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。     

微生物发酵法生产花生四烯酸油脂的研究进展

花生四烯酸(ARA)是一种重要的脂肪酸,现在主要由生物法生产,本文综述了高山被孢霉发酵生产花生四烯酸油脂的菌落形态控制及其代谢途径,以期为相关的研究者提供参考。     

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。     

高山被孢霉的红四氮唑染色程度与菌体油脂中花生四烯酸含量的关系

研究了高山被孢霉菌体被红四氮唑(TTC)染色的条件,并探讨了染色程度与菌体油脂中花生四烯酸含量的关系．高山被孢霉的种子菌体被TTC染色的程度随种龄增加而增加,而种子中的油脂含量和油脂中的花生四烯酸含量也都随种龄增加而增加．在发酵过程中,菌体被TTC染色的程度和菌体中的油脂含量以及油脂中的花生四烯酸含量随培养时间增加而增加．三株具有相似油脂含量、不同花生四烯酸含量的高山被孢霉以及一株不产花生四烯酸的鲁氏毛霉的染色结果显示菌体被红四氮唑染色的程度与菌体油脂中的花生四烯酸含量具有正相关性．该发现有助于花生四烯酸高产菌的快速筛选．     

谷氨酸对花生四烯酸产生菌被孢霉发酵的影响

研究了不同浓度谷氨酸对被孢霉生产花生四烯酸的影响,发现当加入谷氨酸浓度为0.8g/L时总油脂和花生四烯酸产量达最高,选择0.8g/L谷氨酸进行花生四烯酸产生菌被孢霉发酵动力学研究的结果表明,在培养基中加入0.8g/L谷氨酸可以明显促进被孢霉的生长,加速基质代谢,提高单位被孢霉中的油脂和花生四烯酸产量,尤其在发酵第7d时生物量、油脂和花生四烯酸产量达到最大,分别为24.43、9.21、1.41g/L,分别是对照组第7d所得的1.13、1.15和1.69倍。     

过表达亚甲基四氢叶酸脱氢酶对高山被孢霉脂质合成的影响

高山被孢霉是一种富含多不饱和脂肪酸的丝状真菌,但其脂质过程中NADPH的来源还没有研究透彻。以高山被孢霉(尿嘧啶营养缺陷型)作为出发菌株,研究亚甲基四氢叶酸脱氢酶(MTHFD1)对高山被孢霉脂质合成的影响。首先构建了过表达载体pBIG2-ura5s-MTHFD1,采用根癌土壤杆菌介导转化真菌的方法,将二元表达载体转化进高山被孢霉CCFM501中,在筛选培养基SC-CS平板上进行筛选,进而得到稳定遗传MTHFD1基因的过表达菌株(MA-MTHFD1);其次提取MA-MTHFD1菌株基因组进行PCR鉴定,并结合qPCR分析结果,表明MTHFD1基因成功在高山被孢霉中实现了过量表达;最后通过对MA-MTHFD1中的脂肪酸含量、NADPH含量及NADPH合成途径中相关基因转录水平进行分析,研究MTHFD1基因过表达对脂质合成的影响。实验结果表明,过表达MTHFD1基因可以提高高山被孢霉脂质合成能力。与原养型高山被孢霉相比,MA-MTHFD1菌株中脂肪酸含量提高了40.13%,NADPH的含量提高了26.45%,而且NADPH合成途径中其他相关基因苹果酸酶(ME)和异柠檬酸脱氢酶(IDH)的转录水平也发生了上调。这一系列研究结果表明,在高山被孢霉脂质合成还原力形成中,MTHFD1基因起到了关键作用。这为解析高山被孢霉中NADPH来源及深入研究脂质合成机制,从而对其胞内脂肪酸代谢通路进行分子水平上的改建提供了一定的理论依据。     

高山被孢霉Δ5脱饱和酶基因的分离与验证

花生四烯酸(arachidonicacid)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上的关键酶,以二高-γ-亚麻酸为底物,催化其第5位碳脱氢形成花生四烯酸。从花生四烯酸高产菌株高山被孢霉M6(Mortierellaalpina)中通过RT-PCR分离了可能的编码Δ5脱饱和酶完整的cDNA,其大小为1366bp,编码446个氨基酸。推导出的蛋白质含有Δ5脂酰脱饱和酶中保守的特征性结构域,包括该蛋白质N端典型的细胞色素b5结构域,以及3个保守的组氨酸盒。为验证该蛋白质的功能,将该基因片段克隆到穿梭表达载体pPIC9K中,获得的重组载体pPIC9K-D5通过电转化法转化到巴斯德毕赤酵母GS115中,利用G418耐受度筛选出高拷贝数的酵母转化子,在加入外源底物二高-γ-亚麻酸时,利用甲醇诱导酵母转化子表达外源基因。酵母转化子的油脂的气相色谱图谱中出现了一个花生四烯酸的特征峰,进一步对该峰进行气质联谱(GC-MS)分析,证实该峰是花生四烯酸。研究结果表明,从高山被孢霉M6中分离出Δ5脱饱和酶基因。     

