雨生红球藻虾青素合成研究进展

虾青素是一种重要的次级类胡萝卜素,具有高活性的抗氧化功能,广泛应用于食品保健、医药、水产养殖等领域。雨生红球藻是一种在胁迫条件下能够大量积累虾青素的微藻。文中回顾了雨生红球藻虾青素的生物合成研究的进展,包括虾青素生物合成的诱导与调控、虾青素合成与光合作用及脂类代谢的关系等研究现状。     

雨生红球藻营养细胞的虾青素累积

接种于缺氮培养基的雨生红球藻。当置于光强为10—12klx,温度25℃±1.5℃条件下培养时,营养细胞迅速由绿色变成红色。接种4d时,运动细胞占细胞总数的90%,细胞内的虾青素含量(33.0pg/cell)占最终色素累积量的(60.0pg/cell)的55%。接种6d时,虾青素含量已占最终累积量的75%。电镜观察显示,红色的运动细胞除细胞质的大部分区域充满色素颗粒外,细胞的形态结构与绿色细胞基本一致。实验还表明,当接种物置于低光(0.5—1.0klx)下培养时,营养细胞仍有色素沉积,但累积缓慢。当只有高光胁迫(10—12klx,完全培养基培养)时,营养细胞转变成厚壁孢子后(5d后),才大量积累色素。     

活性氧对雨生红球藻生长及虾青素含量的影响

在雨生红球藻培养液中分别添加活性氧1O2、H2O2和·OH的诱生剂,通过测定细胞密度、叶绿素a含量、虾青素含量,研究了这三种活性氧诱生处理对雨生红球藻生长和虾青素含量的影响,初步探索了利用活性氧诱生剂提高雨生红球藻虾青素含量的可行性。实验结果表明,适当浓度的MB能够促进虾青素含量增加,当MB浓度为10-7mol·L-1时,虾青素含量达到5.27μg·mL-1,比对照显著提高。活性氧诱生剂对雨生红球藻生长有抑制作用,但MB的抑制作用小于H2O2和·OH诱生剂。     

营养胁迫对雨生红球藻虾青素累积的影响

通过改变营养条件可诱导雨生红球藻积累虾青素.氮限制实验表明,色素的累积速率与原初氮浓度成反比,也与细胞分裂速率负相关,当BBM培养基中的NaNO3浓度减半时(0.13g@L-1),对细胞增殖及色素累积相对都有利.在高光强下,进一步进行氮、磷饥饿,红球藻细胞分裂明显受抑,但色素的累积作用增强,培养9d,细胞内次生类胡萝卜素的含量分别比对照组提高141.0%和60.5%,色素的累积高峰也比对照组提前2-4d.提高NaCl浓度至0.8%时的盐胁迫,不能诱导虾青素的形成.实验结果还表明,色素的累积与厚壁孢子的形成并不完全相关,游动细胞也能大量积累红色色素.     

雨生红球藻虾青素酰基转移酶的鉴定与功能研究

为研究雨生红球藻(Haematococcus pluvialis)的甘油二酯酰基转移酶(Diacylglycerol acyltransferase, DGAT)是否具有催化虾青素酰基化的功能, 首先通过雨生红球藻的cDNA库克隆得到了一个II型DGAT编码区全长序列(DGTT2)。在甘油三酯(Triacylglycerol, TAG)合成缺陷型酵母Saccharomyces cerevisiae H1246中过表达DGTT2基因发现HpDGTT2不能回补H1246的表型, 即不具有典型的DGAT功能。利用分离得到的雨生红球藻的内质网成功地建立了一个体外的虾青素酰基转移酶酶活测定体系, 添加含有重组HpDGTT2的酵母细胞的微粒体后虾青素酯的含量显著高于对照, 初步表明HpDGTT2具有催化雨生红球藻中虾青素酰基化功能。以上结果为进一步探索雨生红球藻中DGTT2的功能及深入理解虾青素合成在代谢水平的调控奠定了基础。     

雨生红球藻培养基的改良

近年来,雨生红球藻（ＨａｅｍａｔｏｃｏｃｃｕｓｐｌｕｖｉａｌｉｓＦｌｏｔｅｔＷｉｌｌ）作为一种获取天然虾青素的资源被广泛重视ｌ‘,‘１。目前,有关而生红球藻的研究主要集中于虾青素的积累机制、合成调控及其生物学功能‘）。关于雨生红球资生长调控方面的工作报道较少,还没有一个很理想的红球藻培养基【’］,从而在很大程度上阻碍了对雨生红球藻的深人研究与开发利用。雨生红球藻尤其是它的绿色游动细胞对环境ｐＨ值的改变较敏感,其生长状况与培养液的ｐＨ稳定性关系密切［‘”］,而目前较多采用的ＭＣＭ、ＢＢＭ和Ｂｔｈｌ…     

