p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.
对香豆酸是一种具有多种药理活性的天然酚类化合物，也是多种天然药用产物生物合成的前体物质，广泛应用于食品、化妆品、医药等领域。通过微生物合成对香豆酸相对于化学合成和植物提取工艺具有节能减排等优势。但是，目前微生物合成对香豆酸产量较低，难以满足大规模工业发酵生产的要求。为了进一步提高对香豆酸产量，对粘红酵母酪氨酸解氨酶 (Tyrosine ammonia-lyase，TAL) 进行定向进化改造，利用高通量筛选方法从随机突变体文库中筛选TAL 催化活性提高的突变体。通过初筛和复筛两轮筛选，从大约10 000 个突变体中获得1 个TAL 催化活性提高1 倍的突变体。该突变体包含3 个氨基酸突变位点，分别为S9Y、A11N、E518A。进一步通过单点氨基酸饱和突变验证，当S9 位点突变为Y、I、N 和A11 位点突变为N、T、Y 时，TAL 的催化活性提高1 倍以上。通过对S9 和A11 位点3 种类型突变进行组合突变验证，S9Y/A11N 和S9N/A11Y 突变体的TAL催化活力显著高于其他组合。将S9N/A11Y 突变体质粒转入酪氨酸高产菌株CP032。通过摇瓶发酵，该菌株在48 h 时的对香豆酸产量达到394.2 mg/L，比对照菌提高2.2 倍。本研究工作对促进微生物合成对香豆酸的代谢工程研究具有一定的参考价值。.
Keywords: Escherichia coli; biosynthesis; directed evolution; p-coumaric acid; tyrosine ammonia-lyase.