5-氨基乙酰丙酸对NaCl胁迫下番茄幼苗光合特性的影响

为探讨5-氨基乙酰丙酸（ALA）对NaCl胁迫下番茄光合特性的调控作用,以‘金鹏一号’番茄幼苗为试材,研究叶面喷施50 mg·L^-1或根施10 mg·L^-1 ALA对100 mmol·L^-1 NaCl胁迫下番茄幼苗光合及叶绿素荧光参数的影响.结果表明： NaCl胁迫下,番茄幼苗光合气体交换参数（净光合速率P_n、气孔导度g_s、胞间CO₂浓度C_i、蒸腾速率T_r）及叶绿素荧光参数（实际光化学量子产量F_v′/F_m′、F_m′、PSⅡ反应中心实际光化学效率Φ_PSⅡ、表观光合电子传递效率ETR、光化学淬灭q_P、光化学反应Pc）均显著降低,根施或叶施ALA均可以提高NaCl胁迫下番茄叶片的光合能力,但两种处理方式之间存在一定差异.叶面喷施50 mg·L^-1ALA或根施10 mg·L^-1ALA处理均显著提高了番茄叶片P_n、T_r、g_s和C_i,提高了水分利用效率（WUE）,显著增加了NaCl胁迫下叶片的最大净光合速率,减轻了光抑制.根施ALA对叶绿素含量的作用效果较好,而叶施ALA对光合参数的作用效果较好,两处理叶绿素荧光参数差异不显著.叶面喷施或根施ALA可以提高番茄幼苗的耐盐性,其调控作用与促进叶绿素合成与稳定、维持正常气孔开闭、降低气孔限制,进而提高NaCl胁迫下番茄叶片的光合能力和PSⅡ光化学效率有关.
     

微生物发酵生产5-氨基乙酰丙酸研究进展

5-氨基乙酰丙酸是生物体内吡咯生物合成途径的关键中间产物,具有广泛的应用前景。文中从三方面归纳了国内外关于5-氨基乙酰丙酸的最新研究进展:生产5-氨基乙酰丙酸的微生物筛选分离与诱变;基于C4途径的微生物全细胞生物转化合成5-氨基乙酰丙酸;基于微生物代谢工程改造构建高产5-氨基乙酰丙酸的工程菌株。最后,预测了未来5-氨基乙酰丙酸的研究方向和焦点。     

Urinary 5-aminolevulinic acid in lead-exposed children

Lead intoxication can interfere with haem synthesis and alter the concentration of haem precursors, such as the neurotoxin 5-aminolevulinic acid, in plasma and urine. The relationship between blood lead concentration (PbB), a biomarker of lead exposure, and 5-aminolevulinic acid concentration in urine (ALAU), a biomarker of the early biological effect of lead, was examined in lead-exposed children. ALAU was assayed by chemical derivatization and high performance liquid chromatography with fluorescence detection. The study subjects were 79 children with moderate to high lead exposure recruited from a lead-poisoning prevention clinic. Their urine had been previously analysed for creatinine (CR) concentration and the benzene metabolite trans,trans- muconic acid, and their blood had been analysed for lead. We found that ALAU was not correlated with PbB (Spearman r=0.088, p=0.44), but the ratio ALAU/CR was correlated with PbB (Spearman r=0.22, p=0.054). Creatinine and ALAU concentrations were higher in urine samples collected in the afternoon than those collected in the morning, a finding that is consistent with known diurnal variation. However the ratio ALAU/CR was not different in morning and afternoon urines, supporting the use of creatinine adjustment of ALAU analysis of spot urine samples. In view of the neurotoxic properties of ALA, future validation studies of biomarkers of lead exposure and effect in children should include ALAU or ALAU/CR as potential markers of lead effect.     

Urinary 5-aminolevulinic acid in lead-exposed children

Lead intoxication can interfere with haem synthesis and alter the concentration of haem precursors, such as the neurotoxin 5-aminolevulinic acid, in plasma and urine. The relationship between blood lead concentration (PbB), a biomarker of lead exposure, and 5-aminolevulinic acid concentration in urine (ALAU), a biomarker of the early biological effect of lead, was examined in lead-exposed children. ALAU was assayed by chemical derivatization and high performance liquid chromatography with fluorescence detection. The study subjects were 79 children with moderate to high lead exposure recruited from a lead-poisoning prevention clinic. Their urine had been previously analysed for creatinine (CR) concentration and the benzene metabolite trans,trans- muconic acid, and their blood had been analysed for lead. We found that ALAU was not correlated with PbB (Spearman r=0.088, p=0.44), but the ratio ALAU/CR was correlated with PbB (Spearman r=0.22, p=0.054). Creatinine and ALAU concentrations were higher in urine samples collected in the afternoon than those collected in the morning, a finding that is consistent with known diurnal variation. However the ratio ALAU/CR was not different in morning and afternoon urines, supporting the use of creatinine adjustment of ALAU analysis of spot urine samples. In view of the neurotoxic properties of ALA, future validation studies of biomarkers of lead exposure and effect in children should include ALAU or ALAU/CR as potential markers of lead effect.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

Inhibition by light of 5-aminolevulinic acid synthase in extracts from Rhodopseudomonas sphaeroides

Abstract Activity of the key enzyme for tetrapyrrole biosynthesis, 5-aminolevulinic acid synthase, was inhibited upon irradiation of cell-free extracts from Rhodopseudomonas sphaeroides . maximum inactivation was observed after irradiation with light of 422, 522 and 552–556 nm. The relevance of this effect in the control of bacteriochlorophyll synthesis is discussed.     

