Department of Microbiology and Biotechnology, University of Hamburg, Ohnhorststr 18, Hamburg, Germany.
Department of Microbiology and Biotechnology, University of Hamburg, Ohnhorststr 18, Hamburg, Germany.
Metab Eng. 2019 Sep;55:212-219. doi: 10.1016/j.ymben.2019.07.002. Epub 2019 Jul 16.
Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides.
类黄酮具有广泛的生物功能，对制药和医疗保健行业极具吸引力。然而，由于其水溶性低和生物利用度差，其应用往往受到限制，而糖基化通常可以缓解这些问题。糖基转移酶 C（GtfC）是一种来源于微生物组的细菌糖基转移酶，可用于合成各种黄酮类化合物的新型和罕见鼠李糖苷，包括白杨素、大豆黄素、大豆素和橙皮素。其中一些是我们在这项工作中首次描述的。在我们的研究中，我们采用了一种新的代谢工程方法来提高大肠杆菌全细胞生物转化中的鼠李糖基化率。在大肠杆菌 MG1655 中，通过将麦芽糖代谢与糖基化偶联，以橙皮素为模型底物进行了研究。该过程高度依赖于麦芽糖的可用性。通过删除磷酸葡糖变位酶（pgm）和尿苷二磷酸葡萄糖-1-磷酸尿苷转移酶（galU）基因，并同时过表达 dTDP-鼠李糖合成基因（rmlABCD）以及葡萄糖 1,4-α-麦芽糖六糖苷酶，以增加麦芽糖的降解，同时在大肠杆菌 UHH_CR5-A 中过表达 GtfC，可实现最大生产。与在大肠杆菌 MG1655 中表达 GtfC 的 24 小时分批发酵相比，橙皮素鼠李糖苷的产量增加了 3.2 倍。此外，大肠杆菌 UHH-CR_5-A 能够在 48 小时后生产出 2.4 g/L 的橙皮素-3'-O-鼠李糖苷的终产物。为了展示工程大肠杆菌菌株的多功能性，进行了槲皮素和山柰酚的生物转化，分别在 48 小时内生产出 4.3 g/L 的槲皮苷和 1.9 g/L 的圣草酚。到目前为止，这是使用生物转化方法获得的黄酮类化合物鼠李糖苷化的最高产量。这些结果清楚地表明，经过工程改造的大肠杆菌生产宿主具有很高的潜力，可作为高水平生物转化黄酮类鼠李糖苷的平台。
