真菌诱导子对植物次生代谢物的影响及应用

近年来,真菌诱导子在诱导植物次生代谢物生产上已成为一大热点。概述真菌诱导子调节植物次生代谢物的作用机理、影响次生代谢物生产的因素及其在次生代谢物生产上的应用。     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase–driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.     

四个脂肪酸合成酶基因在哺乳动物细胞中的超表达提高长链多不饱和脂肪酸生物合成效率

DHA(22:6n-3)、EPA(20:5n-3)和ARA(20:4n-6)三种长链多不饱和脂肪酸在生物体内活性最强,它们在促进大脑发育和功能维持以及在预防和治疗心血管疾病、炎症、癌症等多种疾病方面有着重要作用。然而,尽管哺乳动物体内有完整的长链多不饱和脂肪酸合成酶系,但哺乳动物合成这些长链多不饱和脂肪酸的效率很低而主要依赖于食物获取。本研究应用转基因方法,将哺乳动物来源的Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶这4种酶的编码基因构建成为一个多基因表达载体,然后转染哺乳动物细胞HEK293T,实现了4个目的基因的超表达,再通过气质联用(GC-MS)分析证实了DHA、EPA和ARA等长链多不饱和脂肪酸的合成效率及水平显著增加,DHA的水平更是提高了2.5倍。由此可见,哺乳动物具有某种抑制长链多不饱和脂肪酸高水平合成的机制,但通过Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶的超表达,能够打破哺乳动物这种抑制机制,从而显著提高DHA、EPA、ARA等的合成水平。同时,本研究的思路也为在转基因动物中生产长链多不饱和脂肪酸提供了重要的启示。     

Elicitor-induced hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase in plant cell suspension cultures

Treatment of Nicotiana glutinosa L. cell suspension cultures with chitosan results in the co-induction of phenylalanine ammonia lyase, 4-eoumarate:CoA ligase, tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase, all involved in the biosynthesis of hydroxycinnamoyltyramines. The highest enzyme activities were observed around 12 h after addition of 0.8 to 1 mg chitosan per g fresh weight of cells. No hydroxycinnamoyltyramines could be detected by TLC or HPLC of extracts made from non-treated or elicited cells. [¹⁴C]-Tyramine was incorporated into insoluble polymeric material at a higher rate in elicitor-treated than in non-treated cells of N. glutinosa. Tyramine hydroxycinnamoyltransferase could be induced in suspension cultured cells of Eschscholtzia califortnca Cham, but not in cells of Phaseolus vulgaris L. or Catharanthus roseus (L.) G. Don by addition of a yeast elicitor to the growth medium.     

植物脂肪酸调控基因工程研究

脂肪酸代谢是植物最重要的代谢途径之一,脂肪酸在人们日常生活及工业生产上有重要用途,作者阐明了植物脂肪酸代谢的基本途径,并对近些年来植物脂肪酸代谢的遗传调控及基因工程的研究进展做了较为详细的总结,同时对植物脂肪酸调控基因工程发展提出了展望。     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

Association analyses of single nucleotide polymorphisms in bovine stearoyl-CoA desaturase and fatty acid synthase genes with fatty acid composition in commercial cross-bred beef steers

Two previously reported non‐synonymous coding single nucleotide polymorphisms (SNPs) of bovine stearoyl‐CoA desaturase (delta‐9‐desaturase) (SCD) (c.878C>T) and fatty acid synthase (FASN) (g:17924A>G) were assessed for their associations with 72 individual and 12 groups of fatty acids in brisket adipose tissue of 223 Canadian commercial cross‐bred beef steers. It was found that the ‘CC’ genotype of the SCD SNP was significantly associated with lower concentrations of saturated fatty acids (SFA) including 10:0, 14:0 and 20:0, higher concentrations of monounsaturated fatty acids including 9c‐14:1, 12c‐16:1 and 13c‐18:1, higher concentrations of polyunsaturated fatty acids (PUFA) including 9c,15c‐18:2, 10c,12c‐18:2, 11c,13t‐18:2 and 12c,14t‐18:2, but lower concentrations of other PUFA of 9c,13t/8t,12c and 20:2n‐6 (P < 0.05). The ‘AA’ genotype of the FASN SNP was significantly associated with higher concentrations of SFAs of 10:0, 12:0, 13:0, 14:0 and 15:0, lower concentrations of unsaturated fatty acids of 9c‐18:1 and 20:3n‐6, and higher concentrations of unsaturated fatty acids of 9c‐14:1 and 12c‐16:1 (P < 0.05). Significant epistatic effects between the SCD and FASN SNP genotypes were also found for several fatty acids including 10:0, 23:0, 6t/7t/8t‐18:1, 12t‐18:1, 13t/14t‐18:1, 16t‐18:1, total trans18:1 and 9c,13t/8t,12c‐18:2 (P < 0.05). These results further suggest that SCD and FASN are strong candidate genes influencing fatty acid composition in beef cattle.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

