【摘要】 本研究利用Red/ET DNA重组技术对实验室已有的含鼠李糖基转移酶基因RhlAB的分泌表达载体pBBR1-genta-RhlAB进行修饰,将3种不同强度组成型启动子(P_apra、P_tac和P_rhaB)成功替换了RhlAB基因在铜绿假单胞菌中的原始启动子,成功获得了表达载体pBBR1-genta-P_apra-RhlAB,pBBR1-kan-P_tac-RhlAB和pBBR1-kanP_rhaB-RhlAB,并将这些表达载体分别在防御假单胞菌Pf-5中异源表达,通过LB培养基发酵42 h后,Pf-5/pBBR1-genta-RhlAB的鼠李糖脂产量为17. 56 mg/L,而Pf-5/pBBR1-genta-P_apra-RhlAB,Pf-5/pBBR1-kan-P_tac-RhlAB和Pf-5/pBBR1-kan-P_rhaB-RhlAB分别是11. 135 mg/L,441. 135 mg/L,557. 764 mg/L,启动子优化后产量分别是原始启动子的0. 63,25. 12和31. 76倍。对发酵产物进行高效液相色谱-质谱联用技术分析,共检出相对含量变化的4类质核比不同的鼠李糖脂同系物。并通过实时荧光定量PCR检测RhlAB基因的表达量,发现启动子替换为P_tac和P_rhaB后RhlAB基因表达量分别是原始启动子的2. 16和2. 77倍。本研究初步实现了RhlAB基因在Pf-5中的表达,发现组成型启动子P_tac和P_rhaB比RhlAB的原始启动子表达效率更高,可为异源合成鼠李糖脂提供重要参考。更多还原

【Abstract】 In this research,the recombinant expression vector pBBR1-genta-RhlAB containing the rhamnosyltransferase gene（ RhlAB） was modified via Red/ET homologous recombineering. Then,three recombinant expression vectors pBBR1-genta-P_apra-RhlAB,pBBR1-kan-P_tac-RhlAB and pBBR1-kan-P_rhaB-RhlAB with three different constitutive promoters( P_apra、P_tacand P_rhaB) were constructed by subcloning technology,and then we electro-transformated the resultant expression vectors into P. protegens Pf-5. When the flask in LB medium was fermented to 42 h,the rhamnolipid concentration of Pf-5/pBBR1-genta-RhlAB supernatant was 17. 56 mg/L,with that of Pf-5/pBBR1-genta-P_apra-RhlAB,Pf-5/pBBR1-kanP_tac-RhlAB and Pf-5/pBBR1-kan-P_rhaB-RhlAB supernatant being 11. 135 mg/L,441. 135 mg/L and 557. 764 mg/L,resulting in 0. 63,25. 12 and 31. 76 fold variation after promoter optimization. High performance liquid chromatographymass spectrometry（ LC-MS/MS） analysis showed that there were total 4 rhamnolipid derivatives in the Pf-5 engineering bacteria. Furthermore,the RhlAB gene expression was later examined by qRT-PCR with the result showed that the expression level of RhlAB gene after P_tacand P_rhaBpromoter was 2. 16 and 2. 77 fold higher than that of the original one.Generally,this study preliminarily realized the expression of RhlAB gene in Pf-5,and found that the constitutive promoter P_tacand P_rhaBwas more efficient than the original promoter which could provide a significant reference with significance for heterologous biosynthesis of rhamnolipid.更多还原