| 其他摘要 | 脂肪酸具有广阔的应用前景，特别是在生物柴油生产中的应用。利用微生物高效转化秸秆等生物质资源生产微生物油脂，将为生物柴油生产提供优质、低成本油脂原料。本课题针对天然微生物生长周期长、油脂含量有限、油脂提取工艺复杂、污染环境等缺陷，通过代谢工程手段，从大肠杆菌脂肪酸合成能力和脂肪酸胞外分泌两个方面开展了脂肪酸代谢定向调控关键技术的研究，并提出利用工程大肠杆菌生产燃料脂肪酸的油脂生产新途径。针对天然大肠杆菌脂肪酸合成受多种因素调控，不能高效合成，本研究定向调节了大肠杆菌脂肪酸合成关键酶活性，调节碳流。结果表明：表达乙酸钙不动杆菌乙酰辅酶 A 羧化酶 ACC 基因，重组菌胞内总脂（IFA）含量是原始菌株的 3 倍（约 177.66mg/g），产率为 1.82%；表达内源苹果酸酶 NADP ME 基因和添加底物苹果酸钠，为脂肪酸合成提供充足的还原力 NADPH，胞内总脂含量提高了 4 倍，约 197.74mg/g；共表达 ACC 和 NADP ME 基因，细胞总脂含量从 50mg/g 增加到 280mg/g，提高了 5.6 倍，产率提高到 2.7%；5L 发酵罐发酵，重组菌 MX32 胞内总脂可达 2.6g/L。针对微生物油脂分离成本及分离过程带来的质量、环境污染等问题，开展了脂肪酸胞外分泌关键技术研究，探索了大肠杆菌脂肪酸转运过程中的酶和蛋白功能。结果表明：通过表达内源硫酯酶基因 tesA 和异源的拟南芥硫酯酶基因AtFatA，解除了产物抑制，获得了胞外游离脂肪酸，（FFA） 占细胞总脂的 14.7%（约 33mg/L）；表达拟南芥硫脂酶基因与 ACC 基因时，FFA 达到 71mg/L；表达两种硫脂酶基因和内膜翻转酶基因 msbA 时，提高了大肠杆菌细胞膜通透性，FFA 含量达到 120mg/L，FFA 与 IFA 比例从 1/25 提高到 1/1.8；经 5L 发酵罐发酵，重组菌 MX36 FFA 含量高达 220mg/L，约占细胞总脂含量的 55%。对大肠杆菌脂肪酸代谢的定向改造，获得了高产游离脂肪酸的工程大肠杆菌，并初步解析了脂肪酸转运与胞外分泌机制。首次在原核中模拟了产油微生物油脂积累过程，为优质化原料脂肪酸生产提供了理想菌株，并为进一步解析原核生物脂肪酸合成机制奠定了理论基础。; Fatty acid is widely used in different fields, especially in the production of biodiesel. Using oleaginous microbiology to produce oil by transforming straw and other biomass resources will provide high quality, low-cost oil raw materials for biodiesel production. However, native oleaginous microorganisms have many disadvantages in biodiesel production, such as long growth cycle, limited fatty acid production, complex extraction process and environmental pollution in the process of microbial oil separation. In this paper, fatty acid synthesis in E. coli was regulated by the method of metobolic engineering from two points: one is the ability of fatty acid synthesis; the other is the section of fatty acid. And then we pointed out a new synthetic process of microbial oil for fatty acid production using E. coli engineered strain.However, increasing the productivity of fatty acids in E. coli is still a challenge. In this work, we tried to construct E. coli engineered strain to enhance fatty acid production by metabolic engineering strategies according to lipid accumulation mechanism in oleaginous microorganism. Acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus was expressed in E. coli to redirect the carbon flux to the generation of malonyl-CoA, which resulted in a 3-fold increase in intracellular lipids. Moreover, providing high level of NADPH by over-expressing malic enzyme and adding malate in culture medium resulted in a 4-fold increase of intracellular lipids (about 197.74 mg/g). Co-expression of ACC and malic enzyme resulted in 284.56 mg/g intracellular lipids, a 5.6-fold increase constrasted to wild-type strain. Under fermentation conditions in 5L fermentor, 2.5g/L fatty acids were produced.Basing on the problem of the cost, quality and environmental pollution in the process of microbial oil separation, we studied the mechanism of fatty acid secretion in E. coli, and discussed the function of regulated enzymes and proteins during the process of fatty acid transportation. Overexpression of native tesA gene and A. thaliana AtFatA resulted in free fatty acid in culture, which was 14.7% of total intracellular fatty acid, and released inhibition caused by product. Furthermore, co-expression of AtFatA and ACC genes improved the ability of fatty acid secretion in E. coli, and resulted in 71 mg/L FFA. The free fatty acid reached 120mg/L due to that cell membrane permeability of E. coli was increased after expressing native msbA gene, and the ratio of FFA to FA was increased from 1/25 to 1/8. Under fermentation conditions in 5L fermentor, 220mg/L of free fatty acids were detected in culture (accounted 55% for total lipids in cell).The E. coli engineered strain with high free fatty acid productivity was constructed in this study, which owned the ability of secreting extracellular fatty acid in culture. Meanwhile, this paper illustrated the mechanism of fatty acid secretion and transportation in E. coli. These results provide some attractive strategies for increasing lipids production in E. coli by simulating the lipids accumulation of oleaginous microorganisms, which could be helpful in developing a prokaryote as a fatty acid producer. |
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