酸簇、具催化功能，另1个位于C-端、参与脱饱和反应中电子传递、类似细胞色素b5。将这个序列申报DDBJ，获得Accession number为：AB024576，推导的蛋白的氨基酸序列的Accession number为：BAA75902。 为验证H-OLE1基因的功能，建立了多形汉逊氏酵母DNA电穿孔实验系统，进行了遗传互补测验。发现完整的H-OLE1基因可互补缺乏Δ59-脂肪酸去饱和酶活性的多形汉逊氏酵母营养缺陷型fadl突变体，却不能互补相应的酿酒酵母olel突变体，而由酿酒酵母GAP表达框架和H-OLE1 ORF组成的嵌合基因可互补上述olel突变体。说明H-OLE1基因编码Δ9-脂肪酸去饱和酶，多形汉逊氏酵母的Δ9-脂肪酸去饱和酶和酿酒酵母的脂肪酸脱饱和系统相亲和，而H-OLE1基因的启动子在异源细胞中没有活性。 为研究H-OLE1基因的转录及其调节规律，通过一系列实验，首次找到了可在研究多形汉逊氏酵母基因表达时用作内标的GAP基因。Northern杂交发现，H-OLE1基因在细胞中以较低水平表达，产生1.5 kb的转录子;基因表达略受不饱和脂肪酸的抑制;在多形汉逊氏酵母HOLE1基因的转录调节中，Choi等在酿酒酵母OLE1基因中发现的脂肪酸调节元件FAR可能不是关键的。 利用基因敲除技术，通过转化H-OLE1∷S-LEU2线性DNA到多形汉逊氏酵母二倍体细胞(fadl／FADl)中，首次构建了多形汉逊氏酵母H-OLE1基因的破坏株。遗传学和分子生物学研究表明，破坏株细胞中线性DNA定位串联多拷贝整合到染色体中并置换了fadl突变部位。利用气相色谱分析了ΔH-OLE1破坏株、fadl-2突变株、野生型菌株及含H-OLE1基因转化子的细胞总脂肪酸，发现多形汉逊氏酵母细胞中除18:0→18:1(Δ9)→18:2(Δ9,12)→18:3(Δ9,12,15)这个脂肪酸去饱和主路外，还可能存在其它几个脱饱和反应与延长反应，如16:1(Δ9)→16:2(Δ9,12)→18:2(Δ11,14);16:1(Δ9)→18:1(Δ11)→18:2(Δ11,15)等。 近年维管组织分化研究进展迅速，取得大量可喜结果，也存在许多不足，如细胞分化调节机理，特别是激素诱导的分子机理研究比较薄弱。为建立研究维管组织分化的理想系统，研究嫁接体发育的激素调节机制，在Parkinson和Yeoman发明的离体茎段嫁接系统的基础上，研究了激素对嫁接体发育特别是维管组织分化的影响。 采用不同的嫁接方法，用试管苗对黄瓜离体茎段自体嫁接、亲和性的黄瓜／黑籽南瓜与不亲和性的黄瓜／绿豆离体茎段嫁接组合进行研究，建立了嫁接过程简单、污染率低的试管苗离体茎段嫁接系统。利用往培养基中添加或不加植物激素研究嫁接体发育，发现通过改变培养基中的植物激素，可使亲和的嫁接体难以形成贯通砧木和接穗的维管束桥，也可诱导非亲和性的嫁接体产生维管束桥。初步研究证明利用植物激素可以克服嫁接不亲和性，这一结果是嫁接基础理论研究的一个重要进展，对揭示嫁接亲和性机制具重要意义。由于黄瓜绿豆嫁接组合中，砧木绿豆是可以固氮的豆科植物，研究结果具有潜在的应用前景。 详细地研究了外源IAA和玉米素(ZT)对黄瓜自体嫁接系统中维管束桥形成时间和数目特别是贯通砧木和接穗的管状分子数的影响。当砧木和接穗培养基中都没有添加植物激素时，嫁接接合部难以产生维管束桥，也难以产生贯通的管状分子。当培养基中添加植物激素时，维管束桥数和贯通的管状分子数随激素浓度和种类的不同而不同。本实验的最佳激素条件是：在接穗培养基中加IAA 1.0 mg／L和ZT 0.25 mg／L，在砧木培养基中加ZT 0.25 mg／L。研究表明在试管苗离体茎段自体嫁接系统中，外源激素是嫁接成功的必要条件。试管苗离体茎段嫁接系统是一个理想的研究植物维管组织分化的新系统。 通过对嫁接体发育期接合部及嫁接体各部分IAA、玉米素及玉米素核苷(Z+ZR)的ELISA分析，发现嫁接接合部维管束的再生受IAA和Z+ZR含量的共同调节;连接接穗和砧木维管束桥的分化比维管束的网联要求更高的IAA水平及LAA(Z+ZR)比率。 上述结果为利用嫁接系统研究维管组织分化机理奠定了基础，使进一步研究嫁接体发育的激素调节机理成为可能。

CHARACTERISTICS OF FATTY ACID METHYL ESTERS (FAMEs) AND ENZYMATIC ACTIVITIES IN SEDIMENTS OF TWO EUTROPHIC LAKES

Enzymatic activities and fatty acid methyl esters (FAMEs) in the sediments of two eutrophic lakes in Wuhan city were investigated. The results showed phosphatase and dehydrogenase activities in the lotus zone and plant floating bed zone were significantly lower than those in other sites, and urease activity was the highest where microorganism agents were put in. Fatty acid group compositions indicated the predominance of aerobic bacteria in the surface sediments in shallow lakes. The ratios of FAMEs specific for bacteria and Gram-positive bacteria exibited significant differences between the two lakes. The results of trans to cis indicated that the microorganisms in Lake Yuehu could adapt themselves to environmental stress better. The enzymatic activities and FAMEs showed differences in different sites, indicating that ecological restoration measures and environmental conditions could affect lake sediment to some extent. But the monitoring, work would be done in series to exactly evaluate the effect of the remediation measures.

Response of growth and fatty acid compositions of Nannochloropsis sp to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO2 at 2,800 mu l l(-1). Increased levels of NaNO3 and KH2PO4 raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l(-1), and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5 omega 3) content based on the dry mass was above 3% under low N (150 mu M NaNO3) or high N (3000 mu M NaNO3) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.

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 mul 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, 20:5omega3) (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.