Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25°C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17–21°C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25°C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.     

红发夫酵母积累虾青素的代谢调控机理研究进展

由红发夫酵母合成的虾青素是一种非常有商业价值的类胡萝卜素.综述了近年来国内外在红发夫酵母中虾青素的生物合成途径、合成代谢调控机理等领域的相关研究进展,并提出了国内应开展的相关创新性研究.     

协同诱变法选育虾青素高产优良酵母菌株

以红法夫酵母(Phaffia rhodozyma)野生型菌株GH1为研究对象,选育虾青素高产优良酵母菌株,并初步研究其生长特性。对野生型菌株GH1用紫外线(UV)和亚硝基胍(NTG)协同诱变,用抗霉素A筛选,选育出虾青素高产突变菌株GH1-7.1。突变菌株GH1-7.1的虾青素最高产量达1200μg/gDCW,比野生菌株GH1的虾青素最高产量271μg/gDCW高出4倍,并且生长加快,达到最高生物量和最高虾青素产量的时间比野生菌株缩短了24h;野生菌株GH1的最适生长温度为20℃,高于28℃则不生长,而突变菌株GH1-7.1的最适生长温度为28～30℃,比野生菌株GH1提高8～10℃。首次采用协同诱变的方法成功选育了虾青素高产酵母菌株,并且生长周期短,最适的生长温度提高,是适宜南方高温环境发酵工程应用的优良菌株。     

红发夫酵母积累虾青素的代谢调控机理研究进展

由红发夫酵母合成的虾青素是一种非常有商业价值的类胡萝卜素。综述了近年来国内外在红发夫酵母中虾青素的生物合成途径、合成代谢调控机理等领域的相关研究进展, 并提出了国内应开展的相关创新性研究。     

高产虾青素红发夫酵母选育研究进展

虾青素是 60 0多种类胡萝卜素中的一种 ,具有抗氧化 ,抗肿癌和增强免疫力等许多重要的生理和生物学功能 ,在水产养殖、食品和医药等领域应用前景广阔。综述了虾青素的生物来源、生物转化途径以及高产虾青素红发夫酵母菌株的选育。     

白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。     

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.     

Citrate, a possible precursor of astaxanthin in Phaffia rhodozyma: influence of varying levels of ammonium, phosphate and citrate in a chemically defined medium.

The influence of ammonium, phosphate and citrate on astaxanthin production by the yeast Phaffia rhodozyma was investigated. The astaxanthin content in cells and the final astaxanthin concentration increased upon reduction of ammonium from 61 mM to 12.9 mM (from 140 microg/g to 230 microg/g and 1.2 microg/ml to 2.3 microg/ml, respectively). Similarly, both the astaxanthin content and astaxanthin concentration increased by reducing phosphate from 4.8 mM to 0.65 mM (160 microg/g to 215 microg/g and 1.7 microg/ml to 2.4 microg/ml, respectively). Low concentrations of ammonium or phosphate also increased the fatty acid content in cells. By analogy with lipid synthesis in other oleaginous yeasts, an examination of the data for varying nitrogen and phosphate levels suggested that citrate could be the source of carbon for fatty acids and carotenoid synthesis. Supporting this possibility was the fact that supplementation of citrate in the medium at levels of 28 mM or higher notably increased the final pigment concentration and pigment content in cells. Increased carotenoid synthesis at low ammonium or phosphate levels, and stimulation by citrate were both paralleled by decreased protein synthesis. This suggested that restriction of protein synthesis could play an important role in carotenoid synthesis by P. rhodozyma.     

一株法夫酵母虾青素高产菌株的生产性能

为了评价虾青素高产菌株-法夫酵母JMU-MVP14的生产性能及建立虾青素高产发酵技术,通过测定糖、生物量、虾青素产量、总类胡萝卜素产量等发酵参数,用摇瓶试验对比了法夫酵母JMU-MVP14和出发菌株的差异,用7 L罐试验对比了pH值调控方式及补料培养基成分对发酵的影响,用1 m3罐试验评估了法夫酵母JMU-MVP14高密度发酵虾青素的产量水平。摇瓶发酵结果表明,法夫酵母JMU-MVP14虾青素及总类胡萝卜素的细胞产率分别达到6.01 mg/g及10.38 mg/g;7 L罐分批发酵试验结果表明,自动流加调     

Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

法夫酵母虾青素合成途径相关基因的研究进展

法夫酵母能合成一种具有很高商业价值的类胡萝卜素——虾青素。它广泛应用于饲料、保健品、医药、化妆品等行业。探索法夫酵母中虾青素合成途径及其调控机理对天然虾青素资源的开发具有重要的意义。虽然许多学者通过各种方法对该途径进行了一系列的研究, 但其机理目前尚未完全阐明。本文综述了法夫酵母虾青素合成途径以及合成途径中相关基因的研究进展, 并对基于基因调控的产量提高策略进行了讨论, 为利用基因工程技术进行定向育种提供了思路。     