Hormonal activities of 5-aminolevulinic acid in callus induction and micropropagation

The role of 5-aminolevulinic acid (5-ALA) as a precursor of chlorophyll and a herbicide is well documented. 5-ALA as a Plant Growth substance is also proven in recent times. In the present report, to elucidate the physiological effects of 5-ALA, the compound was used in in vitro studies using MS medium supplemented with 5-ALA at 2, 5 and 10 mg l^-1. Leaf and cotyledonary node were used as the explants. In vitro studies confirmed the hormonal role of 5-ALA by striking proliferation of callus paripassu induction of rooting and shooting with a profound effect of the former than the latter. Thus, 5-ALA has the dual properties of auxin and cytokinin in the induction of callusing and rhizogenesis, and shooting respectively.     

提高类球红细菌5-氨基乙酰丙酸产率的措施

5-氨基乙酰丙酸是四吡咯化合物生物合成的必需前体物,它作为一种无公害的绿色除草剂、杀虫剂、植物生长促进剂以及治疗癌症的药物而受到广泛的关注。文章综述了类球红细菌5-氨基乙酰丙酸的发酵生产现状以及提高其发酵产率的策略与措施。     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

Roles of phosphate and an enoyl radical in ferritin iron mobilization by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria (AIP) and lead poisoning, undergoes enolization and subsequent iron-catalyzed oxidation at neutral pH. Iron is released from horse spleen ferritin (HoSF) by both ALA-generated O₂^•− and enoyl radical (ALA√), which amplifies the chain of ALA oxidation (autocatalysis). Iron chelators such as EDTA, ATP, but not citrate, and phosphate accelerate this process and ALA-promoted iron release from HoSF is faster in horse spleen isoferritins containing larger amounts of phosphate in the core. ALA (+0.377 V versus standard hydrogen electrode) is less effective in releasing iron from ferritin than are thioglycollic acid, 6-hydroxydopamine, and N,N,N′,N′-tetramethyl-p-phenylenediamine. During electrochemical one electron oxidation of ALA in a nitrogen atmosphere, spin trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid demonstrated the formation of a spin adduct characterized by a six line signal, indicating a secondary carbon-centered radical and attributed to a resonant ALA√ radical. Iron is also released in such anaerobic electrochemical oxidations of ALA in the presence of ferritin, suggesting that, in addition to O₂^•−, ALA√ can promote iron mobilization from ferritin. Hence, ALA√ may amplify the metal-catalyzed oxidation of ALA, damaging ALA-accumulating cells and possibly contributing to the symptoms of porphyria.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Peptide transporter 2 (PEPT2) expression in brain protects against 5-aminolevulinic acid neurotoxicity

The proton-coupled oligopeptide transporter PEPT2 (or SLC15A2 ) is the major protein involved in the reclamation of peptide-bound amino acids and peptide-like drugs in kidney. PEPT2 is also important in effluxing peptides and peptidomimetics from CSF at the choroid plexus, thereby limiting their exposure in brain. In this study, we report a neuroprotective role for PEPT2 in modulating the toxicity of a heme precursor, 5-aminolevulinic acid (5-ALA). Our findings demonstrate that in PEPT2-deficient mice, 5-ALA administration results in reduced survivability, a worsening of neuromuscular dysfunction, and CSF concentrations of substrate that are 8–30 times higher than that in wild-type control animals. The ability of PEPT2 to limit 5-ALA exposure in CSF suggests that it may also have relevance as a secondary genetic modifier of conditions (such as acute hepatic porphyrias and lead poisoning) in which 5-ALA metabolism is altered and in which 5-ALA toxicity is important.     

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...     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。     

Biosynthesis of 5-aminolevulinic Acid via the Shemin Pathway in a Green Sugar Beet Callus

5-Aminolevulinic acid synthase (ALAS) has been detected in a normal (auxin- and cytokinin-dependent) green sugar beet callus under light and under darkness. ALAS activity was lower when the callus was grown under light. The supply of precursors of the Shemin pathway (glycine and succinate) to dark-grown callus enhanced considerably the capacity of the 5-aminolevulinic acid (ALA) formation. Glutamate, -aminobutyrate or -ketoglutarate also increased ALA accumulation. Such an accumulation was also obtained after inhibition of polyamine synthesis. The results show that glutamate or its derivatives might feed the Shemin pathway in conditions preventing glutamate to be used through the Beale pathway.     

Improving salt tolerance of cotton seedlings with 5-aminolevulinic acid

Of 12 different plant growth regulators (PGRs) tested,5-aminolevulinic acid (ALA) was found to improve the salttolerance of cotton seedlings. Cotton seedlings treated with ALAcould grow in soil containing levels as high as 1.5% (wt/wt)NaCl. The analyses of mineral compositions of plant parts revealed that the Na^plus concentrations in the roots of the plantstreated with ALA were suppressed to low concentrations. Fromthese results, it can be presumed that the presence of ALA maycause a reduction of Na^plus uptake.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.     

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.