Isolating Mortierella alpina strains of high yield of arachidonic acid

AIMS: To develop a fast isolation method for arachidonic acid-producing fungi of high yield. METHODS AND RESULTS: Relation between the staining degree of mycelia of Mortierella alpina stained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4 degrees C and the mycelia of these isolates were stained with TTC. CONCLUSIONS: The strain M. alpina M6 that had the highest staining degree had the highest arachidonic acid content (72.3%). The yield of arachidonic acid in this strain reached 4.82 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia of M. alpina was positively correlated with arachidonic acid content in mycelia lipid.     

Monitoring of morphological development of the arachidonic-acid-producing filamentous microorganism Mortierella alpina

Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 microm tip(-1) h(-1) at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 microm tip(-1) h(-1) and at specific hyphal growth rates lower than 0.83 h(-1), but decreased drastically at higher rates of tip extension and hyphal growth.     

Analysis of morphological relationship between micro- and macromorphology of Mortierella species using a flow-through chamber coupled with image analysis

Using a flow-through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro- and macromorphology was investigated. On potato-dextrose-agar plates, 5 species formed rose petal-like colonies, 3 formed large round colonies, and 3 formed donut-like colonies. By observing micromorphology in a flow-through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (q(b): tip/microm/h) and a low tip extension rate (q(tip): microm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet-like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow-specific hyphal growth rate (mu(l)) range (0.35-0.45 h(-1)), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet-like morphology and a pellet core. When the branch formation rate was lower than 2 x 10(-3) tips/microm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h(-1)), low tip extension rate (20 tips/microm/h), and high branch formation rate (8 x 10(-3) tips/microm/h) forms the suitable macromorphology for arachidonic acid production.     

Effect of glutamate on arachidonic acid production from Mortierella alpina

AIMS: To evaluate the effect of glutamate on arachidonic acid production from Mortierella alpina. METHODS AND RESULTS: Cell growth, arachidonic acid production, proportions of poly-unsaturated fatty acids (PuFAs) in fatty acids and glucose-6-phosphate dehydrogenase (G6PDH) activity were analysed when glutamate concentration was 0.8 g l(-1). Biomass and arachidonic acid production were higher in the culture containing glutamate than those in the control culture, and both reached their maximum of 25 g l(-1) and 1.4 g l(-1) after 7 d, respectively. The proportions of some PuFAs, oleic acid, gamma-linolenic acid and dihomo-gamma-linolenic acid were decreased while linoleic acid and arachidonic acid were enhanced by glutamate addition. Glutamate addition enhanced G6PDH activity compared with the control during the whole culture process. CONCLUSIONS: Addition of 0.8 g l(-1) glutamate was beneficial to enhance arachidonic acid production from Mortierella alpina, which was a result of activating the pentose phosphate pathway (PPP). SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the addition of glutamate and regulation of PPP had a positive influence on arachidonic acid synthesis in Mortierella species.     

高山被孢霉产花生四烯酸及其遗传改造的研究进展

花生四烯酸作为一种重要的多价不饱和脂肪酸,因其具有多种生理功能而被认为是潜在的食品添加剂和药物。近年来,利用高山被孢霉合成花生四烯酸已成为研究热点。前期相关研究主要集中在菌种选育及发酵调控方面。随着研究的不断深入,关于高山被孢霉合成花生四烯酸的代谢途径的研究取得了较大进展。以下简要概述前期工作进展,着重论述花生四烯酸合成途径的关键酶及其高山被孢霉的遗传改造的研究情况,包括生物合成花生四烯酸代谢途径、关键酶及其应用、高山被孢霉的遗传操作系统的构建以及遗传改造的应用,并对其研究前景进行了展望。     

Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation

The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l).     

