高山被孢霉GDP-L-岩藻糖合成途径中GDP-甘露糖-4,6-脱水酶的克隆、表达和功能鉴定  

Cloning,expression and function analysis for GDP-D-mannose-4,6-dehydratase in the de novo biosynthesis of GDP-L-fucose from Mortierella alpina

作　　者：王鸿超[1] 张陈[1] 周昕[1] 陈海琴[1] 顾震南[1] 张灏[1] 陈卫[1] 陈永泉[1] 

机构地区：[1]江南大学食品学院,江苏无锡214122

出　　处：《食品与发酵工业》2016年第11期14-19,共6页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31400038)

摘　　要：GDP-L-岩藻糖是糖复合物生物合成和糖类代谢的重要中间产物,作为岩藻糖转移酶的供体参与岩藻糖基化反应,具有重要的生理功能。高山被孢霉是重要的产油真菌,是唯一在分子水平上证明可合成GDP-L-岩藻糖的真菌。GDP-L-岩藻糖从头合成途径中的GDP-甘露糖-4,6-脱水酶(GMD)能催化GDP-D-甘露糖合成GDP-4-酮基-6-脱氧-D-甘露糖。对高山被孢霉中的GMD基因进行克隆、表达和功能鉴定,可进一步阐明GDP-L-岩藻糖体内代谢的分子生物机制。首先对GMD序列进行分析,并以p ET28a+质粒为骨架构建了GMD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用液相色谱-质谱法分析酶反应产物,表明纯化蛋白具有GMD活性。最后对高山被孢霉进行发酵培养,发现GDP-L-岩藻糖产量在氮源耗尽后较高,可达0.10 mg/g。同时GMD的转录水平在氮源耗尽后发生了明显的上调,表明GMD在氮源耗尽后对高山被孢霉体内GDP-L-岩藻糖的合成具有重要作用。这为进一步利用高山被孢霉发酵生产GDP-L-岩藻糖和酶法转化生产GDP-L-岩藻糖奠定了理论基础。GDP-L-fucose is an essential intermediate in glycoconjugate biosynthesis and carbohydrate metabo- lism, which functions as a biological donor for fucosyhransferases involved in glycosylation reactions. Mortierella alpi- na is one of the highest lipid-producing fungi and proved to synthesize GDP-L-fucose at the molecular level. GDP-D- mannose-4,6-dehydratase can catalyze the formation of GDP-4-keto-6-deoxy-D-mannose from GDP-D-mannose in the GDP-L-fucose de novo pathway of M. alpirta. To clarify the molecular mechanism of GDP-L-fucose biosynthesis, the heterologous expression vector for GMD was constructed under the skeleton of plasmid pET28a ＋ and expressed in BL21 （DE3） gold strain of E. coli. The enzymatic activity was investigated by liquid chromatography and mass spec- trometry. The yield of GDP-L-fucose was relatively increased after nitrogen exhaustion （0.10 mg/g）. Additionally, the transcript level of GMD was up-regulated by nitrogen exhaustion, which highlights the functional significance of GMD in the GDP-L-fucose biosynthesis in M. alpina. This study laid the foundation for GDP-L-fucose production in M. alpina and enzyme production of GDP-L-fucose.

关 键 词：功能鉴定 从头合成途径 GDP-甘露糖-4 6-脱水酶 GDP-L-岩藻糖 高山被孢霉 

分 类 号：TQ925[轻工技术与工程—发酵工程]