基因重组与外源铁离子对大肠杆菌合成血红素的影响

【背景】大肠杆菌通过C5途径合成卟啉及血红素,5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)是C5途径中关键的前体物质,血红素由原卟啉IX (protoporphyrin IX, PPIX)螯合一个铁离子所形成,目前5-ALA与PPIX的外泌对卟啉的积累和血红素合成的影响尚不清楚。【目的】构建5-ALA外泌蛋白基因rhtA和卟啉外泌蛋白基因tolC双缺失的大肠杆菌以积累卟啉,同时外源添加铁离子,并过表达亚铁螯合酶基因hemH及参与铁摄取的基因efeB,促进卟啉向血红素的转化。【方法】通过Red同源重组敲除大肠杆菌BL21(DE3)的rhtA和tolC,并外源添加不同浓度的FeSO₄及Fe₂(SO₄)₃,同时构建重组质粒pEHE过表达hemH和efeB,检测卟啉和血红素含量,分析卟啉向血红素的转化。【结果】敲除rhtA和tolC对菌体生长无显著影响,与野生菌WT相比,敲除菌株WT-RT的卟啉含量增加,血红素合成略有提升。外源添加100μmol/L Fe²⁺     

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。     

代谢工程合成5-氨基乙酰丙酸的研究进展

5-氨基乙酰丙酸(5-ALA)是生物体内吡咯类物质生物合成的前体物质,在医药和农业领域显示出广阔的市场潜力。随着代谢工程的发展以及对5-ALA生物合成途径的研究,利用微生物合成5-ALA成为研究的新方向。与传统的化学法合成相比,利用微生物合成5-ALA具有低污染、高产率和低成本等优势。本文中,笔者介绍了5-ALA生物合成的最新研究进展,分析了5-ALA代谢途径的关键影响因素,并对未来的研究方向进行了展望。     

油菜叶绿素b减少突变体Cr3529叶绿素生物合成的研究

利用吸收光谱和荧光光谱法测定了油菜叶绿素b减少突变体Cr3529子叶叶绿素生物合成途径中几种主要前体物质的含量.结果显示:突变体子叶中叶绿素生物合成第一个限速步骤的前体物质δ-氨基乙酰丙酸(ALA)含量与野生型油菜大致相同,饲喂ALA后的突变体及野生型油菜子叶中ALA含量均显著增加,但二者无显著差异;胆色素原含量在突变体中也未降低,而尿卟啉原Ⅲ含量仅为野生型的一半,粪卟啉原Ⅲ、原卟啉Ⅸ、镁原卟啉Ⅸ和原植基叶绿素的含量都明显低于野生型.结果证明,Cr3529突变体中叶绿素生物合成受阻于由胆色素原形成尿卟啉原Ⅲ的步骤,其叶绿素合成缺陷的机制和前体物质的累积与其它叶绿素b减少突变体明显不同.     

大肠杆菌rhtA缺失对血红素合成的影响

【背景】Escherichia coli BL21(DE3)是基因工程的常用宿主,以C5途径合成5-氨基乙酰丙酸(5-Aminolevulinicacid,ALA),ALA是合成血红素的重要前体物质,但ALA分泌对血红素合成的影响尚不清楚。【目的】阐明参与ALA外运的RhtA在血红素合成途径中的作用。【方法】利用Red同源重组,敲除Escherichia coli BL21(DE3)的rhtA,同时构建重组质粒pEA过表达血红素合成途径中的关键酶基因hemA,检测分析血红素及其前体物质含量,以及血红素合成途径中10个关键基因的表达水平。【结果】敲除rhtA对菌体生长没有显著影响,敲除菌株BL21(DE3)Δrht A与原始菌株BL21(DE3)比较,ALA的胞外含量下降23%,血红素含量提高12%,尿卟啉III (Uroporphyrin III,UIII)、粪卟啉III (Coproporphyrin III,CIII)和原卟啉IX (Protoporphyrin IX,PPIX)的含量分别提高25%、15%和18%;敲除rhtA同时过表达hemA的菌株BL21(DE3)ΔrhtA/pEA与仅过表达hemA的菌株BL21(DE3)/pEA比较,胞外ALA减少了16%,血红素含量提高了24%,UIII和CIII含量分别提高55%和64%,PPIX含量显著增加,约为4.7倍。实时定量PCR结果表明,rhtA缺失后,hemC基因转录水平下调,其余9个基因转录水平均有不同程度的上调。【结论】rhtA敲除减少了ALA的外运,使得胞内血红素产量得到提高。     

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。     

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。     

解淀粉芽胞杆菌hemX基因缺失对血红素合成的影响

血红素是一种广泛存在于生物体中的卟啉类化合物,具有多种生理功能。解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有易于培养、分泌表达能力较强等特点,是一种重要的工业菌株。为了筛选血红素合成的最优出发菌株,以不添加和添加5-氨基乙酰丙酸(5-aminolevulinic acid, ALA)的方式,对实验室保藏菌株进行筛选,发现不添加ALA时,菌株BA、BAΔ6、BAΔ6ΔsigF的血红素产量无明显差别;然而添加ALA后,BAΔ6ΔsigF的血红素产量和比生产能力均为最高,分别达到200.77μmol/L和615.70μmol/(L·g DCW)。因此,以BAΔ6ΔsigF为出发菌株,敲除编码细胞色素组装蛋白HemX的hemX基因,探究其在血红素合成途径中的作用,发现敲除菌株发酵液明显变红,且生长未受到明显影响;摇瓶发酵12 h时ALA浓度最高,为82.13 mg/L,略高于对照的75.11 mg/L;不添加ALA时,血红素产量和比生产能力分别为对照的1.99倍和1.45倍;添加ALA后,血红素产量和比生产能力分别为对照的2.08倍和1.72倍;实时定量荧光PCR...     

谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成

【背景】大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hem A)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)和血红素合成的影响。【方法】通过Red同源重组敲除与谷氨酸代谢有关的mscS与aroG,构建hemA表达载体并导入基因缺失菌株中。【结果】mscS单敲除或mscS与aroG双敲除对菌体生长无显著影响。与出发菌株相比,单敲除与双敲除菌株的谷氨酸含量均有所增加,ALA含量略微下降,血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后,胞内血红素含量增至47.603μmol/L。【结论】通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成,该结果为增强C5途径的血红素合成提供了新的思路。     

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Ｂ１２４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。     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

5-Aminolevulinic acid level and dye-decolorizing peroxidase expression regulate heme synthesis in Escherichia coli

Biotechnology Letters - To investigate the level of 5-aminolevulinic acid (5-ALA), a key precursor of heme, and expression of heme-peroxidase on the regulation of heme synthesis in E. coli. A...     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

δ-aminolevulinic acid transport and synthesis,porphyrin synthesis and heme catabolism in chick embryo liver and heart cells

Liver and heart represent two organs with markedly different needs for heme as related to their metabolic roles. To examine these diferences chick embryo heart and liver cells were compared with respect to transport of δ-aminolevulinic acid and activity of δ-aminolevulinic acid synthetase, porphyrin synthesis and heme oxygenase. Heart cells were found to have a low rate of δ-aminolevulinic acid uptake, a high resting level of δ-aminolevulinic acid synthetase activity and a lower level of heme oxygenase activity as compared with liver cells. The hepatic cell uptake of δ-aminolevulinic acid was 6–25-times that of heart cells. The embryonal heart cell appears to be a balanced autonomous system for the synthesis and degradation of heme. The embryonal liver cell represents a cell system permeable to exogenous δ-aminolevulinic acid, which is also responsive to and inducible by external stimuli.     

Tissue-specificity of hemoglobin synthesis: Localization of heme synthesis in the subepidermal fat body of Chironomus thummi (Diptera)

Possible sites of heme synthesis in the fourth instar of Chironomus thummi were investigated by means of autoradiography of specific isotope incorporation. “Body wall” preparations, which include subepidermal and visceral fat body, oenocytes, muscle, epidermis, and cuticle, were cultured for 1 h in a medium containing tritiated-δ-aminolevulinic acid, a specific precursor to heme biosynthesis. Light-microscopic examination of autoradiographs of sections of the body walls indicates that the subepidermal fat body is the major site of incorporation of the precursor into heme. The visceral fat body shows few silver grains. Oenocytes, as well as muscle and epidermis, are characterized by absence of silver deposits. These findings indicate that the subepidermal fat body of Chironomus is the primary site of heme synthesis, and are discussed in relation to specific hemoglobin synthesis.     

Interaction of the inhibitor of the 3',5' cyclic AMP phosphodiesterase of Dictyostelium discoideum with the Affi-Gel blue

Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic δ-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter δ-aminolevulinic acid synthetase activity and had little inductive effect on heme oxygenase activity. Both heme and cobalt/dextran rapidly induced microsomal heme oxygenase by 3–8 fold. Induction of heme oxygenase by heme could be totally blocked by concurrent administration of cycloheximide. These results argue against the hypothesis that iron is the physiological mediator of δ-aminolevulinic acid synthetase activity.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Biosynthetic Preparation of Isotopically Labeled Heme

An efficient method for the preparation of isotopically enriched heme has been developed. This method utilizes a commercially available bacterial host and plasmid, into which a synthetic gene encoding for rat liver outer mitochondrial membrane cytochrome b₅ a heme-binding protein, has been inserted. The method described in this report utilizes the efficient synthesis of the cytochrome b₅ polypeptide together with the enhanced biosynthesis of heme brought about by addition of the first committed precursor in heme biosynthesis, δ-aminolevulinic acid. Apocytochrome b₅ sequesters heme as the macrocycle is being synthesized in order to form holocytochrome b₅, thus avoiding toxic concentrations of free macrocycle in the cell. Relatively high concentrations of free heme in the cell have been shown to stimulate excretion of heme precursors such as coproporphyrinogen and uroporphyrinogen (W. F. Harris III, R. S. Burkhalter, W. Lin and R. Timkovich, (1993) Bioorg. Chem. 21, 209-220), therefore causing isotopic dilution of the labeled material. The heme obtained using this methodology was determined to be >85% enriched. Because the heme in cytochrome b₅ is not covalently attached to the polypeptide, it can be extracted and used in other applications. Use of glutamate, a precursor of δ-aminolevulinate biosynthesis in Escherichia coli, did not result in high levels of isotopic incorporation into heme, thus pointing out to the importance of using a labeled precursor that is committed to heme biosynthesis in order to obtain high levels of isotopic labeling.