基因工程聚球藻 PCC7942 产蔗糖研究
文献类型：学位论文
| 作者 | 梁飞燕 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 吕雪峰 |
| 关键词 | Synechocystis sp. PCC6803 Anabaena sp. PCC7120 Synechococcus elongatus PCC7942 蔗糖 合成生物学 |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 基于可再生生物质资源制备生物燃料与生物基材料化学品，是解决我们所面临的能源、环境、资源问题的有效途径之一。然而，高效、低成本、可持续的糖原料供给，已经成为生物能源与生物炼制发展的主要瓶颈之一。蓝藻作为光能自养原核微生物，能够利用光能和CO2合成有机物，遗传背景和遗传操作相对简单。近年来，运用合成生物学方法修饰蓝藻细胞内天然代谢途径或引入外源代谢途径合成能源化工产品，已经得到广泛关注和快速发展。蓝藻在盐胁迫下可以积累蔗糖，是一种有潜力的糖原料供给模式。基于对目前淀粉类、糖类与纤维类能源植物制备糖原料技术路线的分析，基于对光合微生物蓝藻生物合成及胞外分泌蔗糖技术路线的分析，本研究确立了基因工程蓝藻高效生物合成蔗糖的研究目标，评价了三株代表性蓝藻Synechocystis sp. PCC6803、Synechococcus elongatus PCC7942和Anabaena sp. PCC7120在不同浓度NaCl胁迫下蔗糖的胞内积累；运用合成生物学Biobrick-T载体，构建了系列过表达和敲除Synechococcus elongatus PCC7942蔗糖代谢相关基因的工程藻株；分析了基因工程突变株生物合成蔗糖的效率。本论文取得了如下主要研究结果：1. 在500ml三角烧瓶中培养Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942和Anabaena sp. PCC7120，在指数生长末期用不同浓度NaCl进行胁迫，发现在三株野生藻株中，Synechocystis sp. PCC6803在600mM或900mMNaCl胁迫下24h时迅速积累8 mgL-1 OD730-1的蔗糖，然后迅速降解；Anabaena sp. PCC7120在500mM NaCl胁迫下，48h可积累41mgL-1 OD730-1蔗糖，接下来的2天内蔗糖产量基本稳定；Synechococcus elongatus PCC7942在500mM NaCl胁迫下，48h可积累25 mgL-1 OD730-1蔗糖，接下来的两天内蔗糖产量基本稳定。2. 按照Biobrick规范，在pMD18-T载体的基础上利用XcmI内切酶构建直接用于A/T连接的载体质粒。PCR结果表明阳性克隆率>75%。3. 在Synechococcus elongatus PCC7942中表达大肠杆菌Ptrc和Ptac启动子启动的lacZ基因，结果表明Ptrc和Ptac能够在Synechococcus elongatus PCC7942中被异丙基-β-D-硫代半乳糖苷（IPTG）有效诱导表达，Ptrc的表达活性略高于Ptac，并确定IPTG最佳诱导浓度为200µM。4. 在Biobrick-T载体的基础上构建了系列改造Synechococcus elongatus PCC7942中蔗糖代谢途径的工程藻株。结果发现部分敲除蔗糖水解酶基因（inv,Synpcc_0397）、单独过表达蔗糖合成酶和蔗糖磷酸磷酸化酶基因（sps-spp, Synpcc7942_0808）和质子/蔗糖同向转运蛋白基因（Escherichia coli, cscB）都在一定程度上促进蔗糖的累积，分别提高65%,65%和8.8倍。而过表达葡萄糖-1-磷酸焦磷酸化酶基因（ugp, Synpcc7942_2101）和核酮糖-1,5-二磷酸羧化酶/加氧酶基因（rbcLXS, Synpcc7942_1426 , Synpcc7942_1427）对蔗糖的积累没有影响，对蔗糖的生长也无明显影响。同时过表达sps-spp和cscB获得蔗糖产量最高的基因工程突变株，蔗糖产量是野生型的36倍（424mgL-1 OD730-1, 1.12g/l）。 |
| 英文摘要 | The production of biofuels and biochemicals from renewable biomass is promising stragety to meet the demand of energy and resources as well as to decrease environmental pollution. However, it is one of the bottlenecks to produce sugars with high efficiency, low cost and sustainable supply for bioenergy and biorefinary. Cyanobacteria, photoautotrophic prokaryotic microorganisms, can synthesize organic compounds with solar and CO2. Besides, their genetic background is relatively simple and genetic tools for metabolic engineering are established. Recently, more and more attentions have been attracted by modifying cyanobacterial natural metabolic pathways or transporting new pathways into cyanobacteria to produce biofuels or biochemicals. Cyanobacteria can accumulate sucrose responding to extracellular salt stress and it is promising sugar resource. Based on the comprehensive analysis of traditional technical routes of sucrose production (from starch, sugarcane