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。     

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。     

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。     

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。     

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)。     

破囊壶菌利用工农业废弃物生产脂肪酸的研究进展

破囊壶菌因具有高产脂肪酸的特性而受到广泛关注,然而传统的培养方式需要的原料成本很高,很大程度上阻碍了破囊壶菌的工业化进程,因此,寻找可被破囊壶菌高效利用、来源广泛并且廉价易得的生产原料成为该领域的研究重点。本文以破囊壶菌及其生产脂肪酸为切入点,综述了近年来破囊壶菌利用工农业废弃物生产脂肪酸的研究现状,总结并提出了其发展前景,以期对今后的研究有所启发。     

变温对破囊壶菌FJN-10脂肪酸组分和蛋白质组的影响

[目的]以破囊壶菌Thraustochytrium sp.FJN-10为研究对象,研究不同培养温度和变温条件对菌体生物量、油脂含量、脂肪酸组分、关键基因转录以及蛋白质组的影响。[方法]通过摇瓶实验测定干重研究菌株的生长,提取油脂并经液相色谱分析脂肪酸组分;荧光定量PCR和二维电泳研究脂肪酸合成途径关键的碳链延长酶、脱饱和酶的转录水平和蛋白质的差异表达。[结果]28℃培养时,生物量达13.6 g/L,DHA占总脂肪酸含量达32.41%;15℃培养时,生物量为9.8 g/L,DHA占总脂肪酸含量达56.08%。变温培养下生物量最高可达13.9 g/L、油脂含量及DHA含量在较高的水平。28℃降至15℃时,△4脱饱和酶和△6延长酶基因基因的转录分别提高了3.9和2.5倍。[结论]变温条件下菌株生物量在11.8~13.6 g/L,菌体稳定期延长,DHA的含量可达45%以上。可为今后DHA的产业化提供基础数据。     

添加生物素和浅蓝菌素对裂殖壶菌发酵产DHA的影响

为提高发酵产量,根据裂殖壶菌生物合成二十二碳六烯酸(DHA)的途径,考察添加代谢途径关键酶的辅酶及酶的抑制剂对发酵裂殖壶菌的影响.结果表明:添加生物素可促进油脂积累,添加浅蓝菌素有利于DHA及不饱和脂肪酸含量的提高.添加生物素0.3 mg/L时,DHA占细胞干质量分数达11.26％,相对于对照提高了13％;当添加浅蓝菌素0.1mg/L时,DHA占细胞干质量分数可达12.2％.     

外源添加剂促进裂殖壶菌合成DHA

为提高二十二碳六烯酸（DHA）产量,根据裂殖壶菌生物合成DHA的代谢途径,考察外源添加剂对裂殖壶菌发酵生产DHA影响。研究表明：分生物素、柠檬酸、苹果酸和洛伐他汀均能提高裂殖壶菌DHA的合成能力。同时添加生物素、苹果酸和洛伐他汀时能够显著提高DHA的产量,DHA的最高产量达到11.55 g/L,相比对照提高71.87%。     

裂殖壶菌丙二酸单酰转移酶(MAT)基因的克隆与序列分析

二十二碳六烯酸(DHA)是一种重要的多不饱和脂肪酸,裂殖壶菌(Schizochytrium)因富含DHA成为当前的研究热点。丙二酸单酰转移酶(MAT)是该菌合成DHA的关键酶。以Schizochytriumsp.FJU-512cDNA文库中的EST序列contig417(GenBank accession No.EF483907)为基础,获得了丙二酸单酰转移酶基因的DNA序列和cDNA序列。序列分析表明,cDNA序列与DNA序列完全一致,开放阅读框全长1 176bp,编码391个氨基酸,且具备该类酶氨基酸序列的两个经典保守区。对该蛋白进行系统发育分析,构建进化树,结果显示,该蛋白同Schizochytriumsp.TIO01的丙二酰辅酶A-ACP转移酶(MCAT)亲缘关系最近。     

Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase

Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.     

