集胞藻PCC 6803类铁氧还蛋白Slr1205的功能研究

类铁氧还蛋白 (ferredoxin-like, Fd-like) 在高等植物中具有调控叶绿体发育等多种重要的生理功能,但在蓝藻中的生物功能尚未被发现。通过比较集胞藻PCC 6083编码Fd-like蛋白基因的敲除突变株Δslr1205与野生型 (WT) 在不同碳源和光周期条件下的生理生化表型,分析Slr1205在集胞藻中的功能。结果显示,在高CO2浓度自养、混合营养和光异养时,Δslr1205的生长速率低于WT,而在空气中自养条件下并无差异。与此相对应,混合营养和光异养时Δslr1205比WT的呼吸速率低,与呼吸作用密切相关的NDH-1L复合体的含量少。Δslr1205在所有测试的条件下有较高的类胡萝卜素以及偏黄的表型。这些数据表明,Fd-like蛋白Slr1205的缺失造成在碳源充足条件下的生长速率下降,这可能是由于呼吸作用下调导致供能不足。研究结果为今后深入研究蓝藻Fd-like蛋白奠定了基础,为开展光合作用和呼吸作用的调节机制研究探索了新方向。     

蓝细菌PCC6803染色体上的毒素蛋白Slr0664与抗毒素蛋白Ssr1114的相互作用

【目的】证明蓝细菌PCC6803染色体上的毒素-抗毒素系统(TA,toxin-antitoxin system)ssr1114/slr0664中毒素蛋白Slr0664与抗毒素蛋白Ssr1114之的相互作用。【方法】构建在大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达H6-Ssr1114或共表达H6-Ssr1114和Slr0664的重组质粒,诱导表达后借助亲和捕捉技术在不同的条件下纯化H6-Ssr1114或共纯化重组蛋白H6-Ssr1114和Slr0664,并通过肽谱分析共纯化的重组蛋白,证明H6-Ssr1114与Slr0664之间存在相互作用。【结果】诱导Slr0664表达对细胞产生毒性作用导致生长抑制或细胞死亡,诱导H6-Ssr1114和Slr0664共表达时细胞能能正常生长,在非变性条件下可纯化共表达的重组蛋白H6-Ssr1114和Slr0664,在变性条件下仅H6-Ssr1114被纯化,肽谱分析结果表明共纯化的的重组蛋白为H6-Ssr1114和Slr0664。【结论】ssr1114/slr0664TA系统中抗毒素蛋白Ssr1114与毒素蛋白Slr0664之间存在相互作用。     

蓝细菌PCC6803染色体上的一对毒素-抗毒素基因的鉴定

大肠杆菌细胞染色体上毒素－抗毒素系统(TA, toxin-antitoxin system)基因参与环境胁迫诱导的细胞死亡或生长抑制。在蓝细菌PCC6803染色体上开放阅读框ssr1114和slr0664具有与TA系统相同的遗传结构, slr0664编码产物与RelBE毒素－抗毒素系统中的毒素蛋白RelE同源, 但没有发现有与ssr1114编码产物同源的蛋白。为证明slr0664和ssr1114表达产物的毒性和抗毒性作用, 构建了含有乳糖诱导调控的启动子和阿拉伯糖诱导调控的启动子的表达调控系统, 将slr0664基因编码序列置于Plac启动子控制下, ssr1114编码序列置于PBAD启动子控制下, slr0664诱导表达产物对细胞具有毒性作用, ssr1114诱导表达产物具有抗slr0664表达产物的毒性作用。提示slr0664为毒素基因, ssr1114为抗毒素基因, 二者组成一个TA系统, 但其有关特性和功能尚待进一步证明。     

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PCC6803膜脂循环关键基因酶学性质和生理功能

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索,发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969,但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用,加深对集胞藻PCC6803脂肪酸代谢途径的了解,文中将sll1969基因在大肠杆菌中过表达和体外纯化,得到重组蛋白Sll1969,并对其酶学性质进行初步分析。在30℃条件下,测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为(1.16±0.01)mmol/L和(332.8±10.0)/min;该脂肪酶的最适反应温度为55℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化,发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关,但Sll1969不是细胞中唯一的脂肪酶。     

