基因工程蓝细菌光合生物合成乙醇的研究
发布时间：2019-02-24 09:57
【摘要】：随着世界经济的不断发展,能源短缺与环境污染问题逐渐显现,如何开发可持续、清洁能源并缓解环境污染至为重要。蓝细菌是光合自养微生物,通过光合作用固定二氧化碳合成有机物并释放氧气,蓝细菌合成生物燃料不仅可以缓解能源短缺问题,还可利用化石燃料燃烧释放的二氧化碳进而减轻温室效应。本研究根据蓝细菌代谢途径特点,通过基因工程手段探究合成生物乙醇的优化策略,研究内容和结果如下:(1)以集胞藻PCC 6803的单拷贝工程藻株Syn-ZG25(基因组slr0168位点表达了运动发酵单胞菌的丙酮酸脱羧酶基因PDCzm和集胞藻PCC 6803的乙醇脱氢酶基因slr1192)为基础,在基因组phaAB位点过表达一份PDCzm得到工程藻株Syn-YQ4,其乙醇产量与双拷贝藻株Syn-HZ24(slr0168和pha AB两个位点同时表达PDCzm和slr1192)相当,通过酶活和Western Blot结果分析PDCzm的表达量和活性为乙醇合成途径的限制性因素。(2)聚球藻PCC 7002中构建的乙醇合成藻株通过Western Blot显示slr1192表达量明显高于PDCzm,于是分别在野生型中通过强启动子控制slr1192和产乙醇藻株中过表达一份slr1192,其中强启动子控制方案无效,过表达slr1192后酶活和表达量均明显提高。(3)以目前文献报道倍增时间最短的蓝细菌聚球藻UTEX 2973为基础,在其中性位点NS1分别正向和反向表达一份PDCzm和slr1192,成功构建产乙醇藻株Syn-YQ16(正向插入)和Syn-YQ17(反向插入),其中Syn-YQ16较Syn-YQ17乙醇产量略高,但乙醇合成速率较PCC 6803和PCC 7002单拷贝藻株偏低。
[Abstract]:With the development of the world economy, the problems of energy shortage and environmental pollution appear gradually. How to develop sustainable, clean energy and mitigate environmental pollution is very important. Cyanobacterium is a photosynthetic autotrophic microorganism, which immobilizes carbon dioxide to synthesize organic matter and release oxygen through photosynthesis, and synthesizing biofuel by cyanobacteria can not only alleviate the problem of energy shortage. The burning of fossil fuels can also be used to reduce carbon dioxide emissions in Greenhouse Effect. According to the characteristics of cyanobacteria metabolic pathway, the optimization strategy of biosynthesis of bioethanol was explored by genetic engineering. The main contents and results were as follows: (1) based on the single copy engineering algae strain Syn-ZG25 of PCC 6803, which expressed pyruvate decarboxylase gene (PDCzm) at genomic slr0168 site and ethanol dehydrogenase gene slr1192 (slr1192 gene of PCC 6803). An engineering algae strain Syn-YQ4, was obtained by overexpression of a PDCzm at the genomic phaAB site. The ethanol production of Syn-YQ4, was comparable to that of Syn-HZ24 (slr0168 and pha AB expressed PDCzm and slr1192 at both slr0168 and pha AB loci). The expression and activity of PDCzm were analyzed by enzyme activity and Western Blot results. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. So one slr1192, was expressed in wild type by strong promoter control slr1192 and ethanol producing algae strain respectively, and the strong promoter control scheme was not effective. (3) on the basis of UTEX 2973, which has the shortest doubling time reported in the literature, a PDCzm and slr1192, were expressed in the neutral NS1 of Chlorella cyanobacterium UTEX 2973, respectively. Syn-YQ16 (positive insertion) and Syn-YQ17 (reverse insertion) were successfully constructed. The ethanol production of Syn-YQ16 was slightly higher than that of Syn-YQ17, but the ethanol synthesis rate was lower than that of PCC 6803 and PCC 7002 single copy algae.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ223.122;TQ920.6
本文编号：2429440
[Abstract]:With the development of the world economy, the problems of energy shortage and environmental pollution appear gradually. How to develop sustainable, clean energy and mitigate environmental pollution is very important. Cyanobacterium is a photosynthetic autotrophic microorganism, which immobilizes carbon dioxide to synthesize organic matter and release oxygen through photosynthesis, and synthesizing biofuel by cyanobacteria can not only alleviate the problem of energy shortage. The burning of fossil fuels can also be used to reduce carbon dioxide emissions in Greenhouse Effect. According to the characteristics of cyanobacteria metabolic pathway, the optimization strategy of biosynthesis of bioethanol was explored by genetic engineering. The main contents and results were as follows: (1) based on the single copy engineering algae strain Syn-ZG25 of PCC 6803, which expressed pyruvate decarboxylase gene (PDCzm) at genomic slr0168 site and ethanol dehydrogenase gene slr1192 (slr1192 gene of PCC 6803). An engineering algae strain Syn-YQ4, was obtained by overexpression of a PDCzm at the genomic phaAB site. The ethanol production of Syn-YQ4, was comparable to that of Syn-HZ24 (slr0168 and pha AB expressed PDCzm and slr1192 at both slr0168 and pha AB loci). The expression and activity of PDCzm were analyzed by enzyme activity and Western Blot results. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. So one slr1192, was expressed in wild type by strong promoter control slr1192 and ethanol producing algae strain respectively, and the strong promoter control scheme was not effective. (3) on the basis of UTEX 2973, which has the shortest doubling time reported in the literature, a PDCzm and slr1192, were expressed in the neutral NS1 of Chlorella cyanobacterium UTEX 2973, respectively. Syn-YQ16 (positive insertion) and Syn-YQ17 (reverse insertion) were successfully constructed. The ethanol production of Syn-YQ16 was slightly higher than that of Syn-YQ17, but the ethanol synthesis rate was lower than that of PCC 6803 and PCC 7002 single copy algae.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ223.122;TQ920.6
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