
构建尿嘧啶磷酸核糖转移酶基因缺失菌株实现Gluconobacter suboxydans基因组无痕修饰
杜红燕, 李天明, 刘金雷, 冯惠勇
中国生物工程杂志 ›› 2016, Vol. 36 ›› Issue (7) : 64-71.
构建尿嘧啶磷酸核糖转移酶基因缺失菌株实现Gluconobacter suboxydans基因组无痕修饰
Construct the Uracil Phosphoribosyl Transferase Gene Mutant Strain in Gluconobacter suboxydans for Seamless Genome Editing
弱氧化葡糖酸杆菌(Gluconobacter suboxydans)可高效不完全氧化多种糖类和醇类等多羟基化合物,生成相应的醛类、酮类和有机酸类等产物,是一类重要的工业微生物。利用同源重组技术对基因组进行修饰改造是工业育种的有效手段。传统方法多选用抗生素为筛选标记,存在诸多缺陷。构建尿嘧啶磷酸核糖转移酶基因缺失菌株以实现Gluconobacter suboxydans基因组无痕修饰为目标,将自杀质粒pMD18-Jqupp电转化至野生型Gluconobacter suboxydans J12中,通过四环素抗性和5-氟尿嘧啶双重筛选,获得敲除了编码尿嘧啶磷酸核糖转移酶的基因upp的突变株。经生理验证表明,该突变株在含0.5mg/ml 5-氟尿嘧啶的培养基上生长,而回补upp基因后,在0.5mg/ml 5-F氟尿嘧啶的培养基上不生长。说明获得的G.suboxydans-upp突变株能以upp基因作为负向筛选标记,通过两次同源重组,实现Gluconobacter suboxydans基因组无痕修饰与改造,为今后代谢工程改造Gluconobacter suboxydans获得有价值的工业菌种奠定基础。
Gluconobacter suboxydans is an important industrial microbiology and can incompletely and efficiently oxidize a great variety of carbohydrates, alcohols and other polyols to form the corresponding aldehydes, ketones and organic acids and other products. The use of homologous recombination technology to transform genomic modification is an effective means of industrial breeding. There are many defects when use antibiotic marker as the screening marker in traditional methods. The construction of uracil phosphoribosyl transferase gene deletion strain is to achieve Gluconobacter suboxydans seamless genome editing. The suicide plasmid pMD18-Jqupp is transformed into wild type Gluconobacter suboxydans J12 and the mutant which knocked encoding uracil phosphoribosyl transferase gene was screened by tetracycline resistance and 5-fluorouracil. The physiological validation results show that the mutant strain can grow on medium containing 0.5mg/ml 5-fluorouracil, but the mutant covering upp gene can't grow on medium containing 0.5mg/ml 5-fluorouracil. It proves that the upp gene can be used as a negative selection marker in G.suboxydans-upp mutants to achieve Gluconobacter suboxydans genome modification and transformation by twice homologous recombination. It provides the foundation of changing Gluconobacter suboxydans to obtain valuable industrial strains using metabolic engineering in the future.
尿嘧啶磷酸核糖转移酶 / 基因敲除 / 无痕修饰 / 弱氧化葡糖酸杆菌 {{custom_keyword}} /
Gluconobacter suboxydans / Gene knockout / Scarless modify / Uracil phosphoribosyl transferase {{custom_keyword}} /
[1] De Ley J,Swings J,Gossele F.The Genus Gluconobacter.In:Krieg N R,Holt J G.Bergey's Manual of Systematic Bacteriology,Baltimore:Williams&Wilkins Co,1984:267-278.
[2] Deppenmeier U,Hoffmeister M,Prust C.Biochemistry and biotechnological applications of Gluconobacter strains.Applied and Molecular Biotechnology.2002,60(3):233-242.
[3] Reichstein T,Grü ssner A.Eine ergiebige synthese der 1-ascorbinsäure (C-vitamin).Helvetica Chimica Acta 1934,17(1):311-328.
