摘要
本文旨在以海洋细菌Pseudomonas sp.QDA褐藻多糖(alginate)生物合成操纵元中的algG基因为靶点,利用DNA重组技术改建褐藻多糖的生物合成途径,以期获得产生甘露糖醛酸的突变菌株。首先,利用PCR克隆幽G基因两侧大小分别为1.7kb和1.8kb的DNA片段作为同源片段,用编码Gm抗性的aacCl基因替换algG基因,构建成打靶载体pEX100TUDG。将打靶载体转入QDA,利用Gm抗生素和蔗糖为选择压力进行培养,通过PCR和Southern杂交验证,获得了突变菌株。^1H—NMR、PAGE和GPC分析发现,突变菌株胞外产物为多聚甘露糖醛酸。该突变菌株的获得丰富了具有较高药物开发价值的甘露糖醛酸的来源,为褐藻多糖途径工程的进一步研究提供了理论依据和技术支持。
In order to obtain an△algG::Gm mutant which produces mannuronan,the bacterial alginate biosynthesis pathway was reconstituted by targeted disruption in Pseudomonas sp.QDA.Two homologous fragments adjacent to algG in the alginate biosynthesis gene cluster were amplified and ligated to pEX100T with a non-polar aacC1 gene between them to replace the algG gene,and preserve the transcription of downstream genes.The recombinant plasmid pEX100TUDG was transferred into Pseudomonas sp.QDA by triparental mating.The△algG::Gm mutants were identified by plating on L agar containing Gm and sucrose,and verified by PCR and Southern blotting.^1H-NMR analysis indicated that the product isolated from AalgG::Gm mutant was pure mannuronan.Moreover,analysis of gel-permeation chromatography and carbohydrate electrophoresis showed that there was no difference in molecular weight distribution of the products between wild strain and the mutants.The successful reconstruction of mannuronan biosynthesis pathway expanded the possibilities for making new polysaccharides and discovering valuable drugs through genetic engineering way.
作者
沈洪波
韩峰
林育姿
韩文君
于文功
Shen Hongbo;Han Feng;Lin Yuzi;Han Wenjun;Yu Wengong(Key Laboratory of Marine Drugs of Ministry of Education,Marine Drug and Food Institute,Ocean University of China,Qingdao 266003)
出处
《高技术通讯》
CAS
CSCD
北大核心
2006年第2期175-180,共6页
Chinese High Technology Letters
基金
国家自然科学基金委员会面上基金(30371683)资助项目.
关键词
多聚甘露糖醛酸
途径工程
假单胞菌
差向异构酶
inannuronan
pathway engineering
Pseudomonas strains
mannuronan C-5-epimerase
