大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α-亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。Ω-3脂肪酸脱氢酶（FAD）催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω- 3FAD的功能,用RT-PCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1％,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸（PUFA）底物LA在Δ15位脱氢生成ALA的ω-3 FAD功能,首次实现大豆ω-3 脂肪酸脱氢酶基因在酿酒酵母K601 p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。     

产生5-氨基乙酰丙酸(ALA)光合细菌生物学研究进展

5氨基乙酰丙酸(ALA)是卟啉、血红素和维生素B12的类似物。ALA作为一种无公害绿色除草剂、杀虫剂、抗微生物药剂、植物生长促进剂及治疗癌症与其它疾病等而备受国外研究者及产业界的关注。本文对ALA光合细菌合成、调节途径、分子遗传学的研究作一简要综述。     

南极乔治王岛土壤微生物的生物学特征及生物活性

从南极乔治王岛土壤分离出77株放线菌和丝状真菌,它们大多数是嗜冷菌或兼性嗜冷菌。它们能在0℃生长,适宜温度为5-15℃、15—20℃或l 5—28℃。用多种条件研究了77株菌的生物活性,发现32％的菌株有抗微生物活性(包括革兰氏阳性和阴性细菌、酵母、青霉等)。其中5株菌对精原细胞、10株对KB细胞有抗肿瘤活性。根据形态培养特征、细胞壁化学组分、放线菌大多数属链霉菌(Streptomyces),其次是诺卡氏菌(Nocardia)和类诺卡氏菌(Nocardioides)。 丝状真菌有5种不同的类型,它们都属金孢霉属(Chysosporium)。     

固定化Ralstonia metallidurans CH34降解苯酚的研究*

将既能耐抗重金属又能降解苯酚的细菌Ralstonia m etalliduransCH34固定化以提高其降酚效率。首先通过正交实验,得到了固定化该菌种的最优制备条件,然后对固定化细胞的降酚效果进行了研究。结果表明,固定化R.m etalliduransCH34的降酚效果明显优于游离细胞;抗重金属毒性方面也有较大提高;在加入额外碳源(甲苯,柠檬酸)情况下,固定化R.m etalliduransCH34进行苯酚降解时所受影响明显要小于游离态菌。     

几株光合细菌的分离鉴定及在养殖罗非鱼中的应用*

从渔场底泥样品中分离纯化得到5株紫色非硫光合细菌,根据分离菌株的细胞形态结构、光合内膜结构、活细胞光吸收特征以及生理生化特征,参照伯杰氏细菌分类学手册,5个分离株分别属于沼泽红假单胞菌(Rhodopseudom onas palusteris)和类球红细菌(Rhodobacter spheroides)。利用它们的纯培养物及混合培养物进行的水产养殖试验表明,光合细菌可以明显提高养殖鱼的成活率以及生长速度,罗非鱼经过2周的养殖后,加入光合细菌的试验组比没加入光合细菌的对照组(成活     

褐煤风化过程中微生物群落的演替

对采自辽宁省前屯煤矿的6种不同风化程度的褐煤样品进行扫描电子显微镜观察发现:刚采掘出来的褐煤表面几乎没有微生物存在.经5个月、1年及4年堆积风化的褐煤中也只见到休眠孢子和少量菌丝.将褐煤样品在潮湿状态下培养10天后,扫描电镜观察发现:刚采出来的褐煤及经5个月风化的褐煤表面有大量放线菌生长,而且菌落周围有褐煤被降解迹象.经1年风化的褐煤中除有大量放线菌及细菌生长外,真菌也有所增加.而在经4年风化褐煤中主要是真菌明显增加.平板计数结果同样说明褐煤风化过程中微生物存在演替现象:放线菌为褐煤初期降解的主要微生物,随后是细菌,在风化程度较高的褐煤中,真菌则为优势降解菌.三株优势放线菌为诺卡氏菌(Nocardia Sp.),束丝放线菌(Actinosynnema Sp.)和链霉菌(Streptomyces sp.).两株优势细菌均为节杆菌(Arthrobacter sp.).两株曲霉为栖土曲霉(A.terricola)及褚曲霉(A.ochraceous),为褐煤风化过程的优势真菌.     