高山被孢霉三阶段培养法生产花生四烯酸油脂

为提高高山被孢霉（Mortierella alpina）生物合成花生四烯酸油脂的生产效率,基于花生四烯酸油脂的积累机制,建立了一种三阶段培养法：第一阶段在全培养基中培养促进菌体生物量的快速积累,确定了60 g/L糖初始质量浓度时,最有利于生物量的快速积累;第二阶段在C源丰富而其他营养缺乏的条件下培养,促进油脂快速积累,对糖最佳初始浓度、接种时间、pH和培养时间进行了优化;第三阶段培养诱导油脂中花生四烯酸的高效积累,并确定此阶段培养时间为36 h时为最佳时间。实验结果表明：三阶段培养工艺条件下,菌体生物量、油脂量、花生四烯酸量分别为41.6、26.6和11.4 g/L,本研究相比传统分批发酵工艺在产率和花生四烯酸最终产量方面都有了显著提高。     

毛霉菌油对被孢霉产花生四烯酸的影响

研究了不同浓度毛霉菌油对被孢霉产花生四烯酸（AA）的影响,发现0.3%毛霉菌油可以显著提高花生四烯酸产量,并研究了0.3%毛霉菌油在整个发酵过程中对被孢霉产花生四烯酸的影响。结果表明,毛霉菌油对被孢霉生长和油脂产量基本上没有影响,毛霉菌油可以提高被孢霉的AA产量,主要是因为其能提高AA在油脂中的含量。     

A Novel Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4 and Its Dihomo-gamma-Linolenic Acid Productivity

A novel Delta5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, after treating the parental spores with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-gamma-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28 degrees C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and gamma-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.     

Spore germination in <Emphasis Type="Italic">Mortierella alpina</Emphasis> is associated with a transient depletion of arachidonic acid and induction of fatty acid desaturase gene expression

Mortierella alpina is an oleaginous filamentous fungus whose vegetative mycelium is known to accumulate triglyceride oil containing large amounts of arachidonic acid (ARA 20:4, n − 6). We report that the spores of Mortierella alpina also contain a large proportion of ARA, comprising 50% of total fatty acid. Fatty acid desaturase genes were not expressed in dormant spores but were induced during germination, following a significant drop in the level of ARA (down from 50% of total fatty acid to 12%) prior to germ-tube emergence. We propose that ARA serves as a reserve supply of carbon and energy that is utilised during the early stages of spore germination in Mortierella alpina.     

离子注入花生四烯酸产生菌诱变选育

利用离子束注入生物技术对花生四烯酸产生菌(Mortierella alpina)进行诱变高产菌筛选。筛选到高产菌I₄₉N₁₈,该菌每升培养液可得生物量30.80g(约4%的含水量),干菌体油脂含量为25.8%,其中花生四烯酸的含量占总.脂的45.37%。30L和250L发酵罐发酵试验,该高产菌的花生.四烯酸得率为4.0g/L。     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

微波等离子体溅射诱变选育花生四烯酸高产菌及补料工艺研究

采用微波等离子（N^ ,15w,3min)溅射高山被孢霉T105,筛选到高产菌R254,该菌最高生物量29．3g/L,油脂11．5g/L,花生四烯酸4．20g/L,花生四烯酸产量是原出发菌株1．35倍。通过对突变株R245的代谢情况、继代稳定性以及脂肪酸组成分析,认为微波等离子溅射诱变育种是获得花生四烯酸高产菌株的有效方法。并采用补糖工艺可进一步提高其产量,花生四烯酸产量为7．43g/L,是未补糖时花生四烯酸产量的1．76倍,且为出发菌株花生四烯酸最高产量的2．40倍。     

高山被孢霉遗传操作系统的构建与应用

高山被孢霉是一种重要的产油丝状真菌,其油脂积累量高达细胞干重的50%,其中具有重要生理活性的多不饱和脂肪酸含量较高且种类丰富。近年来,针对高山被孢霉遗传操作系统建立的分子生物学研究进展迅速,这为进一步提高其多不饱和脂肪酸合成水平奠定了基础。对高山被孢霉遗传操作系统的最新应用进展进行了综述,包括高山被孢霉的转化方法、筛选标记的种类、各种方法的优缺点及遗传操作系统的应用。     

利用甘蔗糖蜜发酵生产花生四烯酸的研究

通过培养高山被孢霉利用糖蜜来发酵生产花生四烯酸(ARA),研究了不同甘蔗糖蜜预处理方法对ARA发酵生产的影响。研究表明:H2SO4法是最利于ARA发酵生产的糖蜜预处理方法。利用预处理的甘蔗糖蜜发酵生产ARA,通过单因素实验设计,确定了最优的培养条件,包括初始还原糖80 g/L,N源6 g/L,接种量20%,初始pH6.0和培养温度25℃,在此条件下发酵,干细胞质量、油脂含量、ARA产量和糖利用率分别达到28.5 g/L、11.7g/L、3.68 g/L和94.5%。     

