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A Sense of Balance: Experimental Investigation and Modeling of a Malonyl-CoA Sensor in Escherichia coli.

Fehér T, Libis V, Carbonell P, Faulon JL - Front Bioeng Biotechnol (2015)

Bottom Line: Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose-response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis.In addition, we provide an example of the sensor's use in analyzing the effect of inducer or substrate concentrations on production levels.The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences , Szeged , Hungary.

ABSTRACT
Production of value-added chemicals in microorganisms is regarded as a viable alternative to chemical synthesis. In the past decade, several engineered pathways producing such chemicals, including plant secondary metabolites in microorganisms have been reported; upscaling their production yields, however, was often challenging. Here, we analyze a modular device designed for sensing malonyl-CoA, a common precursor for both fatty acid and flavonoid biosynthesis. The sensor can be used either for high-throughput pathway screening in synthetic biology applications or for introducing a feedback circuit to regulate production of the desired chemical. Here, we used the sensor to compare the performance of several predicted malonyl-CoA-producing pathways, and validated the utility of malonyl-CoA reductase and malonate-CoA transferase for malonyl-CoA biosynthesis. We generated a second-order dynamic linear model describing the relation of the fluorescence generated by the sensor to the biomass of the host cell representing a filter/amplifier with a gain that correlates with the level of induction. We found the time constants describing filter dynamics to be independent of the level of induction but distinctively clustered for each of the production pathways, indicating the robustness of the sensor. Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose-response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis. In addition, we provide an example of the sensor's use in analyzing the effect of inducer or substrate concentrations on production levels. The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.

No MeSH data available.


Fluorescence response of BL21DE3 cells carrying pCFR sensor plasmid and (A) pETM6-PaccABCD, (B) pETM6-MaccABCD, or (C) pMSD8. Colors indicate the concentration of IPTG used for induction: dark blue: 0.01 mM; magenta: 0.1 mM; yellow: 1 mM; cyan: 10 mM.
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Figure 5: Fluorescence response of BL21DE3 cells carrying pCFR sensor plasmid and (A) pETM6-PaccABCD, (B) pETM6-MaccABCD, or (C) pMSD8. Colors indicate the concentration of IPTG used for induction: dark blue: 0.01 mM; magenta: 0.1 mM; yellow: 1 mM; cyan: 10 mM.

Mentions: Since the overexpression of certain members of the accABCD complex (Davis et al., 2000), as well as protein overproduction in general (Flores et al., 2004) have been described to have a toxic effect on the cells, the overexpression of certain constructs was repeated using lower-copy vectors. For the accABCD complex, pMSD8, an ultra-low-copy variant was available expressing the four genes as a T7-driven operon. In addition, two medium-copy alternatives, pETM6-PaccABCD and pETM6-MaccABCD, carrying the genes as a pseudo-operon (separate promoters) and in monocistronic form (separate promoters and terminators), respectively, were also obtained and tested (Xu et al., 2012, 2013). All three plasmids displayed a clear dose-dependent fluorescence/OD pattern, well above the levels of the negative control (Figures 5A–C). For matCmatB, pACYCmatCmatB was readily available as a low-copy alternative that functioned in a quite well-reproducible fashion (Figure 2), when tested with the pBFR1k_RFP_8FapR sensor plasmid.


A Sense of Balance: Experimental Investigation and Modeling of a Malonyl-CoA Sensor in Escherichia coli.

Fehér T, Libis V, Carbonell P, Faulon JL - Front Bioeng Biotechnol (2015)

Fluorescence response of BL21DE3 cells carrying pCFR sensor plasmid and (A) pETM6-PaccABCD, (B) pETM6-MaccABCD, or (C) pMSD8. Colors indicate the concentration of IPTG used for induction: dark blue: 0.01 mM; magenta: 0.1 mM; yellow: 1 mM; cyan: 10 mM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389729&req=5

Figure 5: Fluorescence response of BL21DE3 cells carrying pCFR sensor plasmid and (A) pETM6-PaccABCD, (B) pETM6-MaccABCD, or (C) pMSD8. Colors indicate the concentration of IPTG used for induction: dark blue: 0.01 mM; magenta: 0.1 mM; yellow: 1 mM; cyan: 10 mM.
Mentions: Since the overexpression of certain members of the accABCD complex (Davis et al., 2000), as well as protein overproduction in general (Flores et al., 2004) have been described to have a toxic effect on the cells, the overexpression of certain constructs was repeated using lower-copy vectors. For the accABCD complex, pMSD8, an ultra-low-copy variant was available expressing the four genes as a T7-driven operon. In addition, two medium-copy alternatives, pETM6-PaccABCD and pETM6-MaccABCD, carrying the genes as a pseudo-operon (separate promoters) and in monocistronic form (separate promoters and terminators), respectively, were also obtained and tested (Xu et al., 2012, 2013). All three plasmids displayed a clear dose-dependent fluorescence/OD pattern, well above the levels of the negative control (Figures 5A–C). For matCmatB, pACYCmatCmatB was readily available as a low-copy alternative that functioned in a quite well-reproducible fashion (Figure 2), when tested with the pBFR1k_RFP_8FapR sensor plasmid.

Bottom Line: Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose-response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis.In addition, we provide an example of the sensor's use in analyzing the effect of inducer or substrate concentrations on production levels.The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences , Szeged , Hungary.

ABSTRACT
Production of value-added chemicals in microorganisms is regarded as a viable alternative to chemical synthesis. In the past decade, several engineered pathways producing such chemicals, including plant secondary metabolites in microorganisms have been reported; upscaling their production yields, however, was often challenging. Here, we analyze a modular device designed for sensing malonyl-CoA, a common precursor for both fatty acid and flavonoid biosynthesis. The sensor can be used either for high-throughput pathway screening in synthetic biology applications or for introducing a feedback circuit to regulate production of the desired chemical. Here, we used the sensor to compare the performance of several predicted malonyl-CoA-producing pathways, and validated the utility of malonyl-CoA reductase and malonate-CoA transferase for malonyl-CoA biosynthesis. We generated a second-order dynamic linear model describing the relation of the fluorescence generated by the sensor to the biomass of the host cell representing a filter/amplifier with a gain that correlates with the level of induction. We found the time constants describing filter dynamics to be independent of the level of induction but distinctively clustered for each of the production pathways, indicating the robustness of the sensor. Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose-response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis. In addition, we provide an example of the sensor's use in analyzing the effect of inducer or substrate concentrations on production levels. The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.

No MeSH data available.