Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which res...Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which results in incompatible gene modulation.Here,we expanded the promoter library in O.polymorpha based on transcriptional data,among which 13 constitutive promoters had the strengths ranging from 0–55%of PGAP,the commonly used strong constitutive promoter,and 2 were growth phase-dependent promoters.Subsequently,2 hybrid growth phase-dependent promoters were constructed and characterized,which had 2-fold higher activities.Finally,promoter engineering was applied to precisely regulate cellular metabolism for efficient production ofβ-elemene.The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway(PPP)and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase(PK-PTA)pathway.Coupled with the phase-dependent expression of synthase module(ERG20∼LsLTC2 fusion),the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose)was achieved in strain YY150U under fed-batch fermentation in shake flasks.This work characterized and engineered a series of promoters,that can be used to fine-tune genes for constructing efficient yeast cell factories.展开更多
By using OsRacD cDNA as probe to screen the genomic library of photoperiod sensitive genic male sterile rice line Nongken 58S, a positive clone containing 2 kb promoter and 396 bp coding region of OsRacD was obtained....By using OsRacD cDNA as probe to screen the genomic library of photoperiod sensitive genic male sterile rice line Nongken 58S, a positive clone containing 2 kb promoter and 396 bp coding region of OsRacD was obtained. Compared with the promoter of OsRacD cloned by reverse PCR from normal rice variety Nongken 58 （Nongken 58N）, the homology was 99.8%, and the different nucleotides were outside the predicted response elements in promoter, suggesting that the fertility between rice varieties Nongken 58S and Nongken 58N under the long-day conditions was not attributed to the difference in the structure of OsRacD upstream regulation sequences, but to the developmental regulation of gene differential expression.展开更多
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
Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combina...Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits.In this report,we present an efficient evolutionary approach to build a range of regulatory control elements.The reported method allows for rapid construction of promoter,5'UTR,terminator and trans-activating RNA libraries.Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter.This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix.Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics.Specifically,both the promoter library and 50UTR library exhibits gene expression dynamics spanning across three order of magnitude.The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern.The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements.These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.展开更多
基金This research was supported by the National Key Research and Development Project(2023YFC3503900)Liaoning Distinguished Scholar Program(2023JH6/100500001)。
文摘Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which results in incompatible gene modulation.Here,we expanded the promoter library in O.polymorpha based on transcriptional data,among which 13 constitutive promoters had the strengths ranging from 0–55%of PGAP,the commonly used strong constitutive promoter,and 2 were growth phase-dependent promoters.Subsequently,2 hybrid growth phase-dependent promoters were constructed and characterized,which had 2-fold higher activities.Finally,promoter engineering was applied to precisely regulate cellular metabolism for efficient production ofβ-elemene.The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway(PPP)and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase(PK-PTA)pathway.Coupled with the phase-dependent expression of synthase module(ERG20∼LsLTC2 fusion),the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose)was achieved in strain YY150U under fed-batch fermentation in shake flasks.This work characterized and engineered a series of promoters,that can be used to fine-tune genes for constructing efficient yeast cell factories.
文摘By using OsRacD cDNA as probe to screen the genomic library of photoperiod sensitive genic male sterile rice line Nongken 58S, a positive clone containing 2 kb promoter and 396 bp coding region of OsRacD was obtained. Compared with the promoter of OsRacD cloned by reverse PCR from normal rice variety Nongken 58 （Nongken 58N）, the homology was 99.8%, and the different nucleotides were outside the predicted response elements in promoter, suggesting that the fertility between rice varieties Nongken 58S and Nongken 58N under the long-day conditions was not attributed to the difference in the structure of OsRacD upstream regulation sequences, but to the developmental regulation of gene differential expression.
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The authors would like to acknowledge the Department of Chemical,Biochemical and Environmental Engineering,College of Engineering and Information Technology,Office of the Vice President for Research(gratn number 10145-1113-021-STRT7XUPMAIN)at University of Maryland Baltimore County for funding support.
文摘Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits.In this report,we present an efficient evolutionary approach to build a range of regulatory control elements.The reported method allows for rapid construction of promoter,5'UTR,terminator and trans-activating RNA libraries.Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter.This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix.Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics.Specifically,both the promoter library and 50UTR library exhibits gene expression dynamics spanning across three order of magnitude.The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern.The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements.These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.
