生物合成对香豆酸大肠杆菌工程菌的构建

Construction of Engineering Escherichia coli for p-Coumaric Acid Bioproduction

对香豆酸是一种具有预防心血管疾病、抗氧化和抗菌消炎等生物活性的酚类物质,同时,它也是高价值苯丙烷类保健营养品(如白藜芦醇)的前体。本研究希望创制出一种生物合成对香豆酸的方法,以缓解对香豆酸的供求问题。把带有粘红酵母(Rhodotorula glutinis)酪氨酸解氨酶(Rg TAL)基因的组成型表达载体转化大肠杆菌ATCC31884,并通过PCR鉴定后,获得重组菌株。对重组菌株进行发酵培养,对发酵液进行高效液相色谱检测,确定工程菌具有生物合成对香豆酸的能力。随后通过优化L-酪氨酸的添加量和工程菌的发酵时间,最终确定,底物L-酪氨酸的添加量为0.5 m M,发酵时间为36 h,检测发酵液中的对香豆酸含量最高,为161.23 mg/L。这表明,Rgtal基因在重组大肠杆菌中成功得到了表达,并且能利用自身代谢和外源添加的L-酪氨酸生物合成对香豆酸。

p-Coumaric acid is a bioactive phenolic compound that can prevent cardiovascular disease,with antioxidant,antibacterial,and anti-inflammatory properties.It is also a precursor of high-value phenylpropanes nutraceuticals（such as resveratrol）.A new method for p-coumaric acid bioproduction was developed in this study to increase production levels to meet the growing demand for this compound.The constitutive expression vector containing tyrosine ammonia lyase gene from Rhodotorula glutinis（Rg TAL） was transformed into host strain,Escherichia coli ATCC31884.The recombinant strain was confirmed by polymerase chain reaction（PCR）.Fermentation broth using the recombinant strain was analyzed by high performance liquid chromatography（HPLC） and p-coumaric acid bioproduction by the engineered E.coli strain was confirmed.Optimal concentration of L-tyrosine substrate to be added and fermentation time were identified; highest yield of p-coumaric acid（161.23 mg/L） was achieved with 0.5 m M L-tyrosine and 36 h fermentation.The results indicated that Rgtal gene was successfully expressed in the recombinant engineered E.coli strain,which was capable of p-coumaric acid biosynthesis via its own metabolism,using exogenously supplemented L-tyrosine.