2-羟基异丁酸的微生物法合成研究

发布时间：2018-01-31 01:23

本文关键词： 2-羟基异丁酸大肠杆菌嗜水气单胞菌变位酶发酵　出处：《北京化工大学》2014年硕士论文　论文类型：学位论文

【摘要】：2-羟基异丁酸(2-Hydroxyisobutyric acid,简写2-HIBA)是一种有多用途的重要的化工产品,它可参与多种有机合成反应生成一系列化合物,以它为平台可以衍生出许多重要的其它化工产品,在许多领域有潜在的应用前景。由于其结构特殊,目前为发现其天然的微生物法合成途径。化学方法生产具有成本高、生成条件苛刻、污染大等缺点,随着合成生物学、分子生物学和代谢工程学等迅速发展,利用生物转化的方法生产2-羟基异丁酸成为可能。本论文就微生物法生产2-羟基异丁酸进行了初步的探索研究。以重组大肠杆菌为宿主,构建了bktB、hbd、icmA、icmB和lesB等5个基因的表达载体pEnBHTI,以葡萄糖降解产生的乙酰CoA为前体,经过4步代谢途径,以期能够合成2-羟基异丁酸。实验结果发现,含有pEnBHTI的大肠杆菌能够以葡萄糖为碳源获得3-羟基丁酸,未检测到明显的2-羟基异丁酸积累,提示bktB、hbd和tesB成功表达,但是icmAB表达量可能较低,或是tesB对2-羟基异丁酰辅酶A的催化活性较低,导致以重组大肠杆菌为宿主未能获得产物。在此基础上,以嗜水气单胞菌为宿主,利用其脂肪酸氧化过程中产生的羟基丁酰辅酶A为前体,通过构建icmA和icmB的表达载体pMCSV12,在嗜水气单胞菌单胞菌中构建2-羟基异丁酸的生物合成途径。对重组菌株进行摇瓶培养,发现其能够合成微量的2-羟基异丁酸；进而采用不同的脂肪酸碳源、两步法发酵等对培养条件进行优化,最终获得了46 mg/L的产量。本论文为微生物法合成2-羟基异丁酸提供了初步的研究基础。
[Abstract]:2-hydroxyisobutyric acid2-Hydroxyisobutyric acid2-Hydroxyisobutyric acid2-Hydroxyisobutyric acid (2-H@@. It can participate in a variety of organic synthesis reactions to form a series of compounds, it can be used as a platform to derive many important other chemical products, and has potential applications in many fields, because of its special structure. At present, in order to find its natural way of microbiological synthesis, chemical production has many disadvantages, such as high cost, harsh conditions and pollution. With the rapid development of synthetic biology, molecular biology and metabolic engineering, etc. It is possible to produce 2-hydroxyisobutyric acid by biotransformation. In this paper, the production of 2-hydroxyisobutyric acid by microbial method was studied. The recombinant Escherichia coli was used as host to construct bktB. The expression vector pEnBHTI of 5 genes, including hbddcicmAgnicmB and lesB, was used as precursor of acetyl CoA produced by glucose degradation, and was metabolized by four steps. In order to synthesize 2-hydroxyisobutyric acid, the results showed that 3-hydroxybutyric acid could be obtained by using glucose as carbon source in Escherichia coli containing pEnBHTI, but no obvious accumulation of 2-hydroxyisobutyric acid was detected. These results suggested that bktBnhbd and tesB were successfully expressed, but the expression of icmAB might be low, or the catalytic activity of tesB to 2-hydroxyisobutyryl coenzyme A was lower. On the basis of this, Aeromonas hydrophila was used as host and hydroxy butyryl coenzyme A produced during fatty acid oxidation was used as precursor. By constructing the expression vector pMCSV12 of icmA and icmB, the biosynthesis pathway of 2-hydroxyisobutyric acid was constructed in Aeromonas hydrophila. It was found that it could synthesize 2-hydroxyisobutyric acid. Then different carbon sources of fatty acids and two-step fermentation were used to optimize the culture conditions. Finally, the yield of 46 mg/L was obtained. This paper provides a preliminary research basis for the synthesis of 2-hydroxyisobutyric acid by microbial method.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914.5

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