N-端改造植物P450酶实现工程大肠杆菌合成甜菜黄素

Betaxanthins Bioproduction by Escherichia coli Using N-Terminal-Modified Plant P450 Enzyme

甜菜黄素是安全的植物源水溶性天然色素,主要应用于食品行业.甜菜黄素还具有抗氧化活性和光学特性,在医疗保健、荧光成像等领域具有潜在应用价值.目前,甜菜黄素的主要来源是植物提取分离,但存在甜菜黄素含量低、种类少等问题.本文改造植物P450氧化酶,研发大肠杆菌异源合成甜菜黄素的新方法.首先,对甜菜的P450氧化酶CYP76AD1^(LL)和P450还原酶(cytochrome P450 reductase,CPR)进行N-端截短和改造,研究不同改造序列和表达策略对合成L-多巴的影响.结果表明,截去N-端跨膜序列,利用3种不同N-端序列修饰P450氧化酶,MA序列修饰CPR(MA-tCPR),工程大肠杆菌中均能以L-酪氨酸为底物合成L-多巴.其中亲水性2B1序列(MAKKTSS)修饰P450氧化酶,采用双顺反子表达策略优于单顺反子,工程大肠杆菌BDP10合成L-多巴的产量最高,为40.42 mg/L.进一步表达紫茉莉的4,5-多巴双加氧酶基因,构建了工程大肠杆菌BTA11,利用L-酪氨酸为前体,合成了甜菜醛氨酸.最后,在培养基添加3种氨基酸(对氨基苯甲酸、L-组氨酸、L-亮氨酸)和3种胺类化合物(酪胺、吡咯烷、吲哚啉),工程大肠杆BTA11合成了6种对应的甜菜黄素.研究结果表明,采用N-端改造策略赋予植物P450氧化酶在大肠杆菌中具有催化功能,能合成天然和非天然的颜色多样、结构丰富的多种甜菜素.

Betaxanthins are water-soluble and safe pigments mainly used in the food industry.With rich antioxidant and optical properties,betaxanthins have potential applications in the fields of health care and fluorescence imaging.Presently,the main source of betaxanthins is plant extraction and separation,but the obtained betaxanthin content is low and the betaxanthin varieties are few.In this study,a new method for the heterologous synthesis of betaxanthins by Escherichia coli was developed by the expression of modified plant P450 oxidase.First,to study the effects of modified sequences and expression methods on the conversion of L-tyrosine into L-dopa,beet-derived P450 enzyme CYP76AD1^(LL) and cytochrome P450 reductase(CPR)were truncated and modified at the N-terminal.The results showed that after the truncation of the transmembrane sequences,all three different N-terminal sequence-modified P450 oxidase co-expressing with the MA sequence-modified CPR(MA-tCPR)successfully catalyzed the conversion of L-tyrosine to L-dopa in E.coli.The engineered E.coli strain BDP10 expressing N-terminal hydrophilic sequence 2B1-modified P450 oxidase used in combination with the bicistronic strategy resulted in the highest L-dopa production(40.42 mg/L),which was higher than that obtained with the monocistronic strategy.Furthermore,a betalamic acid-producing strain was constructed through the co-expression of a 4,5-dopa dioxygenase extradiol gene from Mirabilis jalapa,which enabled the conversion of L-tyrosine to betalamic acid.Finally,three amino acids(paraaminobenzoic acid,L-histidine,and L-leucine)and three amines(tyramine,pyrrolidine,and indoline)were fed into the fermentation medium with the engineered E.coli strain,and six betaxanthins could be produced.The results show that N-terminal modification can endow plant P450 oxidase with catalytic function,and the resulting strain can synthesize natural and unnatural betaxanthins with various colors and rich structures.