[4] Michael S.Regioselective oxidation of aminosorbitol with Gluconobacter oxydans,key reaction in the industrial synthesis of 1-deoxynojirimycin syntheis.Journal of Psychiatry&Neuroscience Jpn,2004,29(5):364-382.
[5] Adachi O,Moonmangmee D,Toyama H,et al.New developments in oxidative fermentation.Applied and Molecular Biotechnology,2003,60(6):643-653.
[6] Liu P,Jenkins N A,Copeland N G.A highly efficient recombineering-based method for generating conditional knockout mutations.Genome Research,2003,13(3):476-484.
[7] 杨奇.大肠杆菌DH5α upp基因的敲除及其应用研究.南京:南京理工大学,环境与生物工程学院,2013.Yang Q.Knockout of the upp gene in Escherichia coli DH5α and its application research.Nanjing:Nanjing University of Science&Technology,Environmental and Biological Engineering,2013.
[8] Bailey J E.Toward a science of metabolic engineering.Science,1991,252(5013):1668-1675.
[9] 胡逢雪,丁锐,崔震海,等.大肠杆菌基因无痕敲除技术及策略.生物技术通讯,2013,24(4):552-557.Hu F X,Ding R,Cui Z H,et al.Approaches and strategies of gene scarless knockout in the Escherichia coli genome.Letters In Biotechnology,2013,24(4):552-557.
[10] Hasegawa N,Abeil M K,Yokoyama K,et al.Cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing uracil phosphoribosyltransferase (UPRT) in immunocompetent Syrian hamsters.International Journal of Cancer,2013,133(6):1479-1489.
[11] Andersen P S,Smith J M,Mygind B.Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12.Federation of European Biochemical Societies,1992,204(1):51-56.
[12] Tan Z G,Zhu X N,Chen J,et al.Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production.Applied and Environmental Microbiology,2013,79(16):4838-4844.
[13] Jantama K,Zhang X L,Moore J C,et al.Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C.Biotechnology and Bioengineering,2008,101(5):881-893.
[14] Zhang X L,Jantama K,Moore J C,et al.Production of L-alanine by metabolically engineered Escherichia coli.Applied Genetics And Molecular Biotechnology,2007,77(2):355-366.
[15] Michael E K,Philip H E,Steve D H,et al.Four new derivatives of the broad-host-range cloning vector pBBR1MCS,carring different antibiotic-resistance cassettes.Gene,1995,166(1):175-176.
[16] Prust C,Hoffmeister M,Liesegang H,et al.Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans.Nature Biotechnology,2005,23(2):195-200.
[17] 余华,熊浚智,何晓梅,等.采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因.中国人兽共患病学报,2013,2:129-132,137.Yu H,Xiong J Z,He X M,et al.Generation of a Pseudomonas aeruginosa elastase gene targeted deletion mutant by Red recombination system.Chinese Journal of Zoonoses,2013,2:129-132,137.
[18] Lim J H,Seo S W,Kim S Y,et al.Refactoring redox cofactor regeneration for high-yield biocatalysis of glucose to butyric acid in Escherichia coli.Bioresource Technology,2013,135:568-573.
[19] Koresawa Y K,Miyagawa S J,Ikawa M H,et al.Synthesis of a new cre recombinase gene based on optimal codon usage for mammalian system.The Journal of Biochemistry,2000,127(3):367-372.
[20] Zhu D L,Zhao K,Xu H J,et al.Construction of thyA deficient Lactococcus lactis using the Cre-loxP recombination system.Annals of Microbiology,2015,66(3):1659-1665.
[21] Jager W,Schafer A,Puhler A,et al.Expression of the Bacillus subtilis SacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans.Journal of Bacteriology,1992,174(16):5462-5465.
[22] Hu F,Jiang X,Zhang J J,et al.Construction of an engineered strain capable of degrading two isomeric nitrophenols via a sacB-and gfp-based markerless integration system.Applied and Molecular Biotechnology,2014,98(10):4749-4756.
[23] Tan Y Z,Xu D Q,Li Y,et al.Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum.Plasmid,2012,67(1):44-52.
国家科技支撑计划资助项目(2015BAD15B0501)
/
〈 |
|
〉 |