Huang Fong-Chin, Hinkelmann Jens, Hermenau Alexandra, Schwab Wilfried
Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
J Biotechnol. 2016 Apr 20;224:35-44. doi: 10.1016/j.jbiotec.2016.02.022. Epub 2016 Feb 18.
Glycosyltransferase (GT)-mediated methodology is recognized as one of the most practical approaches for large-scale production of glycosides. However, GT enzymes require a sugar nucleotide as donor substrate that must be generated in situ for preparative applications by recycling of the nucleotide moiety, e.g. by sucrose synthase (SUS). Three plant GT genes CaUGT2, VvGT14a, and VvGT15c and the fungal SbUGTA1 were successfully co-expressed with GmSUS from soybean in Escherichia coli BL21 and W cells. In vitro, the crude protein extracts prepared from four GT genes and GmSUS co-expressing cells were able to convert several small molecules to the corresponding glucosides, when sucrose and UDP were supplied. In addition, GmSUS was able to enhance the glucosylation efficiency and reduced the amount of supplying UDP-glucose. In the biotransformation system, co-expression of VvGT15c with GmSUS also improved the glucosylation of geraniol and enhanced the resistance of the cells against the toxic terpenol. GT-EcW and GTSUS-EcW cells tolerated up to 2mM geraniol and converted more than 99% of the substrate into the glucoside at production rates exceeding 40μgml(-1)h(-1). The results confirm that co-expression of SUS allows in situ regeneration of UDP-sugars and avoids product inhibition by UDP.
糖基转移酶（GT）介导的方法被认为是大规模生产糖苷最实用的方法之一。然而，GT酶需要糖核苷酸作为供体底物，在制备应用中必须通过核苷酸部分的循环利用原位生成，例如通过蔗糖合酶（SUS）。来自大豆的三种植物GT基因CaUGT2、VvGT14a和VvGT15c以及真菌SbUGTA1与GmSUS在大肠杆菌BL21和W细胞中成功共表达。在体外，当提供蔗糖和UDP时，从四种GT基因和GmSUS共表达细胞制备的粗蛋白提取物能够将几种小分子转化为相应的糖苷。此外，GmSUS能够提高糖基化效率并减少UDP-葡萄糖的供应量。在生物转化系统中，VvGT15c与GmSUS的共表达也提高了香叶醇的糖基化水平，并增强了细胞对有毒萜烯醇的抗性。GT-EcW和GTSUS-EcW细胞能够耐受高达2mM的香叶醇，并以超过40μgml(-1)h(-1)的生产率将超过99%的底物转化为糖苷。结果证实，SUS的共表达允许UDP-糖的原位再生，并避免UDP对产物的抑制。
