蓝藻生物燃料中典型萜类物质的代谢工程研究

发布时间：2018-05-17 15:46

本文选题：生物燃料 + 蓝藻　；参考：《福建农林大学》2017年硕士论文

【摘要】：世界人口增长和工业经济的发展对能源的需求越来越大,生物燃料作为一种可替代传统燃料的环境友好型可再生能源受到了人们的关注。目前美国等发达国家和地区对生物燃料的重视程度和研究成绩都远领先于中国,主要的研究方向是以光合微生物为平台生产人们需要的生物燃料和高价值产品。蓝藻是作为第三代生物燃料的重要原料,利用太阳能固定CO2转化成各种形式的生物能,极具应用前景。而萜类物质家族庞大,能作为传统燃料的替代品,还是应用广泛的高价值化合物。因此,如何以蓝藻为代谢平台生产萜类物质是这一领域的研究重点,然而现阶段国内关于这部分的研究仍较空白。本实验以蓝藻为实验研究对象,在了解其MEP代谢路径的基础上,通过代谢工程调控方法,在蓝藻 Cyanobacterium Synechococcus elongatus PCC7942表达高效的外源关键萜类合成酶基因(柠檬烯合成基因和法呢烯合成酶基因),在蓝藻Synechocystissp.PCC 6803中验证鲨烯环化酶基因敲除后的产物累计,以此探索增强或阻碍关键酶的表达对蓝藻细胞萜烯产物累积和细胞生长的影响。目标产物分别包括单萜—柠檬烯,倍半萜—法尼烯和三萜-鲨烯。主要研究结果如下:1.通过构建含有柠檬烯合成酶基因LS的质粒,并成功表达LS基因,我们证明了柠檬烯合成酶基因LS是柠檬烯的合成的关键,Cyanobacterium Synechococcus elongatus PCC7942 具备生产柠檬烯的能力。且不同的启动子对相同基因的表达有影响,同时具有Ptrc启动子和核糖体结合位点(RBS)的菌株产量最高,日平均产率达到53.0μg/L/OD/day,因此要提高产量需要筛选出高效的启动子。2.通过构建含有法呢烯合成酶FS基因的质粒,并成功表达FS基因通,我们证明了法呢烯合成酶基因FS是法尼烯合成的关键,且蓝藻细胞PCC7942同样具备法呢烯合成路径中必要的原料和酶。3.验证了鲨烯下游鲨烯藿烯环化酶基因Shc的敲除的确能积累鲨烯,蓝藻突变株Synechocystissp.PCC 6803△Shc的鲨烯产率可达到2.48mg/L/OD,而在突变株的基础上通过代谢工程进一步提高鲨烯产量,鲨烯合成基因或许是重要突破口。4.本实验成功验证了蓝藻能产生单萜,倍半萜和三萜,蓝藻作为原核光合自养型微生物具有众多优势,完全能通过基因改造来实现萜烯的生产并且达到比较客观的产量,为实现蓝藻生物燃料的工业化生产提供了可能性。
[Abstract]:With the increase of world population and the development of industrial economy, more and more people pay attention to biofuel as a kind of environment-friendly renewable energy which can replace traditional fuel. At present, the United States and other developed countries and regions are far ahead of China in the importance and research achievement of biofuel. The main research direction is to produce biofuel and high-value products that people need on the platform of photosynthetic microbes. Cyanobacteria are important feedstock for the third generation biofuel, which can be converted into various forms of bioenergy by solar fixed CO2. The terpenoid family is so large that it can be used as a substitute for traditional fuels and as a high-value compound in a wide range of applications. Therefore, how to use cyanobacteria as metabolic platform to produce terpenoids is the focus of research in this field, but at present, the research on this part of the domestic is still relatively blank. In this study, cyanobacteria were selected as the experimental objects. On the basis of understanding their MEP metabolic pathway, the metabolic engineering regulation method was adopted. High efficient exogenous key terpene synthase genes (limonene synthase gene and farnene synthase gene) were expressed in cyanobacteria Cyanobacterium Synechococcus elongatus PCC7942. The accumulated products of squalene cyclase gene knockout were verified in cyanobacteria Synechocystissp.PCC 6803. To explore the effect of enhancing or blocking the expression of key enzymes on the accumulation of terpene products and cell growth in cyanobacteria. The target products include monoterpene-limonene, sesquiterpene-farnesene and triterpene-squalene respectively. The main results are as follows: 1. By constructing the plasmid containing LS gene and expressing LS gene successfully, we proved that LS gene is the key factor in limonene synthesis. Cyanobacterium Synechococcus elongatus PCC7942 has the ability to produce limonene. Different promoters affected the expression of the same gene, and the strains with Ptrc promoter and ribosomal binding site (RBS) had the highest yield, and the average daily yield was 53.0 渭 g / L / O ~ (-1) / day. therefore, to improve the yield, we should screen out highly efficient promoter. 2. By constructing a plasmid containing fellene synthase FS gene and expressing FS gene successfully, we have proved that fathene synthase gene FS is the key to farnesene synthesis. In addition, cyanobacteria cell PCC7942 also possesses the necessary raw materials and enzymes in the biosynthesis pathway of farnesene. It was proved that the knockout of squalene cyclase gene Shc in the downstream of squalene actually accumulated squalene. The squalene yield of the cyanobacteria mutant Synechocystissp.PCC 6803 Shc could reach 2.48 mg / L / O, and the squalene yield could be further increased by metabolic engineering on the basis of the mutant. Squalene synthesis gene may be an important breakthrough. 4. This experiment proved that cyanobacteria can produce monoterpenes, sesquiterpenes and triterpenes. Cyanobacteria have many advantages as prokaryotic photosynthetic autotrophic microorganisms. It provides the possibility for the industrial production of cyanobacteria biofuel.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946

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