从头合成赤松素大肠杆菌工程菌的构建

赤松素是高价值芪类保健营养品白藜芦醇的类似物，具有预防心血管疾病、抗癌和治疗关节炎等与其相对应的多种生物活性。本研究采用基因工程手段创制出一种从头生物合成赤松素的方法，以缓解目前赤松素供求不足的问题。把带有苯丙氨酸解氨酶（PAL），肉桂酸辅酶A连接酶（4CL）和赤松素合酶（STS）编码基因的组成型表达载体转化高产L-苯丙氨酸大肠杆菌ATCC31884，并通过PCR鉴定后，获得重组工程菌株。工程菌进行摇瓶培养，对培养液进行高效液相色谱检测，确定工程菌具有生物合成赤松素的能力。随后通过对工程菌在不同培养时间产物生成量的比较，结果表明，赤松素在发酵12 h后增长缓慢，24 h培养液中的赤松素含量最高，为0.32 mg/L，而其中间体肉桂酸的积累最高达到52.92 mg/L。这表明，大肠杆菌工程菌能在不添加任何前体物的情况下，利用自身代谢从头合成赤松素，但中间体肉桂酸的转化能力不足，有待下一步实验进行改善。
Pinosylvin, a stilbenoid analog of the valuable nutrient resveratrol, exhibits many biological effects, such as cardiovascular disease prevention and anticancer and anti-arthritic activities. Herein, genetic engineering procedures were used to develop a de novo method for pinosylvin synthesis in order to ease the current problem of low pinosylvin supply. A constitutive vector containing the phenylalanine ammonia-lyase, cinnamoyl-coenzyme A ligase, and stilbene synthase genes was transformed into the L-phenylalanine-overproducing host strain Escherichia coli ATCC31884. PCR identification confirmed that a recombinant bacterial strain was obtained. The engineered bacterial cells were grown in shaking culture, and the culture medium was measured by HPLC to confirm that an engineered strain with de novo pinosylvin biosynthetic ability had been successfully created. Testing of culture samples taken at different fermentation times revealed that the rate of pinosylvin production increased more slowly after 12 h of culture, the highest pinosylvin yield of 0.32 mg/L was reached after a 24 h fermentation, and the maximum accumulated amount of cinnamic acid was 52.92 mg/L. These results suggest that the engineered E. coli can employ its own metabolism to synthesize pinosylvin de novo without addition of any other substances. However, the conversion efficiency of cinnamic acid was low, and thus further studies are needed to improve the strain.