金黄色破囊壶菌发酵生产DHA的研究

顺 4,7,1 0 ,1 3 ,1 6 ,1 9 二十二碳六烯酸 (ｄｏｃｏｓａｈｅｘａｅｎｏｉｃａｃｉｄ ,简称ＤＨＡ)是ω 3系列多不饱和脂肪酸。近年的研究表明 ,ＤＨＡ是组成大脑和视网膜的重要结构物质 ,如大脑灰质结构脂质中 6 0 %的脂肪酸均为ＤＨＡ[1] 。ＤＨＡ对人体健康有益的生理功能主要表现在[2 ] :调节中枢神经系统功能 ;预防和治疗心血管疾病 ;治疗气喘、关节炎等 ;预防和治疗乳腺癌、结肠癌等。由于ＤＨＡ具有上述生理功能 ,已在医药、食品、保健品等领域得到广泛应用。目前 ,商品ＤＨＡ主要来源于深海鱼油 ,如沙丁鱼、金枪鱼等鱼油 ,由于鱼…     

利用环境胁迫提高破囊壶菌二十二碳六烯酸生产的研究进展

破囊壶菌由于具备生产多种高值天然活性物质的能力,如二十碳五烯酸(eicosapentaenoic acid, EPA)、二十二碳六烯酸(docosahexaenoic acid, DHA)、角鲨烯和类胡萝卜素等,目前已被视为商业脂质生产的优质来源。本文首先对破囊壶菌的生态作用和生物技术价值进行介绍,并概述了脂肪酸的两条生物合成途径;其次重点阐述了NaCl、温度、溶氧和pH这4种环境胁迫因子对破囊壶菌生长、脂质积累、脂肪酸组成和DHA生产的影响;随后总结了当前利用环境胁迫因子的渗透调节策略、分段发酵策略和缓解氧化应激策略提升破囊壶菌DHA生物合成能力的研究现状;最后指出了破囊壶菌在环境胁迫的分子调控机制、分段式发酵策略、菌株进化及代谢工程等方面存在的问题,并对如何改进这些问题以及未来可能的发展方向进行了展望。该综述旨在为破囊壶菌实现高效工业化生产DHA提供有效的参考。     

微藻破囊壶菌产功能性脂肪酸DHA研究进展

二十二碳六烯酸(DHA)作为人体必需的多不饱和脂肪酸,在维护心血管健康、抗癌、支持视觉和脑功能等方面至关重要。传统的深海鱼油提取DHA方法存在鱼腥味重、工艺繁琐等问题,迫使研究者寻求更为高效、环保的替代方案。破囊壶菌(Thraustochytrids)凭借其生长迅速、低重金属污染以及高DHA含量的特性,成为工业化生产DHA的潜力微生物之一。当前在破囊壶菌发酵生产DHA的过程中,依然需要解决一系列关键问题,包括提高发酵产量、降低成本等。本文旨在全面阐述破囊壶菌发酵生产DHA的研究现状,包括菌株筛选与改良、DHA生物合成途径、遗传转化及代谢工程、发酵控制策略等方面。首先,总结归纳了对野生型菌株的自然筛选和诱变改良等方法,不断提高破囊壶菌中DHA产油量。其次,详细介绍了破囊壶菌DHA合成途径的研究进展,着重分析了生物合成途径中关键辅助因子在DHA生产中的作用。此外,概述了外源DNA传递到破囊壶菌细胞的遗传转化技术的应用现状,为提高其遗传转化效率和稳定性提供重要参考。在DHA代谢调控方面,探讨了氮限制对DHA合成的促进作用以及温度和氧气供应对生产效率的影响。最后,对利用破囊壶菌生产DHA存在的主要瓶颈问题和未来发展趋势进行了总结,以推动其在医药、保健品和食品等领域的广泛应用,实现工业规模下的高效生产。     