or cellulose), we aimed to engineer cyanobacteria for efficient sucrose accumulation. The main content contained three aspects. Firstly, we evaluated sucrose accumulation in three respective cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942 and Anabaena sp. PCC7120. Secondly, we designed and constructed Biobrick-T vectors and engineered sucrose metabolic pathways in Synechococcus elongatus PCC7942 for higher sucrose accumulation. Thirdly, we analysed sucrose accumulation capacity of the constructed Synechococcus elongatus PCC7942 mutants. The results are summarized below:1. Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942 and Anabaena sp. PCC7120 were cultured in 500ml flasks with 300ml BG11 medium. The results indicated that Synechocystis sp. PCC6803 accumulated 8mgL-1 OD730-1 sucrose at 24h shocked with 600mM or 900mM NaCl but decreased rapidly after that. Anabaena sp. PCC7120 accumulated 41mgL-1 OD730-1 sucrose at 48h shocked with 500mM NaCl and remained stable for the following 2 days while Synechococcus elongatus PCC7942 accumulated 25mgL-1 OD730-1 sucrose at 48h shocked with 500mM NaCl and also remained stable for the the following 2 days.2. Biobrick-T vectors based on pMD18-T and XcmI were constructed. Their positive frequency of recombination is over 75%.3. The lacZ gene was expressed in Synechococcus elongatus PCC7942 and controlled by Ptrc or Ptac from E.coli. The results indicated that Ptrc and Ptac could be induced by IPTG in Synechococcus elongatus PCC7942. Ptrc activity was a litter higher than Ptac. We also identified the optimal IPTG induction concentration was 200µM.4. A series of Synechococcus elongatus PCC7942 mutants targeting sucrose metabolic pathway were constructed based on Biobrick-T vectors. Sucrose determination experiments indicated that partly knocking out of invertase gene (inv, Synpcc_0397) could increase intracellular sucrose concentration by 65%, while 65% increase for overexpressing sucrose phosphate synthase and sucrose phoaphatase gene (sps-spp, Synpcc7942_0808) and 8.8-fold increase for overexpressing a symporter gene (cscB form Escherichia coli) (extracellular and intracellular). However, overexpressing UDP-glucose phosphatase (ugp, Synpcc7942_2101) and rubisco (rbcLXS, Synpcc7942_1426, Synpcc7942_1427) had no effect on sucrose accumulation as well as cell growth. The highest sucrose production was achieved by co-expressing sps-spp and cscB, which was 424mgL-1OD730-1 (1.12g/l), 35-fold higher than the wild type. |
| 学科主题 | 生物代谢工程 |
| 语种 | 中文 |
| 公开日期 | 2013-07-13 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1518]  |
| 专题 | 青岛生物能源与过程研究所_微生物代谢工程团队 |
| 推荐引用方式 GB/T 7714 | 梁飞燕. 基因工程聚球藻 PCC7942 产蔗糖研究[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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