Tuning genetic control through promoter engineering

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.

Fig. 1.

Generation of the functional promoter library. A variant of the constitutive bacteriophage P_L-λ promoter was mutated through error-prone PCR, used in a plasmid construct to drive the expression of gfp, then screened based on fluorescence of colonies. The chosen constructs have a wide range of fluorescence both on a culture-wide and on a single-cell level, as illustrated by representative flow cytometry histograms at the bottom. All of the selected promoters have a uniform expression level on a single-cell level, as measured by GFP signal.

Fig. 2.

Comprehensive characterization of the promoter library. Several orthogonal metrics were used to characterize the promoter library and ensure the consistent behavior of all its members for various genes and culturing conditions. We show here three metrics that were chosen for quantifying transcriptional of the promoters: (i) The dynamics of GFP production based on fluorescence, (ii) measurement of the relative mRNA transcript levels in the cultures, and (iii) testing of the MIC for chloramphenicol in an additional library of constructs where the promoter drove the expression of chloramphenicol acetyltransferase. The overall strong correlation between the various metrics suggests a broad-range utility of the promoter library for a variety of genes and conditions.

Fig. 3.

Implementation of the promoter library for introducing genetic control. The phenotypes associated with integrating the promoters into the chromosome are tested by using three genes. (a) Selected promoters were integrated into the promoter region of ppc, and strains were cultured in M9-minimal media with only glucose as the carbon source. Although the knockout of ppc is lethal in glucose media, there is a clear maximum yield from glucose and thus an optimal expression level of ppc. (b) Selected promoters were integrated in front of the dxs gene in a recombinant wild-type strain of E. coli, and strains were later assayed for the production of lycopene. A clear maximum in lycopene production was obtained. From the wild-type production level, the native dxs promoter strength can be inferred to be ≈0.26, according to our metric. (c) Selected promoters were integrated in front of the dxs gene in a recombinant strain also overexpressing ispFD and idi. In this case, the linear response of lycopene yield to the promoter strength illustrates a rate-limiting behavior of dxs across all tested promoter strengths.

Fig. 4.

Extension of promoter engineering to other systems. The basic concepts in this paper are further extended to a eukaryotic system (S. cerevisiae) by using the TEF1 promoter. A similar wide range of yECitrine fluorescence is obtained from selected clones of the original promoter library. These results, along with other current work, indicate the ability to select for promoters responsible for tuning precise genetic control.