The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and e...The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.展开更多
[ Objective] This study was to construct an expression vector capable of excising selectable marker gene, further eliminating the effect of marker gene on the functional study of target gene. [ Method] By using Cre/Lo...[ Objective] This study was to construct an expression vector capable of excising selectable marker gene, further eliminating the effect of marker gene on the functional study of target gene. [ Method] By using Cre/LoxP site-specific recombination system, DsRed2-1 vector was modified by introducing LoxP se- quence and multiple cloning site sequence, then the TK gene was ligated into this vector for negative selection. [ Results] The fragments introduced were recom- bined at the molecular level under induced condition, and the specific red fluorescence was also observed in the recombinant vector transfected cells at the cellular level. [ Conclusion] It is feasible to use this Cre/loxP system to excise marker genes, showing a broad application prospect.展开更多
基金This work was supported by the National Natural Science Foundation of China[31870040]the National Key Research and Development Project(2020YFA0907804,2020YFA0907304)+1 种基金the“111”Project of China[B18022]the Fundamental Research Funds for the Central Universities[22221818014],and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.
基金Supported by Doctoral Funds of Xinjiang Production and Construction Corps(2011BB014)Guidance Program of Xinjiang Academy of Agricultural Reclamation Sciences(YYD201110)
文摘[ Objective] This study was to construct an expression vector capable of excising selectable marker gene, further eliminating the effect of marker gene on the functional study of target gene. [ Method] By using Cre/LoxP site-specific recombination system, DsRed2-1 vector was modified by introducing LoxP se- quence and multiple cloning site sequence, then the TK gene was ligated into this vector for negative selection. [ Results] The fragments introduced were recom- bined at the molecular level under induced condition, and the specific red fluorescence was also observed in the recombinant vector transfected cells at the cellular level. [ Conclusion] It is feasible to use this Cre/loxP system to excise marker genes, showing a broad application prospect.
