Metabolic engineering of Escherichia coli for the production of isoflavonoid-4′-O-methoxides and their biological activities
Metabolic engineering of Escherichia coli for the production of isoflavonoid-4′-O-methoxides and their biological activities
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DOI：
10.1002/bab.1452
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发表时间：
2019-07-01
影响因子：
2.8
通讯作者：
Sohng, Jae Kyung
中科院分区：
文献类型：
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作者：
Koirala, Niranjan;Pandey, Ramesh Prasad;Sohng, Jae Kyung
异黄酮代表，如染料木黄酮和大豆苷元，是高效的抗癌、抗菌和抗氧化剂。研究表明，黄酮类化合物的甲基化可增强其转运能力，从而促进吸收，大大提高生物利用度。本论文通过构建大肠杆菌基因工程菌，coliK 12来源的metK（登录号：K 02129）用于增强SAM作为前体的S-腺苷-L-甲硫氨酸（SAM）合酶，以及阿维链霉菌来源的SaOMT 2（O-甲基转移酶，登录号：NP_823558）用于甲基化作为优选底物的大豆苷元和染料木素。通过使用色谱方法如高效液相色谱、液相色谱/飞行时间/质谱（LC-TOF-MS）和核磁共振（NMR）以及NMR（H-1和C-13），分别证实了通过生物转化形成所需产物，包括4 ′-O-甲基-染料木黄酮和4 ′-O-甲基-大豆苷元。此外，底物浓度，孵育时间和培养基参数进行了优化，使用烧瓶培养。最后，在最佳条件下进行补料分批发酵，放大至3L（工作体积），获得最大产量，其中4 '-O-甲基染料木素和4 '-O-甲基大豆苷元的产量分别为164.25 μ M（46.81 mg/L）和382.50 μ M（102.88 mg/L）。特别地，研究并证明了这些二甲双胍甲醇盐对癌细胞系（B16 F10、AGS和HepG 2）和人脐静脉内皮细胞生长的有效抑制活性。本研究对E.大肠杆菌工程，生产的改进，所生产的化合物的表征，以及所生产的黄酮类化合物的初步体外生物活性。
Isoflavonoid representatives such as genistein and daidzein are highly potent anticancer, antibacterial, and antioxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12-derived metK encoding S-adenosine-l-methionine (SAM) synthase (accession number: K02129) for enhancement of SAM as a precursor and Streptomyces avermitilis originated SaOMT2 (O-methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 4 '-O-methyl-genistein and 4 '-O-methyl-daidzein was confirmed individually by using chromatographical methods such as high-performance liquid chromatography, liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS), and nuclear magnetic resonance (NMR), and NMR (H-1 and C-13). Furthermore, substrates concentration, incubation time, and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed-batch fermentation with scale-up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 mu M (46.81 mg/L) and 382.50 mu M (102.88 mg/L) for 4 '-O-methyl genistein and 4 '-O-methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS, and HepG2) and human umbilical vein endothelial cells were investigated and demonstrated. Taken together, this research work described the production of isoflavonoid-4 '-O-methoxides by E. coli engineering, improvement of production, characterization of produced compounds, and preliminary in vitro biological activities of the flavonoids being manufactured.