利用Bio-BglBrick方法表达紫杉醇生物合成酶  

Expression of Taxol Biosynthetic Enzymes via Bio-BglBrick Method

作　　者：金晶[1,2] 王微[2,3] 邵妤[2,4] 陈章[5] 凌焱[2] 李玉霞[2] 宋永波[1] 刘刚[2] 陈惠鹏[2] 

机构地区：[1]沈阳药科大学,辽宁沈阳110016 [2]军事医学科学院生物工程研究所,北京100071 [3]吉林大学,吉林长春130012 [4]安徽大学,安徽合肥230601 [5]成都军区总医院呼吸科,四川成都610083

出　　处：《生物技术通讯》2014年第5期616-620,共5页Letters in Biotechnology

基　　金："十二五"重大新药创制国家科技重大专项"综合性新药研发技术大平台"(2012ZX09301003)

摘　　要：目的:进行紫杉醇药物生物合成前五步催化酶二磷酸盐合酶(GGPPS)、紫杉二烯合酶(TS)、紫杉二烯5α羟化酶(THY5α)、紫杉二烯5α-O-乙酰转移酶(TAT)和紫杉烷10β羟化酶(TDH)基因在大肠杆菌异源生物合成途径的组建及串联,实现单个表达及串联表达,并试图通过连续生物催化获得紫杉醇中间体紫衫二烯。方法:依据合成生物学中Brick基因组装方法,通过对载体pET30a的酶切位点进行定向改造,设计独特的BglⅡ/BamHⅠ和XbaⅠ/SpeⅠ串联表达盒,在大肠杆菌BM Rosetta(DE3)中表达产物。结果:设计多基因表达盒,实现紫杉醇药物生物合成前五步催化酶的单个表达,GGPPS和TS以及THY5α、TAT和TDH的串联表达。结论:利用BglBrick/BioBrick基因组装方法,可以实现紫杉醇生物合成催化酶的快速组装及后续表达。Objective： Genes encoding five sequential pathway steps of taxol biosythesis, which are geranylgeranyl diphosphate synthase （GGPPS）, taxadiene synthase （TS）, taxadiene- 5α- hydroxylase （THY5α）, taxadiene- 5α- O- aeetyl-transferase（TAT） and taxane-10β-hydroxylase（TDH）, were installed in a single E.coli host. Then through biosynthesis, the host can produce an intermediate taxadiene. Methods： Brick gene assembly method is a flexible standard for biological part assembly, through it we mutated the vector pET30a, designing Bgl Ⅱ/BamH Ⅰ and Xba ⅠI/Spe Ⅰ expression cassette. Results： The Bgl Ⅱ/BamH Ⅰ and Xba Ⅰ/Spe Ⅰ muhi-gene expression cassette was designed successfully. The five genes were achieved single genes expressin individually and multi-genes expression si- multaneously, GGPPS and TS, THY5α, TAT and TDH. Conclusion： The use of BglBrick method can help fulfill the rapid assembly of. taxol biosynthesis enzymes and sequential expression.

关 键 词：紫杉醇 生物合成 异源表达 基因组装 

分 类 号：Q78[生物学—分子生物学]