集胞藻PCC6803中S2P同源蛋白Slr0643及Sll0862 金属蛋白酶活性的体外鉴定

【目的】金属蛋白酶S2P在细菌中通过在膜切割转录调控因子、释放δ因子参与胁迫响应是跨膜信号转导的保守机制,但蓝细菌中S2P的功能还未被鉴定,故我们考察集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862的金属蛋白酶活性。【方法】以pET-30b(+)为载体,分别构建重组质粒pF0643和pF0862,在大肠杆菌BL21(CE3)中诱导表达并纯化Slr0643及Sll0862蛋白,以β-酪蛋白为底物检测重组蛋白的酶活性。【结果】体外酶活实验显示重组表达的Slr0643及Sll0862蛋白有内切蛋白酶活性,且其活性受金属螯合剂o-phenanthroline的抑制。体外酶活的鉴定结果为进一步研究Slr0643和Sll0862的体内酶活和生物学功能奠定了基础。【结论】集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862具有金属蛋白酶活性。     

集胞藻PCC 6803蛋白SpPhaB和SpPhaE的原核表达及结晶条件筛选

【目的】克隆蓝藻集胞藻PCC 6803 SpPhaB和SpPhaE的编码基因并构建原核表达载体,优化培养条件以提高在大肠杆菌中可溶蛋白的表达量,筛选SpPhaB结晶条件,为PHB家族蛋白的结构与功能研究提供基础。【方法】克隆集胞藻PCC 6803 PHB合成途径的phaB、phaE基因,将phaB、phaE基因构建到表达载体pET28a中,优化IPTG浓度、诱导温度和诱导时间,提高在大肠杆菌BL21(DE3)中SpPhaB和SpPhaE可溶蛋白的表达产量,经Ni柱亲和层析纯化分别获得His-SpPhaB和His-SpPhaE蛋白,进一步筛选SpPhaB结晶条件。【结果】构建了pET28a-SpPhaB和pET28a-SpPhaE表达载体;优化获得SpPhaB可溶蛋白的最佳表达条件为：诱导温度37 °C、转速220 r/min、IPTG浓度0.1 mmol/L、诱导时间7 h;SpPhaE的可溶性蛋白最佳表达条件为：诱导温度25 °C、转速220 r/min、IPTG浓度0.5 mmol/L、诱导时间7 h,并获得了SpPhaB的结晶条件。【结论】构建了具有高效表达可溶的集胞藻PCC 6803 SpPhaB和SpPhaE的原核表达系统,并筛选优化了SpPhaB的结晶条件,为研究SpPhaB蛋白的结构及功能奠定了基础。     

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中基因slr1761突变体的构建

PCR扩增了集胞藻PCC6803的slr1761基因,进一步以PGEM-T为载体将其克隆到大肠杆菌中,构建了P1761质粒。通过DNA体外重组,以卡那霉素抗性基因插入目的基因片段,构建了既含目的基因上游及下游序列、又携带选择性标记卡那霉素抗性的PK1761质粒。该质粒转化野生型集胞藻PCC6803细胞,利用同源重组原理获得了能在含卡那霉素的培养基上正常生长的基因敲除突变株。对该突变株基因组DNA进行PCR扩增,验证了其基因结构的正确性。     

Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin

Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.     

Slr1122影响Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的色素合成

Synechocystis sp.PCC 6803是一种良好的研究光合作用的模式生物,其中slr1122编码一个250个氨基酸的未知蛋白。据报道Slr1122可能与杂合传感激酶（hybrid sensory kinase）Sll1672（Hik12）相互作用,本研究通过复合物实验证实了Slr1122与Sll1672确实存在相互作用。利用32P标记证明,在加入Slr1122后Hik12的磷酸化受到了明显的影响,推测其可能参与该双组分系统的调控。通过同源双交换,用卡那霉素抗性基因替换slr1122,将slr1122从Synechocystis sp.PCC 6803中敲除,构建了slr1122的缺失体Δslr1122。研究发现在Δslr1122中,编码PSⅡ中核心蛋白D1亚基的slr1181（psbAI）的转录水平明显降低,使PSⅡ光合作用受到影响,导致Δslr1122的生长速率低于野生型（WT）。同时slr1122的缺失使得蓝细菌对光的敏感性增强,在弱光条件下,Δslr1122对光能的利用效率高于WT,其生长速率也较WT高,但与此相反,Δslr1122对强光的耐受力及生长速率则不及WT。Δslr1122体内的藻胆蛋白含量与色素含量均降低,尤其是类胡萝卜素,RT-PCR的结果也显示合成类胡萝卜素过程中的5个关键酶转录水平均下降。这可能是Δslr1122对氧化胁迫变得敏感的原因之一。总之,Slr1122影响杂合传感激酶Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的光合色素合成。     

Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803

We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.     

Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803.

The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.     

Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely ’UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that ’UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO₂ into excreted FAME.