植物激素对破囊壶菌生长与产DHA的影响

研究了植物激素对破囊壶菌(Thraustochytrium roseum) MF2产DHA的影响作用。实验结果表明：植物激素对T.roseum MF2的生长和产DHA有很大影响;赤霉素(GA)能促进DHA的合成,6苄基腺嘌呤(BA)能显著促进T.roseum　MF2的生长,二者的配合使用能明显增加DHA的产量;培养基中适宜的添加量为2mg/L GA和3mg/L BA,可使DHA产量达到982mg/L。     

海洋破囊壶菌Δ4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1．6kb的△^4-脂肪酸脱饱和酶基因（FAD4）。将FAD4酶切后连接到hin dⅢ,Xba I处理过的pYEs2．0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22：5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22：6（占酵母总脂肪含量的41．13％）,△^4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。     

海洋破囊壶菌△4-脂肪酸脱饱和酶基因在酿酒母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6 kb的△4-脂肪酸脱饱和酶基因(FAD4).将FAD4酶切后连接到Hond Ⅲ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4.转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子.添加外源脂肪酸C22:5底物,半乳糖诱导表达.气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),△4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达.     

深圳海域6株破囊壶菌的生长特性及油脂成分分析

【目的】从深圳海域分离得到6株破囊壶菌,对其基本形态特征、生活史和油脂含量等进行研究,开发其应用潜力。【方法】使用松花粉垂钓法对破囊壶菌进行分离,通过18S r RNA基因测序的方法对破囊壶菌进行鉴定,用显微镜观察其基本形态特征,通过使用尼罗红(Nile Red)染色法对油脂含量进行定性检测,并用GC-MS分析菌株的油脂含量和组成情况。【结果】18S r RNA基因鉴定其属于Aurantiochytrium sp.、Schizochytrium sp.和Thraustochytrium sp.三个属。破囊壶菌的脂肪酸主要成分为十六碳饱和脂肪酸和二十二碳六烯酸(DHA),其中Mn11和Mn15的饱和脂肪酸含量达到总脂肪酸含量的70%以上,Mn16和Sw7的DHA产量分别达到1.29 g/L和1.26 g/L。【结论】Mn11和Mn15菌株适合用于生物柴油的生产,Mn16和Sw7是DHA发酵生产的潜力菌株。     

破囊壶菌DNA提取新法

破囊壶菌(Thraustochytrium spp．)是各种海洋环境中常见的异养单细胞真菌。在海洋中,破囊壶菌是有机物质的有效降解者和初级消费者(例如．Raghukumar等,1999)。也有人认为它是食用无脊椎动物的病原体(例如Mass等,1999)。近年来,科学家开始关注破囊壶菌的工业价值,即从其细胞提取多不饱和脂肪酸(PUFA)(例如Boweaes等,1999)。     

海洋破囊壶菌Δ~4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6kb的Δ4-脂肪酸脱饱和酶基因(FAD4)。将FAD4酶切后连接到HindⅢ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22:5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),Δ4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。     

一株具有工业化生产DHA前景的破囊壶菌

多不饱和脂肪酸是保持人体健康不可缺少的营养成分之一,尤其是二十二碳六烯酸(DHA)作为细胞膜磷脂的重要组分,具有非常重要的医药应用和营养价值。目前,在食品工业中,DHA已经添加至牛奶或奶粉中,用作功能性营养强化剂。20世纪80年代,DHA的唯一来源是鱼油,但鱼油的腥味、重金属污染等问题,促使人们探索生产DHA的其他途径如微生物发酵。诱变和筛选是微生物选育过程中比较重要的手段,可以快速使菌株朝着人类所需要的方向突变。UV诱变和化学药物胁迫筛选是使野生株定向突变的一种很好的办法。目前国内外研究主要针对裂殖壶菌属菌株进行诱变育种,许永利[1]用紫外线诱变和喹禾灵筛选方法对裂殖壶菌(Schizochytrium limacinu)进行诱变选育,突变菌株生物量和DHA含量比对照菌株均有提高。吴克刚等[2]利用添加植物激素对Thraustochytriu roseum MF2进行培育诱变,从而获得更高的DHA量,但在脂质含量和DHA在脂质占比率上都不存在明显变化。本期介绍梁园梅、成家杨等[3]发表的论文《高产DHA破囊壶菌Aurantiochytrium sp.PKU#SW7诱变株的筛选》,作者采用紫外线和药物双重诱变胁迫破囊壶菌获得一株突变株,其在生物量、脂质含量和DHA在脂质占比率上都有显著性提高,相比前人的研究,作者获得的突变株有显著优越性,且突变株DHA生产能力传代4次后仍然保持稳定,具有较高的工业价值。在后续的研究中作者若能对筛选条件、培养基(如利用廉价碳源)和培养条件等方面实行进一步优化,提高突变株生物量,降低突变株发酵生产DHA生产成本,将能获得更高的商业价值。     

Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

Correlation analysis between fatty acid compositions of zooplankter individuals, fed on different phytoplankton species by means of pyrolysis–gas chromatography combined with on-line methylation

Pyrolysis–gas chromatography (Py–GC) combined with on-line methylation was applied to a correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and those of ingested algae using principal component analysis (PCA). Py–GC in the presence of organic alkali, tetramethylammonium hydroxide (TMAH), was used to estimate the apparent distributions of fatty acid components contained in a single individual zooplankter weighing several tens of micrograms and a small sample size of ingested algae samples in the order of 10 μg. The observed fatty acid compositions were used as a database for the PCA in order to discriminate the zooplankton and ingested algae samples. The result obtained indicated that the fatty acid compositions of zooplankton individuals used in this work were significantly reflected in those of their ingested food in spite of some contribution from isomerization and/or elongation of fatty acid components during digestion of the ingested algae phytoplankton in living zooplankters.     

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.     

Detection of microbial-derived fatty acids in carious dentin by gas chromatography and gas chromatography-mass spectrometry

The aim of the present study was to analyze the fatty acid content of carious and sound human dentin. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C₁₀-C₁₈ size in the carious dentin, whereas fatty acids of C₁₆ size were present in minute amounts in three samples of the corresponding sound dentine controls. No fatty acids were detected in the other sound dentin control samples. The source of fatty acids was considered to be microorganisms invading the dentin during the progression of the caries lesion. The presence of bacterial fatty acids in carious dentin may serve as a marker for the pathological process and thus contribute to the understanding of the mechanisms involved.     

Effect of pasteurization and storage on some components of pooled human milk

Pooled human milk was subjected to Holder pasteurization and storage at −20°C up to 90 days and examined for its content of fat and

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-lactate and for lipid composition. This treatment reduced fats by 6% and

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-lactate by at least 7%. In addition, pasteurization and storage induced triglyceride hydrolysis. The absolute amount of free fatty acids (FFAs) which was 0.5% after collection, doubled after pasteurization and rose even more after storage. Different FFA compositions were found by several authors using the same analytical method even for milk samples subjected to the same treatment. More detailed information on procedures must be given to explain the different results.     

Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.     

Influences of Culture Temperature on the Growth, Lipid Content and Fatty Acid Composition of Aurantiochytrium sp. Strain mh0186

The growth, lipid content, and fatty acid composition of Aurantiochytrium sp. strain mh0186 at different temperatures were investigated. Strain mh0186 grew well at 15–30°C, but weakly at 10°C. The biomass at 15–30°C was significantly higher than at 10 and 35°C, and the total lipid at 15–35°C was significantly higher than that at 10°C. The amount of DHA in the total fatty acid was highest at 10°C and decreased in response to temperature increase. The content of DHA (mg/g-dry cell weight) at 15–30°C were significantly higher than those at 35°C and those at 15–25°C were significantly higher than those at 10 and 35°C. The DHA yield at 15–35°C was significantly higher than those at 10 and 35°C. Unsaturation of fatty acid was regulated by temperature and was enhanced in response to temperature decrease. The ratio of DHA to DPA varied at different temperatures.     

A genome-wide association study of n-3 and n-6 plasma fatty acids in a Singaporean Chinese population

Polyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. The primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (N = 1361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5 × 10⁻⁸) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88 × 10⁻⁴). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0502-2) contains supplementary material, which is available to authorized users.