Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
Appl Microbiol Biotechnol. 2018 Feb;102(3):1251-1267. doi: 10.1007/s00253-017-8694-6. Epub 2018 Jan 7.
Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 μM) and 11% (9 mg/L; 22 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 μM) and 14 mg/L (17%, 34 μM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48 h upon supplementation of 200 μM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 μM) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin.
从 Glycine max 中提取的两种植物来源的 C-葡萄糖基转移酶（CGTs）UGT708D1 和 Gentiana triflora 的 GtUF6CGT1 被用于对选定的黄酮查尔酮和木樨草素进行葡萄糖基化反应。通过在多顺反子载体中引入异源 UDP-葡萄糖生物合成基因，即葡萄糖激酶（glk）、磷酸葡糖变位酶（pgm2）和葡萄糖 1-磷酸尿苷酰转移酶（galU），以及来自不同生物体的葡萄糖易位蛋白扩散蛋白（glf），可以在大肠杆菌细胞胞质溶胶中增强 UDP-葡萄糖库。该载体具有单个 T 启动子、核糖体结合位点和每个基因的终止子。通过高效液相色谱-光电二极管阵列、高分辨率四极杆飞行时间电喷雾电离质谱和一维核磁共振分析对 C-葡糖苷化产物进行了分析。补料分批摇瓶培养显示，UGT708D1 和 GtUF6CGT1 将查尔酮分别转化为查尔酮 6-C-β-D-葡萄糖苷的转化率分别为 8%（7mg/L；16μM）和 11%（9mg/L；22μM）。此外，具有外源 UDP-葡萄糖生物合成基因和葡萄糖易位蛋白扩散蛋白的工程大肠杆菌菌株使查尔酮 6-C-β-D-葡萄糖苷的产量提高了约 1.4 倍，分别产生 10mg/L（12%，24μM）和 14mg/L（17%，34μM）。UGT708D1 和 GtUF6CGT1 不需要在培养基中额外添加 UDP-葡萄糖。当工程菌株在 3L 体积的实验室规模发酵罐中放大时，生物转化进一步提高。发酵液提取物的 HPLC 分析表明，在 48 小时内补充 200μM 查尔酮时，查尔酮向查尔酮 6-C-β-D-葡萄糖苷的转化率为 50%（42mg/L，100μM）。在 48 小时的 50-mL 摇瓶发酵中，木樨草素的最大转化率为 38%（34mg/L，76μM）。查尔酮的 C-葡糖苷衍生物比查尔酮的 O-葡糖苷衍生物更具溶解性和对高温、不同 pH 值范围和β-葡萄糖苷酶的稳定性。