炼油厂冷却器管道生物垢中铁细菌、硫细菌及硫酸盐还原菌的分离和鉴定

炼油厂污水经活性污泥处理及砂滤后,在冷却水系统中循环使用,冷却器管壁上有深棕色软垢形成。从垢中分离到11株铁细菌,经鉴定其中5株为锈色纤毛菌(Leplothrix ochracea);3株为浮游球衣细菌(Sphaerotilus natans);1株为大单鞘铁细菌(Sideromonas major);2株为有鞘丝状铁细菌,按其所具氧化铁及氧化锰的能力属于纤毛菌属,但其培养及形态特征与该属中已知种比较有较显著的差别。     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸(5-aminolevulinate,ALA)由5-氨基乙酰丙酸合酶(5-aminolevulinate synthase,ALAS)催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg/L培养基。纯化的ALA纯度达到90%。     

几株细菌的重金属抗性水平和吸附量*

从堆积时间为80~100 a的铅锌矿渣中分离了6株细菌,通过测定部分16S rRNA基因序列确定了它们的系统发育地位。结果表明有3株细菌属于节杆菌属(Arthrobacter),同A.nicotinovorans和A.histidinolovorans两个种关系密切。另外3株属于壤霉菌属(Agromy-ces),同Ag.m ediolanus具有较近的亲缘关系。总体来看,这些菌株都对检测的5种重金属有高的最低抑菌浓度(minimal inhibitory conc     

代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸

5-氨基乙酰丙酸 (5-aminolevulinic acid,5-ALA) 在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成。为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造。首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸合成酶ALAS,选择酶活相对较高的沼泽红假单胞菌的RphemA基因作为关键合成酶基因,并筛选到能显著增强RphemA的酶活性的核糖体结合位点RBS5。重组菌株ALAS的比酶活可达 (221.87±3.10) U/mg,且5-ALA产量提高了14.3%;随后通过敲除α-酮戊二酸脱氢酶抑制蛋白基因 (odhI) 和琥珀酸脱氢酶基因 (sdhA),促进了前体琥珀酰CoA向5-ALA途径的流动;通过sRNA抑制hemB表达减少了5-ALA的降解;并且过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了5-ALA的输出效率;使用重组菌株C. glutamicum 13032/?odhI/?sdhA-sRNAhemB-RBS5RphemA-eamA摇瓶发酵,5-ALA最高产量达11.90 g/L,较出发菌株提高了57%。最后,在5 L发酵罐中进行补料分批发酵,48 h内5-ALA的产量达25.05 g/L,为目前以葡萄糖为碳源发酵的最高产量。本研究构建了高产5-ALA重组谷氨酸棒杆菌,具有良好的工业应用前景。     

Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid

Microbial production of 5-aminolevulinic acid (ALA) by photosynthetic bacteria compared to other bacteria and algae is reviewed. During aerobic-microaerobic cultivation of Rhodobacter sphaeroides mutant strain CR520, control of the redox potential was effective for producing large amounts of extracellular ALA. ALA has been practically applied in agriculture as an herbicide, an insecticide and a growth-promoting factor for plants. New agricultural applications including salt tolerance and cold temperature tolerance of plants are also described. Finally, recent medical applications for cancer treatment, tumor diagnosis and other clinical uses are discussed.     

光合细菌嗜酸柏拉红菌5-氨基乙酰丙酸合成酶基因的克隆与原核表达

5-氨基乙酰丙酸(ALA)可作为除草剂、杀虫剂和植物生长调节剂在农业上应用,但由于其成本较高而限制了它的大面积使用。利用常规基因工程操作方法结合载体介导PCR法(Vecterette PCR)克隆了嗜酸柏拉红菌(Rhodoblastus acidophilus)的5-氨基乙酰丙酸合成酶(ALAS)基因。并将编码ALAS的基因插入到原核表达载体pQE30中,在大肠杆菌不同菌株(E.coli JM109、M15及BL21(DE3))中进行诱导表达。对产物进行SDS-PAGE分析表明,ALAS基因已在细菌中成功表达。使用Ni-NTA亲和层析法对表达的ALAS进行分离、纯化,得到大小约为44kD的ALAS蛋白。通过优化工程菌株的培养条件,建立了发酵生产ALA的方法,其胞外分泌ALA产量达5.379g/L,ALAS酶活力高达333U/min.mg。这是目前国内外利用生物法生产ALA产量最高的报道,为ALA的产业化应用打下了良好的基础。     

The role of 5-aminolevulinic acid in the response to cold stress in soybean plants

