Abstract
BackgroundMetabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model.ResultsOur method was successfully applied to fine tune the ppc gene of Escherichia coli. While developing the promoter knock-in methodology, we showed the importance of conserving the natural leader region containing the ribosome binding site (RBS) of the gene of interest and of eliminating upstream regulatory elements (transcription factor binding sites). The gene expression was down regulated instead of up regulated when the natural RBS was not conserved and when the upstream regulatory elements were eliminated. Next, three different promoter knock-ins were created for the ppc gene selecting three different artificial promoters. The measured constitutive expression of the ppc gene in these knock-ins reflected the relative strength of the different promoters as predicted by the PSP model. The applicability of our PSP model and promoter knock-in methodology was further demonstrated by showing that the constitutivity and the relative levels of expression were independent of the genetic background (comparing wild-type and mutant E. coli strains). No differences were observed during scaling up from shake flask to bioreactor-scale, confirming that the obtained expression was independent of environmental conditions.ConclusionWe are proposing a novel methodology for obtaining appropriate levels of expression of genes of interest, based on the prediction of the relative strength of selected synthetic promoters combined with an optimized promoter knock-in strategy. The obtained expression levels are independent of the genetic background and scale conditions. The method constitutes therefore a valuable addition to the genetic toolbox for the metabolic engineering of E. coli.
Highlights
Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound
We describe an optimized procedure for the fine tuning of gene expression, using as proof of concept the ppc gene of Escherichia coli
We developed a method based on the use of a previously constructed library of promoters [3] in combination with a mathematical model (Promoter Strength Predictive, promoter strength predictive (PSP)) which can be applied for the fine tuning of gene expression, using the ppc gene as model system
Summary
Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. To ensure the constant (over) expression of a certain gene, the endogenous promoter can be replaced by a constitutive promoter with a desired strength In this context, a synthetic promoter library is useful for the fine tuning of genes. We describe an optimized procedure for the fine tuning of gene expression, using as proof of concept the ppc gene of Escherichia coli (coding for phosphoenolpyruvate carboxylase). The advantage of this procedure over the state-of-the art procedures is the universal applicability of the created artificial promoter library in combination with a promoter strength predictive (PSP) model [3], making it unnecessary to create a new promoter library for each targeted gene. The proposed strategy is less time consuming and cheaper
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.