法夫酵母高密度培养及虾青素的高产研究*

本文对法夫酵母Phaffia rhodozyma的不同流加培养模式进行了研究。实验结果表明,采用指数流加,虾青素产率和细胞干重具有较大值,分别达到14.52mg/l和32.56g/l;其次是恒pO2流加和恒速流加培养,虾青素产率分别达到8.89mg/l和6.70mg/l; 恒pH流加方式更有利于法夫酵母细胞的生长(14.62g/l DCW)。但是,不同流加培养模式所得的μmax和qasta具有较大的差距。恒pH、恒pO2流加培养及间歇培养有较大值,分别为0.0613 h-1、0.056 h-1、0.053 h-1;指数流加的μmax较小。间歇培养中虾青素生成比率最大,qasta=0.048×10-3h-1。     

烷化剂NTG诱变虾青素产生菌红法夫酵母的研究

虾青素是一种很有效的生物抗氧化剂和某些生物的天然着色剂,应用前景广阔。红法夫酵母是 生产虾青素的一个来源,优点颇多。天然菌株虾青素产量较少,缺少实用价值。实验采用烷化剂ＮＴＧ 诱变红法夫酵母,筛选出类胡萝卜素产量高的诱变株。用薄层层析对红法夫酵母产生的色素及其皂 化产物进行分析,并对各个成分的扫描光谱进行了比较。认为红法夫酵母产生的类胡萝卜素成分主 要是虾青素及虾青素二酯,还有一部分β－胡萝卜素。同时,还对虾青素产生的时相和ＢＨＴ对虾青素 光分解的保护作用进行了初步研究。     

Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2)

We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.     

INHIBITION OF ASTAXANTHIN SYNTHESIS UNDER HIGH IRRADIANCE DOES NOT ABOLISH TRIACYLGLYCEROL ACCUMULATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)1

Under stress conditions, Haematococcus pluvialis Flotow accumulates fatty acid–esterified astaxanthin, in extraplastidial lipid globules. The enhanced accumulation of fatty acids, mainly in triacylglycerols (TAG), among which oleic acid predominates, is linearly correlated with that of astaxanthin. We used inhibitors of either carotenoid or lipid biosynthesis to assess the interrelationship between carotenogenesis and TAG accumulation under high light irradiance as the stress factor. The two carotenogenesis inhibitors used—norflurazon, an inhibitor of phytoene desaturase, and diphenylamine (DPA), an inhibitor of β‐carotene C‐4 oxygenase—suppressed the accumulation of astaxanthin in a concentration‐dependent manner. Concurrently, the accumulation of neutral lipids was significantly less affected. The lipid biosynthesis inhibitor sethoxydim, which inhibits acetyl‐CoA carboxylase, significantly decreased de novo fatty acid synthesis and, in concert, drastically inhibited astaxanthin formation. In the presence of various concentrations of the three inhibitors, the inhibition of astaxanthin was not accompanied by a proportional decrease in oleic acid, which was used as a marker for TAG fatty acids. When astaxanthin synthesis was completely inhibited, the volumetric content of oleic acid was about 60% of the control value when the two carotenogenesis inhibitors (0.05 μM norflurazon or 20 μM DPA) were used and 27% of the control when the lipid‐synthesis inhibitor (50 μM) was used. We suggest therefore that TAG accumulation under high irradiance is not tightly coupled with astaxanthin accumulation, although the correlation between these two processes was demonstrated earlier. Furthermore, we propose that the accumulation of a certain amount of TAG is a prerequisite for the initiation of fatty acid–esterified astaxanthin accumulation in lipid globules.     

高温湿热酸法破壁提取法夫酵母胞内虾青素

法夫酵母是一种能积累虾青素的红酵母, 对其进行破壁是当前虾青素工业化提取生产的瓶颈工艺。研究在高温湿热条件下,低浓度盐酸对法夫酵母破壁而提取其胞内虾青素的工艺。探讨了不同破壁温度、盐酸浓度、液料比与破壁处理时间等因素对法夫酵母破壁后虾青素及类胡萝卜素提取率的影响, 确定了高温湿热酸法破壁提取虾青素的最佳条件为: 饱和蒸汽压力 0.1 MPa (121°C), 盐酸浓度0.5 mol/L, 液料比 (V/W)30 mL/g, 破壁时间2 min。在最佳条件下进行中试放大实验, 可得到虾青素与类胡萝卜素的提取率分别为: (84.8±3.2)%, (93.3±2)%。经优化后的新破壁工艺安全高效, 不会有毒性残留, 具有良好的工业应用前景。     

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.