基于Staphylococcus carnosus来源的D-果糖-1,6-二磷酸醛缩酶在大肠杆菌合成稀有酮糖的研究

摘要

将Staphylococcus carnosus来源的FruAS.car醛缩酶基因fda和大肠杆菌来源的YqaB磷酸酶基因yqaB分别插入到表达质粒CDFDuet-1得到重组质粒CDF-fda-yqaB,并转化该重组质粒到大肠杆菌BL21 Star(DE3).以同时过量表达FruAS.car醛缩酶和YqaB磷酸酶的大肠杆菌BL21 Star(DE3)/CDFDuet-1-fda-yqaB作为发酵菌株,以葡萄糖为碳源通过糖酵解途径在胞内生成供体磷酸二羟基丙酮,分别在培养基中添加丙醛、丁醛为受体,进行相应稀有酮糖的合成,从而成功地将羟醛缩合反应在大肠杆菌中实现,酮糖产物用HPLC检测并进行纯化和1HNMR鉴定.最后,以13C同位素全标记的葡萄糖为碳源,添加丙醛为受体进行了同位素标记实验,证实了产物从DHAP而来的3个碳原子最终来自葡萄糖.%fda gene encoding FruAS.car from Staphylococcus carnosus and yqaB gene encoding YqaB phosphatase from Escherichia coli were inserted into the expression vector CDFDuet-1 to obtain the recombinant plasmid CDF-fda-yqaB.The plasmid CDF-fda-yqaB was transformed into E.coli BL21Star (DE3).The engineered E.coli BL21Star (DE3)/CDFDuet-1-fda-yqaB with capacity of co-expression of aldolase FruAS.car and phosphatase YqaB was used as the fermentation strain.The rare ketoses were synthesized with dihydroxyacetone phosphate (DHAP) generated in cells via glycolytic pathway as the the donor molecular and the propionaldehyde (butyraldehyde) as the acceptors.The ketose products were analyzed by HPLC and confirmed with 1H NMR after purification.In addition,the isotope experiment was performed using [U-13C6] labeled glucose as the sole carbon source with propionaldehyde as the acceptor.The result showed that C1,2,3 of the product derived from DHAP were ultimately from glucose.