| 基因工程集胞藻PCC6803合成乙醇的研究 |
| 高政绪
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| 导师 | 吕雪峰
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| 2012-11
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| 学位授予单位 | 中国科学院研究生院
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| 学位授予地点 | 北京
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| 学位专业 | 生物化学与分子生物学
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| 关键词 | 集胞藻pcc6803
丙酮酸脱羧酶
乙醇脱氢酶
生物乙醇
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| 其他摘要 | 随着世界对能源需求量的日益增加和石化燃料燃烧排放的二氧化碳等温室气体而导致的环境问题日益严重，人们开始积极寻找可再生的清洁能源作为替代能源。生物能源作为其中之一，它的发展越来越受到人们的重视。而生物乙醇作为一种主要的生物燃料，受到了工业界和学术界越来越多的关注。目前生物乙醇的生产由于存在“与人争粮”（粮食基生物乙醇）或生产成本太高（纤维素基生物乙醇）的问题，其工业化生产受到严重制约。光合微生物蓝藻可以通过光合作用固定二氧化碳直接合成目的有机化合物，因此通过基因工程手段改造蓝藻，将乙醇的代谢途径导入蓝藻中，可以实现由二氧化碳到乙醇在生物体内的一步合成。在本研究中，作者采用了一个整合生物加工过程的策略，将光合作用生成生物质和微生物转化合成乙醇整合到一个宿主蓝藻Synechocystis sp. PCC 6803中进行，该藻株可以通过光合作用利用二氧化碳直接生成乙醇，可以大大提高生产效率。该研究的主要内容和结果如下：第一，在集胞藻PCC6803中表达了来自于运动发酵单胞菌Zymomonas mobilis的丙酮酸脱羧酶基因pdc和乙醇脱氢酶基因adhⅡ，Western blot结果显示两个蛋白都得到了很好的表达，在集胞藻PCC6803中成功构建了运动发酵单胞菌的乙醇合成途径，该菌株Syn-XT43在摇瓶中通气培养乙醇的最高产量为0.4 g/L。第二，在集胞藻PCC6803中表达了来自于运动发酵单胞菌Zymomonas mobilis的丙酮酸脱羧酶基因pdc和来自于集胞藻PCC6803自身的乙醇脱氢酶基因slr1192，Western blot结果显示两个蛋白也都得到了很好的表达，该菌株Syn-ZG25在摇瓶中通气培养乙醇的最高产量为0.65g/L。第三，在集胞藻PCC6803基因组的slr0168和phaAB两个位点同时整合了丙酮酸脱羧酶基因pdc和来自于集胞藻PCC6803自身的乙醇脱氢酶基因slr1192，Western blot结果显示在该菌株Syn-HZ24中，两个蛋白的表达量比只在slr0168位点整合两个基因的菌株Syn-ZG25提高了一倍左右，在柱式光反应器中培养，并接上乙醇冷凝回收装置，乙醇产量大幅度提高，最高达到5.5 g/L。第四，筛选了9个来自于蓝藻自身的乙醇脱氢酶基因，分别是来自于来自于集胞藻PCC6803的slr1192、slr0942和sll0990，鱼腥藻7120的all0879、alr0895、alr0897、all2810和all5334，聚球藻7942的Synpcc7942_0459，将它们分别与丙酮酸脱羧酶基因pdc在E.coli中共表达检测其催化合成乙醇的能力，结果表明slr1192的催化活性最高。第五，成功纯化了乙醇脱氢酶slr1192，并作了动力学参数测定，它更倾向于用NADPH作为还原力。slr1192催化反应的最适pH值是8，Zn2+和Co2+对slr1192的活性影响最大，但是高pH值以及高浓度的金属离子浓度对Syn-ZG25的乙醇产量没有显著影响。第六，检测了20℃培养条件下Prbc、Prnpb、Prbp1和PnrtA四个启动子对乙醇合成能力的影响，结果表明效果最好的两个启动子是Prnpb和Prbc，其次是PnrtA和Prbp1；含有PnrtA启动子的Syn-ZG74可以用硝酸盐诱导乙醇的合成，但是产量低，Prbp1没有诱导效果。第七，检测了不同水质和缺氧的培养条件对Syn-ZG25乙醇产量的影响，结果表明这两种培养条件对乙醇产量影响不大，但是在缺氧的培养条件下乙醇的生产周期相对延长。; Rapidly growing demand for energy and environmental concerns about carbon dioxide emissions make development of renewable biofuels more and more attractive. Tremendous academic and industrial efforts have been paid to produce bioethanol, which is one major type of biofuels. Current production of bioethanol is limited for commercialization because of food competitive issue (from food-based biomass) or cost effective issue (from lignocellulose-based biomass). Photosynthetic bacteria, e.g. cyanobacteria, are potential candidates as they harbor photosynthetic capability to convert carbon dioxide to organic carbon metabolites and can be genetically modified to assemble ethanol-producing pathway. In this study, we intrinsically applied a consolidated bioprocessing strategy to integrate photosynthesis producing biomass and microbial conversion producing ethanol together into photosynthetic bacteria, Synechocystis sp. PCC6803, which can directly convert carbon dioxide to ethanol in one single biological system. This strategy can strongly improve the efficiency of producing bioethanol. The main researching contents and results are summarized as follows:First, a Synechocystis sp. PCC6803 mutant strain Syn-XT43 harboring the ethanol-producing pathway was constructed by genetically introducing exogenous pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. The two protein successfully expressed in Syn-XT43 was confirmed by Western blot analysis, and the maximal ethanol production was 0.4 g/L in flask.Second, a Synechocystis sp. PCC6803 mutant strain Syn-ZG25 harboring the ethanol-producing pathway was constructed by genetically introducing exogenous pyruvate decarboxylase from Zymomonas mobilis and endogenous alcohol dehydrogenase from Synechocystis sp. PCC6803. The two protein successfully expressed in Syn-ZG25 was confirmed by Western blot analysis, and the maximal ethanol production was 0.6 g/L in flask.Third, a Synechocystis sp. PCC6803 mutant strain Syn-HZ24 harboring the ethanol-producing pathway was constructed by genetically introducing exogenous pyruvate decarboxylase from Zymomonas mobilis and endogenous alcohol dehydrogenase from Synechocystis sp. PCC6803 through homologous recombination at slr0168 site and phaAB site. In this strain the expression of two proteins were highly improved about 1 fold by Western blot analysis. The ethanol production was also improved and the maximal ethanol production was 5.5 g/L in photo-bioreactor with a condensing device.Fourth, totally nine alcohol dehydrogenases slr1192 sll0990 slr0942 all0879 all5334 alr0895 all2810 alr0897 and Synpcc7942_0459 from different cyanobacteria strains were cloned and expressed in E. coli to test ethanol-producing efficiency. The result suggested that the capacity of synthesizing ethanol of slr1192 was the highest.Fifth, the kinetic characteristic of purified slr1192 was tested and the enzyme was inclined to use NADPH as the reducing power. The optimal pH of slr1192 was pH 8, and the in vitro slr1192 activity was sharply inhibited by very low concentration Zn2+ or Co2+. However, the high pH and high concentration of Zn2+ or Co2+ in cultivation culture can not affect the ethanol production in Syn-ZG25.Sixth, effects of four promoters Prbc Prnpb Prbp1 and PnrtA on the capacity of ethanol production in cyanobacteria were evaluated. Results suggested that Prnpb and Prbc were stronger than Prbp1 and PnrtA when these promoters were used to produce ethanol in cyanobacteria at 20℃. Ethanol can be induced to produce by NO3- in Syn-ZG74 harboring the promoter PnrtA, but the ethanol production is very low. The promoter Prbp1 did not have the inductive effects in ethanol production. Seventh, effects of water quality (tap water and distilled water) and aeration condition on ethanol production in Syn-ZG25 were evaluated. Results showed that there was no significant difference in ethanol production when different water was used and the ethanol production period could be prolonged under anoxic condition. |
| 作者部门 | 生物代谢工程 |
| 学科领域 | 生物代谢工程
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| 公开日期 | 2013-01-24
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| 学位类型 | 博士
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| 语种 | 中文
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| 文献类型 | 学位论文
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| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/1451
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| 专题 | 微生物代谢工程研究组
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推荐引用方式
GB/T 7714 |
高政绪. 基因工程集胞藻PCC6803合成乙醇的研究[D]. 北京. 中国科学院研究生院,2012.
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