黄腐酸对雨生红球藻虾青素的积累和CHY基因表达量影响

实验以雨生红球藻Haematococcus pluvialis LUGU为对象,研究了不同浓度的黄腐酸对微藻细胞生长、虾青素积累以及β-胡萝卜素羟化酶(CHY)基因表达量的影响。结果表明,FA浓度为5 mg/L,藻细胞生物量产率达到了79.39 mg/(L·d),虾青素产量达到了20.82 mg/L,分别比对照组提高了4.25%和86.89%;FA浓度为10 mg/L,藻细胞的生物量产率和虾青素产量分别比对照组提高了5.44%和9.78%。RT-PCR分析显示,虾青素合成的关键基因CHY的表达受FA的诱导,当添加5和10 mg/L的黄腐酸时,CHY基因最大的表达量分别为对照的18.1倍和7.3倍,当添加20 mg/L的黄腐酸时CHY基因的最大的表达量仅为对照的3.2倍,FA诱导下的雨生红球藻虾青素的积累含量和CHY基因表达量呈正相关。实验表明,适当浓度的黄腐酸不仅能够显著提高虾青素合成关键酶基因CHY的表达水平,并且明显促进了藻细胞内虾青素的积累,因此黄腐酸可作为虾青素生产的一种有效诱导子。     

褪黑素对非生物胁迫下雨生红球藻中虾青素积累的影响

以雨生红球藻Haematococcus pluvialis LUGU株为研究对象, 研究在高光照和缺氮胁迫条件下, 添加不同浓度褪黑素(melatonin, MLT)对雨生红球藻生长、虾青素积累、活性氧(ROS)、信号分子及dxs基因表达量的影响。结果表明, 外源添加10 μmol/L MLT可有效提高藻细胞中虾青素的含量, 最高可达31.32 mg/g, 是对照组(13.27 mg/g)的2.36倍; 抑制了细胞内ROS水平, 上调了信号分子一氧化氮(NO)和水杨酸(SA)的含量; 此外, dxs基因表达水平比对照组明显提高, 最高达11.3倍。研究表明, 在非生物胁迫条件下, 雨生红球藻中虾青素的大量积累可能与外源MLT调控细胞内ROS、信号分子及基因表达有关。     

UV-B辐射对雨生红球藻光合特性和虾青素含量的影响及其响应

实验研究了不同强度的UV-B(280-320 nm)辐射对雨生红球藻(Haematococcus pluvialis)的光合活性、生物量、色素含量、活性氧(ROS)含量和抗氧化酶活性等的影响, 以探讨利用UV-B辐射诱导虾青素生物合成增强的可能性。结果发现, 经UV-B辐射处理后,雨生红球藻的光合活性降低、生物量增长被抑制。UV-B辐射对叶绿素影响不大, 但会改变细胞的类胡萝卜素和虾青素含量:0.1和0.3 W/m2强度的UV-B辐射使细胞中的这两种色素含量升高, 0.5 W/m2组的色素含量短暂升高后恢复到对照水平。中低强度的UV-B可以促进雨生红球藻单细胞虾青素含量的增加, 但由于其对细胞生长的抑制作用, 并不能使虾青素大量积累。随辐射时间延长, 细胞内ROS含量未明显增加,但抗氧化酶(过氧化氢酶和超氧化物歧化酶)活性下降, 雨生红球藻可能主要依靠虾青素来淬灭ROS。以上结果表明, UV-B辐射对雨生红球藻的主要生理生化过程有抑制作用, UV-B辐射既可以诱导虾青素的合成又会消耗一部分虾青素, 对虾青素含量的影响与其强度有关, 而利用虾青素来清除细胞内的ROS可能是雨生红球藻抵御这种不利环境条件的最重要的途径。       

光照强度对雨生红球藻合成虾青素的影响

雨生红球藻（Haematococcus pluvialis）是一种单细胞绿藻,在强光照射条件下能够合成并大量积累具有强抗氧化性的次生类胡萝卜素-虾青素。然而,其合成机理目前还不明确,导致虾青素诱导合成过程中操作的盲目性,限制了虾青素产量的提高。     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis： Effects of Cultivation Parameters

The green alga, Haematococcus pluvlalis Flotow is used as a source of the ketocarotenoid astaxanthin for application in fish aquaculture, pharmaceutical and cosmetic industries. Ceils of the green alga were induced by the application of different light and starvation conditions to evaluate the effect in astaxanthin accumulate. The conditions used for the Induction were high light intensity （170 μmol·m^-2·s^-1）, iron starvation, sulfur starvation and phosphate starvation. The results show that stresses applied in culture, which interfere with cell division, trigger the accumulation of astaxanthin. Notably, sulfur starvation results in a massive accumulation of this commercially important carotenoid.     