Development of Candida albicans hyphae in different growth media--variations in growth rates, cell dimensions and timing of morphogenetic events

In six liquid culture media, all of which stimulated Candida albicans to grow in the hyphal form, the rates of hyphal extension and increase in cellular ATP concentration, hyphal diameters, times of evagination of hyphae, times of septum formation and positions of septa in the hyphae appeared to vary independently. There were no discernible associations between properties such as length or volume of hyphal compartments at the time of septation and temporal parameters of hyphal growth. The results suggest that growth environment influences many of the processes contributing to hyphal development, but that these processes are not necessarily interrelated.     

高山被孢霉淬灭方法及胞内代谢产物的气相色谱-质谱分析比较

在花生四烯酸生产菌高山被孢霉代谢组学研究中,需利用胞内代谢物的提取手段并基于气相色谱-质谱（GC-MS）分析方法对其进行检测。比较了3种胞内代谢物提取方法及不同色谱柱条件下GC-MS分析结果。研究表明：采用冷甲醇淬灭分别较液氮直接淬灭及真空过滤后,减少了胞内代谢物的泄露并更好地实现了胞外及胞内代谢物的分离。在对代谢物分析的比较中,极性色谱柱（DB-FFAP）检出的代谢物仅为11种,主要为有机酸、醛类;而代谢物经衍生化后采用非极性色谱柱（DB-5）共检出32种化合物,主要为糖、糖苷及醇类。     

高山被孢霉发酵生产花生四烯酸的宏观形态

采用图像处理技术对高山被孢霉(Mortierella alpina)发酵过程中的不同形态进行分析,并对其产花生四烯酸(ARA)能力进行了比较。研究发现:复合N源中蛋白胨与酵母粉比例是影响高山被孢霉宏观形态的重要因素,球形形态生长的菌体中ARA产量较分散,菌丝体中ARA产量高;在球形形态中,空心球的菌体生物量低,ARA比例低,蓬松球可以兼顾菌体高生物量、高油脂比例及高ARA比例。产ARA能力由生物量、油脂比例及油脂中ARA比例共同决定。结果表明:直径大约4 mm、成核区域面积大约为43.6%、紧密度为71.36的蓬松球形态,是高山被孢霉一种相对较佳的发酵形态,其菌体产ARA能力分别是空心球和分散丝状菌体产ARA能力的2.01和2.70倍。     

实时PCR分析△5脱饱和酶在花生四烯酸合成中的作用

花生四烯酸是人体的必需脂肪酸,具有独特的生物活性。 Δ5脱饱和酶是花生四烯酸生物合成途径中催化二高-γ-亚麻酸脱饱和为花生四烯酸的酶。本研究通过实时定量PCR技术,检测了Δ5脱饱和酶在不同花生四烯酸产量的高山被孢霉(Mortierella alpina)菌株M10,M6和M23中的mRNA表达水平,以及在高产花生四烯酸菌株M6培养过程中的mRNA表达水平的动态变化,发现Δ5脱饱和酶基因的mRNA表达水平与花生四烯酸的产生之间存在明显的线性关系,表明Δ5脱饱和酶是高山被孢霉中花生四烯酸合成途径中起到了非常重要的作用。     

Extraction of lipids from Mortierella alpina and enrichment of arachidonic acid from the fungal lipids

Mortierella alpina is known as an arachidonic acid (AA) producing oleaginous fungus. Extraction of lipids from wet and dry M. alpina biomass was compared. Lipids yield of extraction from dry cells was higher than that of extraction from wet. Wet extraction mainly extracted lipid bodies and lipids in membranes did not extract effectively. Enrichment of AA from the fungal lipids by a urea inclusion method was studied. Most of the saturated and monounsaturated fatty acids, 93.0% and 84.6%, respectively, were removed by forming urea inclusion compounds. AA was concentrated after urea inclusion. Its content in total fatty acids increased 6.2-folds and reached 57.1% with a recovery of 81.9%.