Over-expression of UDP-glucose pyrophosphorylase increases validamycin A but decreases validoxylamine A production in Streptomyces hygroscopicus var. jinggangensis 5008.
Over-expression of UDP-glucose pyrophosphorylase increases validamycin A but decreases validoxylamine A production in Streptomyces hygroscopicus var. jinggangensis 5008.
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DOI：
10.1016/j.ymben.2011.10.001
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发表时间：
2011-11
影响因子：
8.4
通讯作者：
Xiaoping Zhou;Hang Wu;Zhong Li;Xiu-fen Zhou;Linquan Bai;Z. Deng
中科院分区：
文献类型：
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作者：
Xiaoping Zhou;Hang Wu;Zhong Li;Xiu-fen Zhou;Linquan Bai;Z. Deng
吸水链霉菌TL 01在发酵生产井冈霉素A（18 g/L）的过程中，积累了大量的中间产物井冈羟胺A（4.0g/L）。在发酵过程中未观察到井冈霉素A的化学或酶水解。TL 01中葡萄糖基转移酶ValG的过表达并没有增加糖基化的效率。然而，增加井冈霉素A和降低井冈羟胺A生产中观察到的无细胞提取物和发酵液中的TL 01补充了高浓度的UDP-葡萄糖。发现TL 01中催化UDP-葡萄糖形成的UDP-葡萄糖焦磷酸化酶（Ugp）的酶活性远低于参与UDP-葡萄糖生物合成的其他酶和葡糖基转移酶ValG的活性。从S.吸水菌5008并验证为Ugp编码。在TL 01中，Ugp的转录增加了1.5倍，Ugp活性增加了100%。此外，产生了22 g/L井冈霉素A和2.5g/L井冈羟胺A，并且井冈霉素A/井冈羟胺A的比率从TL 01中的3.15增加到5.75。这些数据证明井冈霉素A的生物合成受到UDP-葡萄糖供应的限制，这可以通过Ugp过表达来缓解。
During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18g/L), a considerable amount of an intermediate validoxylamine A (4.0g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22g/L validamycin A and 2.5g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.