In this study, the possibility of enhancing cold stress tolerance of soybean plants (Glycine max L.) by exogenous application of 5-aminolevulinic acid (ALA) was investigated. ALA was added to the Hoagland solution at various concentrations ranging from 0 to 40 μM for 12 h. After ALA treatment, the plants were subjected to cold stress at 4°C for 48 h. ALA at low concentrations (5-10 μM) provided significant protection against cold stress compared to non-ALA-treated plants, enhancing chlorophyll content (Chl) as well as relative water content (RWC). Increase of thiobarbituric acid reactive species (TBARS) levels was also prevented, whereas exposure to higher ALA concentrations (15-40 μM) brought about a dose dependent increase of these species, reaching a maximum of 117% in plants pre-treated with 40 μM ALA compared to controls. ALA pre-treatment also enhanced catalase (CAT) and heme oxygenase-1 (HO-1) activities. These findings indicate that HO-1 acts not only as the rate limiting enzyme in heme catabolism, but also as an antioxidant enzyme. The highest cold tolerance was obtained with 5 μM ALA pre-treatment. Results show that ALA, which is considered as an endogenous plant growth regulator, could be used effectively to protect soybean plants from the damaging effects of cold stress by enhancing the activity of heme proteins, e.g., catalase (CAT) and by promoting heme catabolism leading to the production of the highly antioxidant biliverdin and carbon monoxide, without any adverse effect on the plant growth.     

Regulation in Plant Stress Tolerance by a Potential Plant Growth Regulator, 5-Aminolevulinic Acid

Exogenous application of different plant growth regulators is a well-recognized strategy to alleviate stress-induced adverse effects on different crop plants by regulating a variety of physiobiochemical processes such as photosynthesis, chlorophyll biosynthesis, nutrient uptake, antioxidant metabolism, and protein synthesis, which are directly or indirectly involved in the mechanism of stress tolerance. Of various environmental factors, salinity, drought, and extreme temperature (low or high) considerably diminish plant growth and yield by modulating endogenous levels as well as signaling pathways of plant hormones. Of various plant hormones/regulators, a potential plant growth regulator, 5-aminolevulinic acid (ALA), is known to be effective in counteracting the injurious effects of various abiotic stresses in plants. Until now the mechanisms behind ALA regulation of growth under stress have not been fully elucidated. It is also not yet clear how far growth and yield in different crops can be promoted by exogenous application of ALA and whether this ALA-induced growth and yield promotion is cost-effective. Thus, in this review we discuss at length the effects of ALA in regulating growth and development in plants under a variety of abiotic stress conditions, including salinity, drought, and temperature stress. Furthermore, advances in the functional and regulatory interactions of this plant growth regulator with plant stress tolerance, as well as the effective mode of exogenous application of ALA in inducing stress tolerance in plants are also comprehensively discussed in this review. In the future, overaccumulation of ALA in plants through manipulation of gene(s) could enhance plant stress tolerance. Thus, genetic manipulation of plants with the goal of attaining increased synthesis/accumulation of ALA and hence improved stress tolerance under stress conditions is an important area for research.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

ALA Pretreatment Improves Waterlogging Tolerance of Fig Plants

5-aminolevulinic acid (ALA), a natural and environmentally friendly plant growth regulator, can improve plant tolerance to various environmental stresses. However, whether ALA can improve plant waterlogging tolerance is unknown. Here, we investigated the effects of ALA pretreatment on the waterlogging-induced damage of fig (Ficus carica Linn.) plants, which often suffer from waterlogging stress. ALA pretreatment significantly alleviated stress-induced morphological damage, increased leaf relative water content (RWC), and reduced leaf superoxide anion (O2⋅¯) production rate and malonaldehyde (MDA) content in fig leaves, indicating ALA mitigates waterlogging stress of fig plants. We further demonstrated that ALA pretreatment largely promoted leaf chlorophyll content, photosynthetic electron transfer ability, and photosynthetic performance index, indicating ALA significantly improves plant photosynthetic efficiency under waterlogging stress. Moreover, ALA pretreatment significantly increased activities of leaf superoxide dismutase (SOD) and peroxidase (POD), root vigor, and activities of root alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH), indicating ALA also significantly improves antioxidant ability and root function of fig plants under waterlogging stress. Taken together, ALA pretreatment improves waterlogging tolerance of fig plants significantly, and the promoted root respiration, leaf photosynthesis, and antioxidant ability may contribute greatly to this improvement. Our data firstly shows that ALA can improve plant waterlogging tolerance.     

Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli

5-Aminolevulinic acid (ALA) is a value-added compound with potential applications in the fields of agriculture and medicine. Although massive efforts have recently been devoted to building microbial producers of ALA through metabolic engineering, few studies focused on the cellular response and tolerance to ALA. In this study, we demonstrated that ALA caused severe cell damage and morphology change of Escherichia coli via generating reactive oxygen species (ROS), which were further determined to be mainly hydrogen peroxide and superoxide anion radical. ALA treatment activated the native antioxidant defense system by upregulating catalase (CAT) and superoxide dismutase (SOD) expression to combat ROS. Further overexpressing CAT (encoded by katG and katE) and SOD (encoded by sodA, sodB, and sodC) not only improved ALA tolerance but also its production level. Notably, coexpression of katE and sodB in an ALA synthase expressing strain enhanced the biomass and final ALA titer by 81% and 117% (11.5 g/L) in a 5 L bioreactor, respectively. This study demonstrates the importance of tolerance engineering in strain development. Reinforcing the antioxidant defense system holds promise to improve the bioproduction of chemicals that cause oxidative stress.     

