| 题名 | 基因工程改造集胞藻 PCC6803产蔗糖潜力研究 |
| 作者 | 杜伟 |
| 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 | 生物能源 碳源 集胞藻PCC6803 代谢工程 |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 碳源在整个利用生物质能制备生物燃料与生物基材料化学品过程中占据关键地位，可持续、廉价、非地域性的生产微生物易利用的碳源将会加速整个生物能源与生物炼制的研发进程。传统碳源平台的建立基于粮食作物、非粮纤维素以及微藻生物质，在应用过程中存在各自核心障碍，限制了其进一步发展的空间。蓝藻作为一种光合微生物，具有其独特的易于遗传改造的优势，可以直接合成微生物易于利用的碳源——蔗糖，近年来引起包括学术界和工业界的广泛研究和重视。本研究通过基因工程手段，对集胞藻PCC6803从胞内蔗糖代谢途径和蔗糖胞外分泌两个方面进行了改造，探索其高效分泌产蔗糖的潜力。该研究主要内容和结果如下：第一，构建突变体，对集胞藻PCC6803蔗糖合成途径中三个关键基因：sps （slr0045）、spp（slr0953）、ugp（slr0207）分别进行单独过量表达或进行融合过量表达，结合盐胁迫的条件测定各突变体胞内蔗糖积累量，确定了SPS催化蔗糖合成过程中的关键步骤，过量表达sps基因后，胞内蔗糖积累量较野生型提高了约2倍；第二，通过敲除集胞藻PCC6803胞内蔗糖合成主要竞争性途径——甘油葡糖苷（GG）合成途径中的第一步基因ggpS（sll1566）后，在盐胁迫下细胞胞内积累的蔗糖量较野生型提高了约1.5倍；第三，构建了WD21突变体菌株（PCC6803 Δslr0168::Omega PpetE sps-spp-ugp ΔggpS:: Kmr），即整合过量表达蔗糖合成途径中的关键基因，同时又敲除了蔗糖合成主要竞争性途径的基因，并在不同的盐浓度胁迫下测定WD21胞内蔗糖的积累量。结果表明，600 mM NaCl胁迫下，单位OD细胞胞内蔗糖的积累量为35 mg-1 L-1 OD-1，总蔗糖积累量约为290 mg L-1，较野生型提高了约4倍，但和900 mM NaCl胁迫下无显著性差异；第四，我们应用了酸洗法对WD21突变体菌株的蔗糖胞外分泌能力进行了评估。实验结果表明，WD21在盐胁迫下胞内积累的蔗糖至少80%以上被分泌到了胞外。单从分泌的效果来看非常理想，但由于中途需要酸的处理，规模进一步放大受到一定限度上的限制；第五，在WD21的基础上，敲除了细胞编码胞外回收利用蔗糖的转运体的基因，并在该位点过量表达外源蔗糖转运体（CscB）基因后，蔗糖可以分泌到胞外，但其分泌效率较低。通过实时定量PCR对cscB基因的表达量进行分析，发现其表达量相对于sps低了约50倍。实验结果表明cscB的低表达量可能是导致蔗糖可以分泌到胞外，但分泌的效率较低的原因。 |
| 英文摘要 |
Carbon resource represents one of the most important factors for biofuel production from biomass. Manufacturing optimal carbon source for microbial fermentation under sustainable way with competitive cost and region-independence would speed up the whole research process of biofuels and biorefinery. Conventional carbon resource platforms were established based on food crops, cellulosic biomass and microalgae biomass. Each one has its core abstacles which limited the further development. Cyanobacteria, photosynthetic bacteria with the capability to directly accumulate sucrose--optimal carbon resource for microbes, can be genetically modified for biofuels production, drawing much attention recently both in the field of academia and industry. In this study, intracellular sucrose metabolic pathway modifications and sucrose export methods were evaluated in Synechocystis sp. PCC6803 (Synechocystis) for exploring its sucrose production capacity. The main research contents and results are summarized as follows:Firstly, we constructed strains individually overexpressing sps (slr0045), spp (slr0953), ugp (slr0207), three genes which are the most relevant to the sucrose biosynthesis in Synechocystis, and co-overexpressing sps-spp and sps-spp-ugp using fusion PCR, in order to find the most important step for sucrose accumulation under salt shock conditions based on the sucrose final accumulation amounts. These results indicated that the proposed most important step to accumulate sucrose is catalyzed by SPS. And the intracellular sucrose accumulation in the mutants containing overexpression of sps was nearly two times more than the wild type. Secondly, ggpS (sll1566), encoding the first step enzyme during the biosynthesis of glucosylglycerol (GG)--the main competitive product for sucrose, was knocked out. Mutant deficient for GG biosynthesis accumulated sucrose nearly 1.5 times more than the wild type under salt shock.
Thirdly, the mutant WD21, combining co-overexpression of sps-spp-ugp with ggpS knock-out, was generated for sucrose accumulation determination under a range of NaCl concentrations. The results indicated that WD21 exhibited a higher sucrose accumulation under 600 mM NaCl, at an amount of up to 35 mg-1 L-1 OD-1 and final yield 290 mg l-1, nearly four times more than that of wild type but almost identical to that under 900 mM NaCl.
Fourthly, acid-wash technique was evaluated on WD21 for exporting the intracellular sucrose into the culture medium. The results showed that more than 80% of the intracellular sucrose of WD21 accumulated under salt shock was exported into the culture medium, which indicated that this technique was relatively effective for sucrose secretion. However, the industrial-scale might be restricted considering the additional acid treatment.Finally, WD25 mutant was constructed on the basis of WD21 to overexpress sucrose transporter gene cscB from E.coli at the genome sites of the knocking-out ggtCD, genes encoding protein transport system for active uptake of sucrose in the medium. Sucrose has been transported extracellularly, though the total amount was rather low. After monitoring the expression level of cscB by real-time PCR, the expression of cscB was 50 times less than sps, which might lead to the low sucrose yield and sucrose export efficiency in Synechocystis.
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| 语种 | 中文 |
| 学科主题 | 生物代谢工程 |
| 公开日期 | 2013-07-13 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1521]  |
| 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 | 杜伟. 基因工程改造集胞藻 PCC6803产蔗糖潜力研究[D]. 北京. 中国科学院研究生院. 2013. |
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