集胞藻PCC6803 priA基因的突变体

目前对蓝藻的高温耐受性研究主要集中在热激蛋白和光系统II放氧蛋白复合体的外周蛋白基因,如放氧蛋白复合体的3个外周蛋白基因psbO、psbU和psbV[1-4],热激蛋白基因htpG[5]和hsp17[6],而对于是否存在其他耐受高温所需基因尚未进行系统的筛选.     

集胞藻PCC6803 EGY同源基因破坏突变体的构建及表型分析

摘要：拟南芥中近来发现的定位于叶绿体的膜嵌合金属蛋白酶EGY1影响叶绿体发育与脂肪酸合成,经生物信息学分析,集胞藻PCC6803 (Synechocystis sp. PCC6803)中slr0643、sll0862基因编码同源蛋白。【目的】为了鉴定这两个基因的功能,【方法】本文通过同源重组插入卡那霉素抗性基因、切断目的基因,分别构建了slr0643::km和sll0862::km两种突变体,检测突变体的生理生化表型。【结果】在30℃,20 μE/m2s自养培养下,slr0643::km与野生型相比,早期     

集胞藻PCC6803 中relA/spoT 同源基因syn-rsh (slr1325)的鉴定

[目的]四磷酸或五磷酸鸟苷(Guanosine 3′,5′-bispyrophosphate,(p)ppGpp)是细菌在遭遇环境胁迫时细胞产生应激反应的信号分子,(p)ppGpp由其合成酶RelA或具有合成酶或水解酶双重催化功能的RelA/SpoT合成.本文证明了集胞藻PCC6803(Synechocystis sp.)中唯一编码RelA/SpoT同源蛋白(命名为Syn-RSH)的基因slr1325(syn-rsh)的功能.[方法]通过互补试验证明syn-rsh表达产物的生物学功能;以纤维素薄层层析检测不同条件下Escherichia coli(p)ppGpp合成缺陷突变株及集胞藻PCC6803细胞中的(p)ppGpp.[结果]诱导Syn-RSH表达可使(p)ppGpp合成酶和水解酶基因缺失的E.coli突变株回复野生型表型,并在细胞中积累一定水平的ppGpp;在实验室培养条件下,集胞藻PCC6803细胞中可检测到低水平的ppGpp,氨基酸饥饿可诱导ppGpp水平升高并维持在相应水平.[结论]Syn-RSH具有(p)ppGpp合成酶和水解酶的双重功能,(p)ppGpp是集胞藻PCC6803在实验室生长条件下细胞生长所必需的.     

集胞藻PCC 6803高产精氨酸藻株的紫外诱变选育

精氨酸在医药和食品工业上具有广泛用途。集胞藻PCC 6803是单细胞蓝藻, 能利用工业废气(主要成分是氮氧化物NO_x)与水反应生成的硝酸盐和亚硝酸盐合成氨基酸等化合物, 因而选育高产精氨酸藻株, 不仅能提高精氨酸产量, 而且能去除工业废气中的NO_x, 具有潜在的应用前景。研究在集胞藻PCC 6803中利用紫外诱变, 筛选抗0.8 g/L D-精氨酸和抗0.2 g/L 6-氮尿嘧啶的突变株, 选育到了一株精氨酸产量显著提高的突变株#13807-111-55, 它每OD₇₃₀值细胞的胞外精氨酸产量相比出发株提高了62.3倍, 达到(0.76±0.1) mg/(L·OD₇₃₀), 总精氨酸产量相比出发株提高了6.0倍, 达到(0.82±0.08) mg/(L·OD₇₃₀)。该突变株每OD₇₃₀值细胞的胞外精氨酸产量明显高于胞内, 表明该突变藻株是精氨酸分泌型, 因而具有潜在的应用前景。     

RNA解旋酶基因crhR对于集胞藻PCC6803低温适应的作用

蓝藻对低温胁迫的适应涉及许多基因的表达调控,RNA解旋酶基因crhR即是其中之一。研究检查了该基因在集胞藻PCC6803从30℃转到15℃后的转录情况,观察到在2h内有瞬时诱导表达。该基因失活导致集胞藻在15℃下几乎不能生长,光合作用和呼吸速率大幅下降,脂质过氧化物不能被有效清除。以PrbcL过量表达crhR基因可互补突变株表型,并且在低温下生长略优于野生型。在野生型中,低温诱导脂肪酸脱饱和酶基因desA、desB、desD表达上调,膜脂不饱和度增加;而在crhR突变株中,低温诱导的desB基因的上调表达显著削弱,同时多不饱和脂肪酸含量没有显著增加。推测crhR基因可能影响蓝藻在低温胁迫下的蛋白合成或通过伴侣蛋白发挥影响。       

敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响

探究在集胞藻PCC 6803中引入外源乙醇合成基因并敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响。在集胞藻PCC 6803中引入来源于运动型发酵单胞菌的丙酮酸脱羧酶基因（pdc）与大肠杆菌的NADPH依赖型醛还原酶基因（yqhD）光强启动子PrbcL的驱动下组合表达,生物合成乙醇。在此基础上进一步敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因,以提高乙醇合成前体丙酮酸含量,促进乙醇的生产。结果显示敲除slr1556基因可以提高丙酮酸含量并显著增加乙醇的产量。竞争性丙酮酸转化乳酸代谢途径的阻断可以有效促进丙酮酸的累积,进而促进乙醇的生产。     

ggpS基因过表达对集胞藻PCC 6803甘油葡萄糖苷和甘油合成的影响

为了研究甘油葡萄糖苷磷酸合成酶(GgpS)在集胞藻PCC 803甘油葡萄糖苷和甘油合成中的作用,本研究在前期获得高产甘油葡萄糖苷藻株的基础上分别过量表达来自于集胞藻PCC 6803自身和聚球藻PCC7002的甘油葡萄糖苷磷酸合成酶基因ggpS,并测定了在不同浓度NaCl胁迫时突变藻株的甘油葡萄糖苷和甘油积累量。结果发现获得的突变株甘油葡萄糖苷合成没有提高,但是甘油合成显著增强。此外,当培养基NaCl浓度从600 mmol/L提高到900 mmol/L时,集胞藻PCC 6803自身ggpS过表达藻株的甘油合成进一步提高75%。这些结果显示了GgpS在将碳代谢流导入集胞藻甘油合成途径中的作用。研究成果也为进一步通过基因工程改造提高集胞藻甘油葡萄糖苷和甘油合成效率奠定了基础。     

集胞藻PCC6803高效基因表达平台的构建与评价

针对蓝细菌代谢工程改造的需求,成功构建了可以在模式蓝细菌菌株集胞藻PCC6803中高效表达外源基因的3个基因组整合表达平台,以及1个可以在多株蓝细菌中表达的广宿主穿梭表达平台。该表达平台通过选用集胞藻PCC6803中1,5-二磷酸核酮糖缩化酶/氧化酶的启动子驱动外源基因的表达,应用“SD-AUG”翻译融合的策略提高外源蛋白翻译效率,以及加入终止子序列Trbc以提高转录终止效率,实现了对外源基因的高效表达。利用lacZ作为报告基因,检测了所构建表达平台pFQ20在集胞藻中的基因表达效率,结果显示β-半乳糖苷酶的活性为109 Miller。同时,基于pFQ20表达平台在集胞藻PCC6803中表达了来自大肠杆菌的硫酯酶基因tesA’,蛋白印迹实验结果显示了硫酯酶的成功表达。该表达平台为在蓝细菌中开展遗传研究及基因工程改造提供了有用的遗传工具,其构建策略为在蓝细菌中构建高效稳定的外源基因表达元件提供了借鉴。     

集胞藻Synechocystis sp.PCC 6803脂质组的分析

以集胞藻Synechocystis sp.PCC 6803为研究对象,研究建立了基于超高效液相色谱耦合串联质谱技术脂质组学分析方法.鸟枪法脂质组学通过电喷雾离子化有效分离油脂粗提物中所含单个脂质分子,在三重四极杆扫描碎片离子,能够利用特征片段离子鉴定光合甘油酯的种类和酰基组成,具有高效、灵敏度高和质量准确度高等优点.对...     

集胞藻PCC6803中ORF sll0260对于维持基本生命活动的必要作用

集胞藻(Synechocystis sp.)6803的未知功能基因中有很多是细胞的基本生命活动所需要的,这些基因插入失活往往会导致细胞死亡,因而得不到分离完全的突变株,难以进行遗传学研究.构建突变株以铜离子调控的启动子PpetE来控制此类未知功能基因的表达则可能获得完全分离.构建PPpetE-sll0260突变株并对sll0260必要作用进行研究.在完全分离的突变株中,去除铜离子可关闭sll0260的表达.此时,突变株生长受到严重抑制,色素含量大为降低,类囊体膜结构破坏,光合作用消失,呼吸能力下降.这些结果表明该基因对于维持集胞藻6803的基本生命活动来说是必需的.亚细胞定位研究显示sll0260编码一个膜蛋白,位于质膜和外膜混合物中.sll0260可能作为某些离子的转运蛋白起作用,或者直接与类囊体膜的发生过程相关.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.     

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10⁵ molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.     

Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-D-xylulose 5-phosphate reductoisomerase mutants

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.     

集胞藻6803光合自养生长突变株的筛选与鉴定

集胞藻(Synechocystis sp.)PCC6803(以下称集胞藻6803)是一种单细胞蓝藻,既可进行自养生长,也可在光合系统失活的情况下利用葡萄糖进行异养生长[1],具有天然的DNA转化系统,为筛选光合自养生长突变株和基因功能的鉴定提供了便利.其全基因组序列已于1996年公布[2].     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.