雨生红球藻的紫外、激光复合诱变育种

用紫外线和激光复合诱变生产虾青素的雨生红球藻,以适宜条件下的生长速率和亚适宜条件下的虾青素累积能力为筛选指标。结果表明,与原始出发株比,紫外线诱变后,色青累积速率提高37.8％,但生长速率有所下降,紫外线,激光复合诱变结果,生长速率提高11.1%,培养1个月时虾青素累积量提高52.2%。电镜观察结果表明,激光可刺激叶绿体发育,从而改善了紫外线诱变后的生长抑制状况,展示复合诱变是筛选高产虾青素藻株的有效方法。     

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L^?1 day^?1) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L^?1 day^?1 by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

雨生红球藻FACHB-712营养细胞和厚壁孢子低温保藏效果及优化

以高产虾青素的雨生红球藻（Haematococcus pluvialis Flotow）FACHB-712藻株为材料,研究2种细胞形态（营养细胞和厚壁孢子）在低温保藏下的复苏率及其差异原因。结果显示,采用两步法（先预冻降温后再投入液氮中）冻存其营养细胞,在不同冻存条件下,其存活率均低于5%,以10%甘油作为保护剂、冻存速率为0.5℃/min、预冻温度为-40℃、保留30 min,然后再投入液氮罐（-196℃）中保藏,其存活率可达到13.3%。采用两步法冻存厚壁孢子,其复苏存活率高达66.13%,复苏萌发后细胞的生长特性、虾青素含量与液氮保藏前无明显差异（P > 0.05）。对液氮保藏前后藻细胞形态和超微结构观察结果表明,超低温保藏后,营养细胞的结构受到较大损伤,而厚壁孢子受到的损伤相对较小。当添加不同保护剂后,直接将厚壁孢子分别冻存在-20℃、-80℃低温及液氮中,发现-80℃低温冻存处理组的复苏存活率相对较高,可达27%。研究表明采用两步法先预冻降温后再投入液氮中冻存厚壁孢子,是长期保藏雨生红球藻FACHB-712的最佳方法,也可采用一步法将厚壁孢子冻存于-80℃冰箱中。     

氮胁迫对雨生红球藻色素积累与抗氧化系统的影响

选用雨生红球藻CG-06为试验藻株,以BBM为基础培养基,分别设置了0、13.7、27.5、41.2mg·L-1四个硝态氮浓度梯度,分析并探讨在不同硝态氮浓度条件下雨生红球藻生长、生理特性、细胞内主要色素含量的变化以及抗氧化酶活性。结果表明：细胞中色素的积累量和积累速率与初始硝态氮浓度成反比,与抗氧化酶活性呈负相关。缺氮时,培养到第3天的藻细胞中虾青素含量达到4.95μg·mg-1,而对照组在培养到第9天的细胞中才开始产生虾青素,而且在整个培养周期内细胞中的虾青素最大含量仅为4.17μg·mg-1。酶活测定结果显示,虾青素含量较高的红色细胞中,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性明显高于绿色细胞,且GSH-Px活性最高。研究表明,雨生红球藻可能有两种过氧化防御机制,绿色细胞阶段以抗氧化酶作用为主,在培养后期启动虾青素保护机制,两种机制具有协同作用。     

Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis

The singlet oxygen quenching activities of carotenoids, -carotene, free astaxanthin, its monoester and its diester, were examined in vitro by a simple and rapid method for the measurement of Methylene Blue-sensitized photooxidation of linoleic acid in the hexane/ethanol solvent system. The concentrations of carotenoids, -carotene, free astaxanthin, its mono- and di-ester, required for 50% inhibition of lipid oxidation were 40, 8, 9, and 0 M in 100% ethanol and, 14, 16, 10, and 7 M in 50% (v/v) hexane in ethanol, respectively. Astaxanthin esters function as powerful antioxidant agents under both hydrophobic and hydrophilic conditions.     

Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors

The feasibility of a one-step method for the continuous production of astaxanthin by the microalga Haematococcus pluvialis has been verified outdoors. To this end, influence of dilution rate, nitrate concentration in the feed medium, and irradiance on the performance of continuous cultures of H. pluvialis was firstly analyzed indoors in bubble column reactors under daylight cycles, and then outdoors, using a tubular photobioreactor. At the laboratory scale, the behavior of the cultures agreed with that previously recorded in continuous illumination experiences, and attested that the major factors determining biomass and astaxanthin productivity were average irradiance and specific nitrate supply. The rate of astaxanthin accumulation was proportional to the average irradiance inside the culture, provided that a nitrate limiting situation had been established. The accumulation of astaxanthin under daylight cycles was maximal for a specific nitrate input of 0.5 mmol/g day. The recorded performance has been modeled on the basis of previously developed equations, and the validity of the model checked under outdoor conditions. Productivity values for biomass and astaxanthin of 0.7 g/L day and 8.0 mg/L day respectively, were obtained in a pilot scale tubular photobioreactor operating under continuous conditions outdoors. The magnitude of the experimental values, which matched those simulated from the obtained model, demonstrate that astaxanthin can be efficiently produced outdoors in continuous mode through a precise dosage of the specific nitrate input, taking also into consideration the average irradiance inside the culture.     

光诱导雨生红球藻虾青素积累的信号通路转录组分析

雨生红球藻Haematococcus pluvialis在逆境胁迫条件下可大量积累虾青素,被认为自然界最理想的虾青素生产者.高光能有效诱导其虾青素的合成与积累,但藻细胞感知和转导光信号进而调控虾青素积累的机制尚不清楚.文中采用Illumina Hiseq 2000高通量测序技术分别获得正常、高白光及高蓝光处理组4.0 ...