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。     

Biosynthesis of docosahexaenoic acid (DHA, 22:6-4, 7,10,13,16,19): two distinct pathways

Docosahexaenoic acid (DHA) has long been recognized for its beneficial effect in humans, but its biosynthetic pathway has not been clearly established until recently. According to Sprecher, in mammals, DHA is synthesized via a retro-conversion process in peroxisomes-the aerobic delta4 desaturation-independent pathway. Recent identification of a Thraustochytrium delta4 desaturase indicates that delta4 desaturation is indeed involved in DHA synthesis in Thraustochytrium. More interestingly, an alternative pathway for DHA biosynthesis-the anaerobic polyketide synthase pathway was also reported recently to occur in Schizochytrium, another member of the Thraustochytriidae. This mini-review attempts to assess the latest research on these distinct pathways for DHA biosynthesis.     

Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea

The existence of Delta 4 fatty acid desaturation in the biosynthesis of docosahexanoic acid (DHA) has been questioned over the years. In this report we describe the identification from Thraustochytrium sp. of two cDNAs, Fad4 and Fad5, coding for Delta 4 and Delta 5 fatty acid desaturases, respectively. The Delta 4 desaturase, when expressed in Saccharomyces cerevisiae, introduced a double bond at position 4 of 22:5(n-3) and 22:4(n-6) resulting in the production of DHA and docosapentanoic acid. The enzyme, when expressed in Brassica juncea under the control of a constitutive promoter, desaturated the exogenously supplied substrate 22:5(n-3), resulting in the production of DHA in vegetative tissues. These results support the notion that DHA can be synthesized via Delta 4 desaturation and suggest the possibility that DHA can be produced in oilseed crops on a large scale.     

真菌发酵生产鱼油特征脂肪酸

DHA和EPA是鱼油的特征脂肪酸,具有调整血脂、改善大脑功能和维持正常视力等重要作用.一直以来,其来源主要是海产鱼油.近年研究发现许多微生物的脂质中也含有DHA和EPA,特别是一些低等真菌含量甚为丰富,利用其发酵生产DHA和EPA替代鱼油资源具有广阔的前景.阐述了能产生EPA,DHA的真菌及其合成途径和发酵影响因素.     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.     

Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production

An isolation program targeting Thraustochytrids (marine fungoid protists) from 19 different Atlantic Canadian locations was performed. Sixty-eight isolates were screened for biomass, total fatty acid (TFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) content. Analysis of fatty acid methyl ester results discerned four distinctive clusters based on fatty acid profiles, with biomass ranging from 0.1 to 2.3 g L⁻¹, and lipid, EPA, and DHA contents ranging from 27.1 to 321.14, 2.97 to 21.25, and 5.18 to 83.63 mg g⁻¹ biomass, respectively. ONC-T18, was subsequently chosen for further manipulations. Identified using 18S rRNA gene sequencing techniques as a Thraustochytrium sp., most closely related to Thraustochytrium striatum T91-6, ONC-T18 produced up to 28.0 g L⁻¹ biomass, 81.7% TFA, 31.4% (w/w biomass) DHA, and 4.6 g L⁻¹ DHA under optimal fermentation conditions. Furthermore, this strain was found to produce the carotenoids and xanthophylls astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene. Given this strain’s impressive productivity when compared to commercial strains, such as Schizochytrium sp. SR21 (which has only 50% TFA), coupled with its ability to grow at economical nitrogen and very low salt concentrations (2 g L⁻¹), ONC-T18 is seen as an ideal candidate for both scale-up and commercialization.     

海洋破囊壶菌Thraustochytrium sp. FJN-10 DHA生物合成 途径相关延长酶的克隆与表达

二十二碳六烯酸(Docosahexaenoic acid,DHA)是具有各种重要生理功能的高度不饱和脂肪酸.以海洋真菌Thraustochytrium sp.FJN-10为研究对象,利用RT-PCR结合RACE,获得了两个碳链延长酶(TFD6和TFD5)的完整基因,其中TFD6 cDNA全长816 bp,编码271个氨基酸;TFD5 cDNA全长831 bp,编码276个氨基酸.将TFD6、TFD5酶切后分别连接到HindⅢ/Xba Ⅰ处理过的pYES2载体,醋酸锂法转化酿酒酵母感受态细胞,成功构建了延长酶酵母表达系统.气相色谱分析表明TFD6可延长C18:3n-6至C20:3n-6,TFD5可延长C20:5n-3至C22:5n-3.