透明颤菌血红蛋白基因的研究与应用

总结了近 15年来透明颤菌血红蛋白的研究结果 ,包括它的分布、结构、功能、合成等分子生物学以及在基因工程中的应用。透明颤菌的血红蛋白是 2 0世纪 70年代被发现的 ,由于它具有结合氧的特性 ,可使透明颤菌这一专性好氧的革兰氏阴性菌在贫氧环境中生长。透明颤菌血红蛋白是同型二聚体形式的可溶性血红蛋白分子 ,每分子透明颤菌血红蛋白由两个分子量为 15 775的亚基和两个b型血红素组成。透明颤菌血红蛋白的功能是为透明颤菌强大的呼吸膜增加氧的流量。完整的有功能的血红蛋白由血红蛋白亚基和血红素组成 ,血红蛋白亚基由基因vgb编码 ,血红素由生化代谢合成。透明颤菌血红蛋白基因在野生透明颤菌中是以单拷贝的方式随染色体一起复制表达的。透明颤菌血红蛋白基因已经被克隆和测序。同时也讨论了将透明颤菌血红蛋白基因整合到异源宿主菌中增加重组蛋白产量和发酵产量方面的研究。最后 ,概述了当透明颤菌血红蛋白在植物中表达时 ,转基因植物表现出生长增加以及代谢物产量发生变化的情况。     

利用PCR法扩增透明颤菌血红蛋白基因条件的优化

以透明颤菌染色体基因组DNA为模板,利用PCR技术获取透明颤菌(Vitreoscilla)血红蛋白基因(vgb)。在多聚合酶链式反应中采用碱变性模板与热启动等方法进行透明颤菌血红蛋白基因体外扩增,成功地扩增出约0.5 kb的透明颤菌血红蛋白基因,并对vgb的PCR条件进行了研究。获取理想的目的基因PCR反应的综合参数比十分重要。     

透明颤菌血红蛋白基因在金色链霉菌中的克隆与表达

分别用质粒pJJ699与pUC19(vhb),pIJ702与pBR322(vhb),构建大肠杆菌链霉菌穿梭质粒,将透明颤菌血红蛋白基因转入金色链霉菌。在低溶解氧浓度下,透明颤菌血红蛋白的表达,可提高金色链霉菌氧的利用效率,产物合成比原始菌株提高40%～60%。在局部低氧的环境中,采用四环素抗性基因启动子带动血红蛋白基因表达,可有效发挥透明颤菌血红蛋白的氧传递效率,优于透明颤菌血红蛋白基因受溶解氧调控的天然启动子。     

透明颤菌(沈阳株)血红蛋白基因克隆与序列分析

目的：以透明颤菌（Vitreoscilla）基因组DNA为模板,扩增透明颤菌血红蛋白基因（vgb）。方法：采用PCR81与摹甲季缉彗术,将PCR产物插入到克隆载体PET28a中,测序应用DNA测序仪。结果：获得了约0．5kbDNA片段,测序结果与已发表的透明颤菌血红蛋白基因核苷酸序列比较,同源性为97％。结论：所扩增的DNA片段确认是透明颤菌血红蛋白基因。     

在链霉菌中表达透明颤菌血红蛋白需要异源启动子

构建了质粒pIJ4083Mpro、pIJ4083\|pro\,pWLD8和pFW3。在浅青紫链霉菌TK24中,启动子探针质粒pIJ4083上的邻苯二酚双加氧酶基因(xylE)不能被透明颤菌血红蛋白基因(vgb)的启动子带动转录,表明vgb启动子在链霉菌中无作用。TK24中,pWLD8和pFW3均能表达透明颤菌血红蛋白(VHb),pWLD8上可能是由Plac带动vgb的表达;pFW3上vgb基因去掉了非必要部分,克隆在PCR扩增得到的glnA启动子下游,两者连成嵌合基因。     

利用ORF438启动子在链霉菌中表达透明颤菌血红蛋白

利用ＯＲＦ４３８启动子在链霉菌中表达透明颤菌血红蛋白崔风文杨胜利（中国科学院上海生物工程研究中心上海２００２３３）１９８８年,由原核的透明颤菌（Ｖｉｔｒｅｏｓｃｉｌａｓｐｐ．）克隆到血红蛋白基因（ｖｇｂ）［１］,其后Ｍａｇｎｏｌｏ等在天蓝链霉菌及变青...     

透明颤菌血红蛋白基因表达对金色链霉菌生长代谢的影响

利用四环素抗性基因启动子在金色链霉菌中表达透明颤菌血红蛋白基因。在1m3发酵罐中研究了工程菌株的生长代谢特性。在溶解氧充足的条件下,透明颤菌血红蛋白表达,对金色链霉菌生长代谢未产生明显影响,工程菌株与参比菌株的生长代谢特性基本一致,工程菌株和参比菌株金霉素最终浓度分别为22905u/mL、22896u/mL。在低溶解氧条件下,透明颤菌血红蛋白的表达,可促进金色链霉菌菌体生长、菌丝活力保持和金霉素的合成：工程菌菌体浓度比参比菌株高5％～10％,产物合成提高11.4％。     

透明颤菌血红蛋白与耐低氧抗生素生产菌株构建

透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)是由透明颤菌属细菌产生的氧结合蛋白。通过引入透明颤菌血红蛋白基因(vgb)从降低需氧角度定向改造抗生素生产菌是近年来研究的热点。vgb在宿主菌中的异源表达能有效提高宿主菌在低氧环境下的代谢效率,促进代谢产物积累,基于这一特性,采用基因工程育种策略构建具有低氧耐受性的生产菌种,将有效降低抗生素生产成本。简述了近年来VHb的研究进展,重点总结了耐低氧抗生素生产菌株构建的研究及其应用现状,并对其在抗生素工业生产中的应用前景进行展望。     