高山被孢霉亚甲基四氢叶酸脱氢酶的克隆、表达和功能鉴定

叶酸代谢途径中的亚甲基四氢叶酸脱氢酶（MTHFD）可将5,10-亚甲基四氢叶酸氧化为5,10-甲炔基四氢叶酸,此过程会生成NADH或NADPH。对高山被孢霉中的MTHFD基因进行克隆、表达和功能鉴定,可进一步阐明脂质合成所需还原力NADPH的来源。首先对MTHFD序列进行分析,并以pET28a（+）质粒为载体构建了MTHFD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用比色法分析酶反应产物,表明纯化蛋白质具有MTHFD活性。高山被孢霉MTHFD对NAD⁺和NADP⁺均具有催化能力,但更偏好于将NADP⁺转化为NADPH。最后对高山被孢霉进行发酵培养,发现MTHFD的转录水平在脂质开始积累后发生了明显的上调,表明MTHFD在高山被孢霉脂质合成过程中发挥重要作用,很可能是脂质合成所需NADPH的关键来源。这为对高山被孢霉进行分子改造,使之成为高产各种多不饱和脂肪酸的细胞工程提供了理论依据。     

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。     

高山被孢霉△5脱饱和酶基因的分离与验证

花生四烯酸（arachidonic acid）是人体的必需脂防酸,具有独特的生物活性。△5脱饱和酶是花生四烯酸生物合成途径上的关键酶,以二高-γ-亚麻酸为底物,催化其第5位碳脱氢形成花生四烯酸。从花生四烯酸高产菌株高山被孢霉M6（Mortierella alpina）中通过RT—PCR分离了可能的编码△5脱饱和酶完整的cDNA,其大小为1366bp,编码446个氨基酸。推导出的蛋白质含有△5脂酰脱饱和酶中保守的特征性结构域,包括该蛋白质N端典型的细胞色素b。结构域,以及3个保守的组氨酸盒。为验证该蛋白质的功能,将该基因片段克隆到穿梭表达载体pPIC9K中,获得的重组载体pPIC9K-D5通过电转化法转化到巴斯德毕赤酵母GS115中,利用G418耐受度筛选出高拷贝数的酵母转化子,在加入外源底物二高-γ-亚麻酸时,利用甲醇诱导酵母转化子表达外源基因。酵母转化子的油脂的气相色谱图谱中出现了一个花生四烯酸的特征峰,进一步对该峰进行气质联谱（GC—MS）分析,证实该峰是花生四烯酸。研究结果表明,从高山被孢霉M6中分离出△5脱饱和酶基因。     

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.     

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.     

Sesamin inhibits lysophosphatidylcholine acyltransferase in Mortierella alpina

The filamentous fungus, Mortierella alpina, accumulates complex lipids relatively rich in arachidonic acid (C(20:4) Delta(5,8,11,14)). The lignan, sesamin, has been used to reduce arachidonic acid production by specifically inhibiting Delta(5)-desaturation [Shimizu, Akimoto, Shinmen, Kawashima, Sugano and Yamada (1991) Lipids 26, 512-516]. Microsomal membrane preparations from M. alpina exhibit acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase (LPCAT) activity. LPCAT is an enzyme involved in channelling fatty acid substrates to phosphatidylcholine for subsequent desaturation. Sesamin was found to inhibit this enzyme as measured in both spectrophotometric and radioactive assays. The inhibitory effect of sesamin on LPCAT was only evident in species of Mortierella and could not be demonstrated in other organisms.     

Genome characterization of the oleaginous fungus Mortierella alpina

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.     

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).     

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.     

Effects of aeration on metabolic profiles of <Emphasis Type="Italic">Mortierella alpina</Emphasis> during the production of arachidonic acid

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden–Meyerhof–Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography–mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.     

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。