Tuning genetic control through promoter engineering

@article{Alper:2005aa, abstract = {Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli. Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs, which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms (Saccharomyces cerevisiae) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.}, author = {Alper, Hal and Fischer, Curt and Nevoigt, Elke and Stephanopoulos, Gregory }, citeulike-article-id = {1570057}, doi = {10.1073/pnas.0504604102}, journal = {Proc Natl Acad Sci USA}, keywords = {bacterial, cerevisiae, coli, engineering, escherichia, expression, foundationalsb, fungal, gene, genetic, genetics, library, promoter, regions, regulation, saccharomyces, syntheticbiology, transcription}, local-url = {file://localhost/Users/macowell/Documents/Papers/2005/Alper/Proc\%20Natl\%20Acad\%20Sci\%20USA\%202005\%20Alper.pdf}, month = {Sep}, number = {36}, pages = {12678--83}, posted-at = {2007-08-16 21:20:59}, priority = {2}, title = {Tuning genetic control through promoter engineering}, url = {http://www.pnas.org/cgi/content/abstract/102/36/12678}, volume = {102}, year = {2005} }

TY - JOUR ID - Alper:2005aa L3 - citeulike-article-id:1570057 TI - Tuning genetic control through promoter engineering JF - Proc Natl Acad Sci USA VL - 102 IS - 36 SP - 12678 EP - 83 N2 - Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli. Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs, which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms (Saccharomyces cerevisiae) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors. KW - bacterial KW - cerevisiae KW - coli KW - engineering KW - escherichia KW - expression KW - foundationalsb KW - fungal KW - gene KW - genetic KW - genetics KW - library KW - promoter KW - regions KW - regulation KW - saccharomyces KW - syntheticbiology KW - transcription AU - Alper, Hal AU - Fischer, Curt AU - Nevoigt, Elke AU - Stephanopoulos, Gregory PY - 2005/Sep// UR - http://www.pnas.org/cgi/content/abstract/102/36/12678 ER -