大肠杆菌-链霉菌穿梭载体的构建及应用

pIJ6021和pIJ4123是链霉菌的高拷贝表达载体,它们携带有受硫链丝菌素诱导的强启动子P_tipA。分别在它们的合适位点插入大肠杆菌质粒的复制子和在大肠杆菌中选择用的抗性标记基因(bla),得到了两个能在大肠杆菌和链霉菌中穿梭复制、并保持结构稳定的链霉菌表达载体：pHZ1271和pHZ1272。将透明颤菌(Vitreoscillia sp.)血红蛋白基因(vhb)克隆到pHZ1272中,用它转化变铅青链霉菌(Streptomyces lividans),经Western blotting分析和CO结合实验表明,在变铅青链霉菌中表达出了有生物活性的透明颤菌血红蛋白,从而证明所构建的pHZ1272载体具有在链霉菌中表达外源基因的功能。     

透明颤菌血红蛋白基因在产PHB重组大肠杆菌中的引入

将透明颤菌血红蛋白基因 (vgb)采用插入染色体的方式引入产聚 β 羟基丁酸酯(PHB)重组大肠杆菌VG1 (pTU1 4)中 ,以从分子水平上提高克隆菌对氧的利用率 ,解决PHB发酵生产过程中的供氧矛盾 ,透明颤菌血红蛋白的一氧化碳差光谱分析明表 ,vgb基因可以在VG1 (pTU1 4)中成功表达 ,且其表达量受溶氧水平的调控。Vgb基因的引入可以同时促进菌体生长和PHB产品的积累 ,且溶氧水平越低 ,VHB表达量越高 ,这种促进作用就越明显     

Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering

In hypoxic conditions, bacteria express a kind of hemoglobin, which is proposed to enhance respiration and energy metabolism by promoting oxygen delivery. Bacteria hemoglobin from Vitreoscilla stercoraria - Vitreoscilla hemoglobin (VHb), when expressed in various hosts in oxygen-limited conditions, has been shown to improve growth, protein secretion, metabolite productivity and stress resistance of hosts, thus rendering the protein promising in metabolic engineering, especially in plant metabolism optimization. In this review, many well-studies areas are presented to illustrate the potential of VHb application in biotechnology industry, to discuss the cellular mechanisms of VHb function and to show the wide variety of approaches taken within the field.     

透明颤菌血红蛋白的研究与发展前景

在缺氧条件下,透明颤菌血红蛋白(VHb)通过促进氧输送增强呼吸和能量代谢,并在各种宿主中表达,显示出改善增长,蛋白质分泌,代谢产物的生产力和提高宿主抗逆性等的生理效应,从而使蛋白质在代谢工程尤其在优化植物代谢中应用前景广阔.概括了VHb在生物技术工业的诸多研究领域中的应用潜力,探讨VHb功能的细胞机制,并显示出各领域所采取的各种方法.     

Expression of vitreoscilla hemoglobin improves growth and levels of extracellular enzyme in Yarrowia lipolytica

Enhancement in oxygen uptake by high-cell-density cultivations has been achieved previously by expression of the bacterial hemoglobin gene from Vitreoscilla. The Vitreoscilla hemoglobin (VHb) gene was expressed in the yeast Yarrowia lipolytica to study the effect of expression in this commercially important yeast. The expression of VHb in this yeast was found to enhance growth, contrary to reported observations in wild-type Saccharomyces cerevisiae in which there was no significant growth enhancement. VHb-expressing Y. lipolytica exhibited higher specific growth rate, enhanced oxygen uptake rate, and higher respiratory activity. We report the beneficial effects of VHb expression on growth under microaerobic as well as under nonlimiting dissolved oxygen conditions. Earlier studies in Y. lipolytica have demonstrated inhibition of mycelia formation by respiratory inhibitors and poor nitrogen source, conditions poor for growth. VHb(+) Y. lipolytica cells were more efficient at forming mycelia, indicating better utilization of available oxygen as compared with the VHb(-) cells. Expression of VHb was also found to increase the levels of enzyme ribonuclease secreted into the medium, a property that may be beneficial for producing heterologous proteins in Y. lipolytica.     

Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth and oxygen uptake

The hemoglobins found in unicellular organisms show a greater chemical reactivity, protect cells against oxidative stress and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than VHb counterparts.     

透明颤菌血红蛋白的应用进展

透明颤菌血红蛋白（VHb）具有在限氧条件下促进异源宿主细胞生长和增加产物产量的作用,已在发酵、环保、转基因动植物、重组蛋白表达等方面得到了广泛应用。将VHb与酶或蛋白融合表达可提高酶的活性、稳定性或蛋白的分离效率。对VHb进行改造有助于获得性能更良好的"新"蛋白。     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (VHb): role of Tyr95 (G5) and Tyr126 (H12)

Although Vitreoscilla hemoglobin (VHb) carries a conventional globin fold, its proximal site geometry is unique in having a hydrogen-bonding network between proximal site residues, HisF8-TyrG5-GluH23 and TyrG5-TyrH12. TyrG5 and TyrH12 were mutated to study their relevance in VHb function. VHb G5 mutants (Tyr95Phe and Tyr95Leu showed no stable oxyform and nitric oxide dioxygenase activity, whereas, VHb H12 mutants (Tyr126Phe and Tyr126Leu) displayed little change in their oxygen affinity indicating a crucial role of Tyr95 in protein function. The VHb H12 mutant, Tyr126Leu, enhanced the intracellular pool of oxygen and cell growth better than VHb. Molecular modeling suggests that the replacement of tyrosine with leucine in Tyr126Leu creates an opening on the protein surface that may facilitate oxygen diffusion and accumulation.     

Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases

The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.