PDF(Pubmed)
Abstract：
Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.
摘要：
阿魏酸（对羟基-3-甲氧基肉桂酸，FA）是存在于植物细胞壁中的天然活性物质，抗氧化剂，抗癌，抗血栓和其他特性;它被广泛用于医学，食物,和化妆品。通过生态友好型生物工艺生产FA具有巨大的潜力。在这项研究中,FA由代谢工程大肠杆菌生物合成。作为第一步,基因tal(编码酪氨酸氨裂解酶，RsTAL）来自球形红细菌，sam5(编码对香豆酸3-羟化酶，SeSAM5）来自espanaensis和comt（编码咖啡酸O-甲基转移酶，将来自小麦的TaCM）克隆到pET质粒骨架上的操纵子中，证明含有该构建体的大肠杆菌菌株成功地从L-酪氨酸生产FA,并证实了TaCM作为咖啡酸O-甲基转移酶的功能。发酵结果表明，JM109（DE3）是比BL21（DE3）更适合FA生产的宿主细胞。之后，通过调节启动子强度（T7启动子或T5启动子）和拷贝数（pBR322或p15A）来优化FA途径的基因表达强度，p15a-T5的组合效果最好。为了进一步提高FA产量，大肠杆菌天然pntAB，编码吡啶核苷酸转氨酶，从五个NADPH再生基因中选择补充氧化还原辅因子NADPH，用于在FA生物合成过程中将对香豆酸转化为咖啡酸。按顺序,将咖啡酸进一步转化为FA，也过表达非天然甲硫氨酸激酶(来自标准链霉菌的MetK)。根据烧瓶发酵数据显示，工程大肠杆菌菌株产生212mg/L的FA和11.8mg/L的咖啡酸残留物，可以得出结论，据我们所知，这是大肠杆菌K-12菌株获得的最高FA产量。