哺乳动物细胞多不饱和脂肪酸合成酶系的重建将亚油酸代谢转变为二十二碳六烯酸

二十二碳六烯酸(DHA,22:6n-3)是一种长度为22个碳原子且含有6个双键的ω-3系多不饱和脂肪酸,在人体中具有重要生物学功能。人体及其他哺乳动物体内只能合成少量的DHA,更多的需求必须从食物中获取。然而,DHA的天然资源(主要是深海鱼类等海洋产品)日趋枯竭,开发新型资源以满足不断扩大的市场需求势在必行。本研究利用转基因技术,在哺乳动物细胞中使Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶超表达,同时表达来源于秀丽隐杆线虫Caenorhabditis elegans的Δ15去饱和酶和小眼虫Euglena gracilis的Δ4去饱和酶,结果表明,这6种酶的表达或超表达能将ω-6系的亚油酸(LA,18:2n-6)有效地转化为DHA(22:6n-3),后者的含量从对照组的16.74%提高到实验组的25.3%。本研究的策略及技术路线为将来利用遗传改造的哺乳动物生产珍稀的DHA(22:6n-3)等长链多不饱和脂肪酸产品提供了重要的启示。     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Apocryphal FADS2 activity promotes fatty acid diversification in cancer

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Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

A Dictyostelium long chain fatty acyl coenzyme A-synthetase mediates fatty acid retrieval from endosomes

We have identified a subset of Dictyostelium endosomes that carry a long chain fatty acyl coenzyme A-synthetase (LC-FACS 1) on their cytosolic surface. Immunofluorescence studies and observations using GFP-fusion proteins collectively suggest that LC-FACS 1 associates with endosomes a few minutes after their formation, remains bound through the acidic phase of endocytic maturation and dissociates early in the phase where the endosomal content is neutralised prior to exocytosis. Mutants in the fcsA gene, encoding the LC-FACS 1 protein, were constructed by homologous recombination. These cells show a strong defect in the intracellular accumulation of fatty acids, either taken up together with the liquid medium or bound to the surface of particles. Because the mutant cells are otherwise fully competent for macropinocytosis and phagocytosis, we conclude that the LC-FACS 1 protein mediates the retrieval of fatty acids from the lumen of endosomes into the cytoplasm.     

Myocardial fatty acid oxidation during ischemia and reperfusion

Inhibition of fatty acid oxidation is an early event in myocardial ischemia that most likely contributes to tissue injury by the accumulation of potentially toxic intermediates such as acylCoA and acylcarnitine. After reperfusion both myocardial oxygen consumption and fatty acid oxidation may rapidly recover to preischemic levels, even when contractile function remains depressed. The mechanisms underlying the apparent dissociation between contractile function and oxidative metabolism early during reperfusion are still controversial. In isolated rat hearts subjected to 60 min of no-flow ischemia myocardial oxygen consumption and oxidation of palmitate were lowered during reperfusion by 3 mM of NiCl₂ and by 6 µM of ruthenium red. The results provide indirect evidence for the hypothesis that intracellular calcium transport may be involved in the mechanisms responsible for the high oxidative metabolic rate early after reperfusion     

Fungal resistance of rubber wood modified by fatty acid chlorides

Decay resistance of Rubber wood (Hevea brasiliensis) esterified with three fatty acid chlorides (hexanoyl chloride (C6), decanoyl chloride (C10) and tetra-decanoyl chloride (C14)) was evaluated. Unmodified and modified wood samples were exposed to a brown rot (Polyporus meliae) and a white rot (Coriolus versicolor) fungus for 12 weeks. Unmodified rubber wood was severely decayed by P. meliae and C. versicolor, which was indicated by significant weight loss. The rate of decay by brown rot was higher than white rot. Modified wood samples exhibited very good resistant to brown and white-rot fungi. The degree of protection increased with increase in degree of modification. P. meliae, a brown rot fungus, removed structural carbohydrate component in unmodified wood selectively whereas, C. vesicolor showed preference to lignin. The FTIR spectra of modified wood exposed to fungi show no significant changes in relative peak intensities of lignin/carbohydrates indicating effectiveness of chemically modified wood in restricting chemical degradation. Chemical modification occurred more efficiently at carbohydrate portion of the wood. Therefore, it is more effective in retarding decay due to P. meliae.