Role of 5-aminolevulinic acid on growth,photosynthetic parameters and antioxidant enzyme activity in NaCl-stressed <Emphasis Type="Italic">Isatis indigotica</Emphasis> Fort.

5-aminolevulinic acid (ALA) is a key precursor for the biosynthesis of porphyrins such as heme and chlorophyll. ALA alleviates salinity stress damage in germinating seeds and improves seedling growth. Exogenous application of ALA at low concentrations has been shown to enhance salt tolerance in a number of plants. In the present study, we studied the effect of exogenous application of ALA on enhancing salt stress tolerance in Isatis indigotica Fort. (Anhui population as S1, Shanxi population as S2). A foliar application of 0, 12.5, 16.7, 25.0, and 50.0 mg/L ALA was given to the leaves of I. indigotica plants treated with 100 mmol/L NaCl. The fresh weight of leaves and roots; chlorophyll relative content (SPAD value); photosynthetic parameters, such as net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci) and water use efficiency of the treated plants were determined. The third leaf of each treated plant was used to determine the activities of antioxidant enzymes. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutamate synthase (GOGAT), nitrate reductase (NR) activities and the malondialdehyde (MDA) content increased in response to 100 mmol/L NaCl in both S1 and S2 plants. However, the fresh weight of leaf and root, chlorophyll relative content, Pn, Gs, Ci decreased in response to salt stress in both S1 and S2 plants. In all foliar application of ALA in S1 plants, the MDA content, and the activities of SOD and POD were the highest in response to 50.0 mg/L foliar application of ALA. GOGAT and NR activities were the highest in response to 16.7 mg/L foliar ALA. Chlorophyll content and Pn were the highest in S1 plants treated with by 25.0 mg/L ALA. In S2 plants, plant fresh weight, chlorophyll relative content, SOD, CAT, NR activities and Pn treated with 16.7 mg/L ALA were higher than that of the control (CK0). POD, MDA, GOGAT activities in S2 plants treated with 25.0 mg/L ALA were the highest among all treatments. Thus, our results showed that the optimal concentration of ALA (16.7 ~ 25.0 mmol/L) increases the activity of antioxidant enzymes, which in turn helps to abate the damage caused by salt stress in I. indigotica seedlings. Furthermore, ALA also results in an increase in chlorophyll content, Pn and the activities of GOGAT and NR.     

5-Aminolevulinic acid improves photosynthetic gas exchange capacity and ion uptake under salinity stress in oilseed rape (Brassica napus L.)

Salinity is one of the major constraints in oilseed rape (Brassica napus L.) production. One of the means to overcome this constraint is the use of plant growth regulators to induce plant tolerance. To study the plant response to salinity in combination with a growth regulator, 5-aminolevulinic acid (ALA), oilseed rape plants were grown hydroponically in greenhouse conditions under three levels of salinity (0, 100, and 200 mM NaCl) and foliar application of ALA (30 mg/l). Salinity depressed the growth of shoots and roots, and decreased leaf water potential and chlorophyll concentration. Addition of ALA partially improved the growth of shoots and roots, and increased the leaf chlorophyll concentrations of stressed plants. Foliar application of ALA also maintained leaf water potential of plants growing in 100 mM salinity at the same level as that of the control plants, and there was also an improvement in the water relations of ALA-treated plants growing in 200 mM. Net photosynthetic rate and gas exchange parameters were also reduced significantly with increasing salinity; these effects were partially reversed upon foliar application with ALA. Sodium accumulation increased with increasing NaCl concentration which induced a complex response in the macro-and micronutrients uptake and accumulation in both roots and leaves. Generally, analyses of macro- (N, P, K, S, Ca, and Mg) and micronutrients (Mn, Zn, Fe, and Cu) showed no increased accumulation of these ions in the leaves and roots (on dry weight basis) under increasing salinity except for zinc (Zn). Foliar application of ALA enhanced the concentrations of all nutrients other than Mn and Cu. These results suggest that under short-term salinity-induced stress (10 days), exogenous application of ALA helped the plants improve growth, photosynthetic gas exchange capacity, water potential, chlorophyll content, and mineral nutrition by manipulating the uptake of Na⁺.