Tuning genetic control through promoter engineering.
نویسندگان
چکیده
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.
منابع مشابه
A synthetic library of RNA control modules for predictable tuning of gene expression in yeast
Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control module...
متن کاملEscherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning.
System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-n...
متن کاملRapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression
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 circu...
متن کاملTuning the Dragon Promoter Finder System for Human Promoter Recognition
Discovery of new genes through the identification of their promoters in anonymous DNA and the study of transcriptional control make promoter and transcription start site recognition an important issue for Bioinformatics. The biological process of transcription activation is very complex and is not completely understood. Hence computer systems for promoter recognition may not perform well if we ...
متن کاملCloning and expression of rhl AB operon under the control of tac promoter in E. coli
Today, efforts go towards the replacement of chemical surfactants by natural biological biosurfactants (biosurfactant), as these materials are not carcinogenic and highly compatibile with the environment. One of the main classes of biosurfactants is rhamnose containing glycolipid biosurfactant (rhamnolipids). This type of biosurfactants can be applied in many industries such as oil industry, ph...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 102 36 شماره
صفحات -
تاریخ انتشار 2005