15 research outputs found
Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data
Accurate
characterization of promoter activity is important when
designing expression systems for systems biology and metabolic engineering
applications. Promoters that respond to changes in the environment
enable the dynamic control of gene expression without the necessity
of inducer compounds, for example. However, the dynamic nature of
these processes poses challenges for estimating promoter activity.
Most experimental approaches utilize reporter gene expression to estimate
promoter activity. Typically the reporter gene encodes a fluorescent
protein that is used to infer a constant promoter activity despite
the fact that the observed output may be dynamic and is a number of
steps away from the transcription process. In fact, some promoters
that are often thought of as constitutive can show changes in activity
when growth conditions change. For these reasons, we have developed
a system of ordinary differential equations for estimating dynamic
promoter activity for promoters that change their activity in response
to the environment that is robust to noise and changes in growth rate.
Our approach, inference of dynamic promoter activity (PromAct), improves
on existing methods by more accurately inferring known promoter activity
profiles. This method is also capable of estimating the correct scale
of promoter activity and can be applied to quantitative data sets
to estimate quantitative rates
Synthetic Promoter Library for Modulation of Actinorhodin Production in <i>Streptomyces coelicolor</i> A3(2)
<div><p>The objective of this study was the application of the synthetic promoter library (SPL) technology for modulation of actinorhodin production in <i>Streptomyces coelicolor</i> A3(2). The SPL technology was used to optimize the expression of a pathway specific positive transcriptional regulator ActII orf4, which activates the transcription of the <i>S. coelicolor</i> actinorhodin biosynthetic gene cluster. The native <i>actII orf4</i> promoter was replaced with synthetic promoters, generating a <i>S. coelicolor</i> library with a broad range of expression levels of <i>actII orf4</i>. The resulting library was screened based on the yield of actinorhodin. Selected strains were further physiologically characterized. One of the strains from the library, ScoSPL20, showed considerably higher yield of actinorhodin and final actinorhodin titer, compared to <i>S. coelicolor</i> wild type and <i>S. coelicolor</i> with <i>actII orf4</i> expressed from a strong constitutive promoter. ScoSPL20 demonstrated exceptional productivity despite having a comparatively weak expression from the promoter. Interestingly, the ScoSPL20 promoter was activated at a much earlier stage of growth compared to the wild type, demonstrating the advantage of fine-tuning and temporal tuning of gene expression in metabolic engineering. Transcriptome studies were performed in exponential and actinorhodin-producing phase of growth to compare gene expression between ScoSPL20 and the wild type. To our knowledge, this is the first successful application of the SPL technology for secondary metabolite production in filamentous bacteria.</p></div
Comparative table describing physiological parameters of <b><i>S. coelicolor</i></b><b> WT, </b><b><i>S. coelicolor</i></b><b> oxp-</b><b><i>actII orf4</i></b><b> and ScoSPL20. Results are the mean values with standard deviations from three biological replicates.</b>
ā <p>Did not display a defined RED production phase, onset of RED and ACT production was simultaneous and growth associated.</p>ā”<p>r<sub>S</sub> and Y<sub>SX</sub> were calculated in the exponential growth phase.</p
Hierarchical clustering (Pearson correlation, average distance) of the genes.
<p>Genes with a significant change in expression level between the WT and the SPL20 strain during the stationary phase. The rows and columns represent genes and samples respectively. The scale of the color-bar is based on the z-score.</p
Summary of transcriptome analysis.
<p>Categories of genes that are up-regulated or down-regulated in the comparison of gene expression profiles of the WT and the ScoSPL20 strains in the exponential and stationary phases.</p
Initial screening of Synthetic Promoter Library.
<p>Yield of ACT (g/g) on biomass for 200 selected SPL strains at the end of 120 h of cultivation. <i>S. coelicolor</i> WT strain and <i>S. coelicolor</i> oxp-<i>actII orf4</i> were used as reference strains. Arrows and numbers on the yield graph indicate the position of 11 ScoSPL strains that were selected for detailed physiological characterization. Number of experimental replicates: 2.</p
Comparative table representing specific growth rate (Ī¼), volumetric production rate for AC (q<sub>P</sub>ACT) and volumetric production rates for undecylprodigiosin (q<sub>P</sub>RED) represented as mg l<sup>ā1</sup> h<sup>ā1</sup> and yield of biomass over substrate (Y<sub>SX</sub>) from one of the two biological replicates.
ā¦<p>- Y<sub>SX</sub> was calculated in the exponential phase of growth.</p
Expression levels of different SPL promoters ā determined using <i>xeg</i> as reporter gene.
<p>WT-ACT refers to the native <i>actII orf4</i> promoter. Number of experimental replicates: 2.</p
Promoter sequences for selected ScoSPL strains and that of the wild type <i>actII orf4</i> promoter.
<p>Promoter sequences for selected ScoSPL strains and that of the wild type <i>actII orf4</i> promoter.</p
Presentation_1_Glucose-Dependent Promoters for Dynamic Regulation of Metabolic Pathways.PDF
<p>For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers. A common cell factory Saccharomyces cerevisiae is a Crabtree-positive yeast and is typically fermented at industrial scale under glucose-limited conditions to avoid the formation of ethanol. In this work, we aimed to identify and characterize promoters that depend on glucose concentration for use as dynamic control elements. Through analysis of mRNA data of S. cerevisiae grown in chemostats under glucose excess or limitation, we identified 34 candidate promoters that strongly responded to glucose presence or absence. These promoters were characterized in small-scale batch and fed-batch cultivations using a quickly maturing rapidly degrading green fluorescent protein yEGFP3-Cln2<sub>PEST</sub> as a reporter. Expressing 3-hydroxypropionic acid (3HP) pathway from a set of selected regulated promoters allowed for suppression of 3HP production during glucose-excess phase of a batch cultivation with subsequent activation in glucose-limiting conditions. Regulating the 3HP pathway by the ICL1 promoter resulted in 70% improvement of 3HP titer in comparison to PGK1 promoter.</p