Production of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli
Production of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli
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DOI：
10.1007/s00253-006-0730-x
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发表时间：
2007-03-01
影响因子：
5
通讯作者：
Seo, Jin-Ho
中科院分区：
文献类型：
--
作者：
Byun, Seong-Goo;Kim, Myoung-Dong;Seo, Jin-Ho
利用重组大肠杆菌菌株生产鸟苷5'-二磷酸（GDP）-L-岩藻糖，L-岩藻糖是合成岩藻糖基寡糖的必需底物。将GDP-D-甘露糖-4，6-脱氢酶（GMD）和GDP-4-酮-6-脱氧甘露糖-3，5-差向异构酶4-还原酶（WcaG）这两种GDP-L-岩藻糖生物合成的关键酶在重组E. coli中表达。GMD和WcaG在重组E. coli BL21（DE3）的最适温度为25 ℃，最适浓度为0.1 mM异丙基-β-D-硫代吡喃葡萄糖苷。在葡萄糖限制的分批补料培养中获得了38.9 +/-0.6 mg l（-1）的最大GDP-L-岩藻糖浓度，并且通过zwf基因编码的NADPH再生葡萄糖-6-磷酸脱氢酶的共表达进一步提高了该浓度，达到55.2 +/-0.6 mg l（-1）在相同的培养条件下，L-岩藻糖的浓度。
A recombinant Escherichia coli strain was developed to produce guanosine 5'-diphosphate (GDP)-L-fucose, donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-D-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25 degrees C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 +/- 0.6 mg l(-1) was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 +/- 0.5 mg l(-1) GDP-L-fucose under the same cultivation condition.