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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.


Related in: MedlinePlus

Reproducibility of measurements of malonyl-CoA production in BL21DE3 cells using the fluorescent sensor plasmid pCFR and various production plasmids. The IPTG-dependence of fluorescence measured at OD = 0.6 is shown in every case. (A) Two cultures harboring pMSD8, originating from two distinct colonies after transformation are depicted by open squares and open triangles. Open diamonds represent the re-measurement of the strain corresponding to the open squares, 1 day later. (B) Six cultures harboring ETM6-MaccABCD measured on different days. (C) Two cultures of the same clone of pACYCmatCmatB originating from the same starter culture, measured in the same run, in the presence of Na-malonate.
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Figure 7: Reproducibility of measurements of malonyl-CoA production in BL21DE3 cells using the fluorescent sensor plasmid pCFR and various production plasmids. The IPTG-dependence of fluorescence measured at OD = 0.6 is shown in every case. (A) Two cultures harboring pMSD8, originating from two distinct colonies after transformation are depicted by open squares and open triangles. Open diamonds represent the re-measurement of the strain corresponding to the open squares, 1 day later. (B) Six cultures harboring ETM6-MaccABCD measured on different days. (C) Two cultures of the same clone of pACYCmatCmatB originating from the same starter culture, measured in the same run, in the presence of Na-malonate.

Mentions: Prior to comparing the performance of various malonyl-CoA-producing constructs, we investigated the reproducibility of our measurements at three different levels. First, we compared the fluorescence values of parallel clones, resulting from the transformation of a given producer construct. These displayed a strong variation in their response to IPTG, which was not unexpected taking into account their heterologous colony morphologies (Figure 7A). Second, we re-tested the best performing clones after their storage in the form of glycerol stocks. Reproducibility was substantially better, being the best for the lowest copy-number pMSD8 plasmid (Figure 7A). For other constructs, the performance seemed to fall into two distinct clusters: it either matched that of earlier measurements, or deteriorated to a lower, near-zero value, as demonstrated by the dose–response curves of pETM6-MaccABCD (Figure 7B). This phenomenon was reminiscent of a burden-relieving mutation that swept through the population early after induction. The third level of reproducibility corresponds to comparing the performance of a given strain in the same microtiter plate in the same measurement-run, starting from the same overnight seed-culture (Figure 7C). The high reproducibility of such measurements was sufficient to draw conclusions on the effect of providing the substrates for malonyl-CoA production (see below).


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)

Reproducibility of measurements of malonyl-CoA production in BL21DE3 cells using the fluorescent sensor plasmid pCFR and various production plasmids. The IPTG-dependence of fluorescence measured at OD = 0.6 is shown in every case. (A) Two cultures harboring pMSD8, originating from two distinct colonies after transformation are depicted by open squares and open triangles. Open diamonds represent the re-measurement of the strain corresponding to the open squares, 1 day later. (B) Six cultures harboring ETM6-MaccABCD measured on different days. (C) Two cultures of the same clone of pACYCmatCmatB originating from the same starter culture, measured in the same run, in the presence of Na-malonate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Reproducibility of measurements of malonyl-CoA production in BL21DE3 cells using the fluorescent sensor plasmid pCFR and various production plasmids. The IPTG-dependence of fluorescence measured at OD = 0.6 is shown in every case. (A) Two cultures harboring pMSD8, originating from two distinct colonies after transformation are depicted by open squares and open triangles. Open diamonds represent the re-measurement of the strain corresponding to the open squares, 1 day later. (B) Six cultures harboring ETM6-MaccABCD measured on different days. (C) Two cultures of the same clone of pACYCmatCmatB originating from the same starter culture, measured in the same run, in the presence of Na-malonate.
Mentions: Prior to comparing the performance of various malonyl-CoA-producing constructs, we investigated the reproducibility of our measurements at three different levels. First, we compared the fluorescence values of parallel clones, resulting from the transformation of a given producer construct. These displayed a strong variation in their response to IPTG, which was not unexpected taking into account their heterologous colony morphologies (Figure 7A). Second, we re-tested the best performing clones after their storage in the form of glycerol stocks. Reproducibility was substantially better, being the best for the lowest copy-number pMSD8 plasmid (Figure 7A). For other constructs, the performance seemed to fall into two distinct clusters: it either matched that of earlier measurements, or deteriorated to a lower, near-zero value, as demonstrated by the dose–response curves of pETM6-MaccABCD (Figure 7B). This phenomenon was reminiscent of a burden-relieving mutation that swept through the population early after induction. The third level of reproducibility corresponds to comparing the performance of a given strain in the same microtiter plate in the same measurement-run, starting from the same overnight seed-culture (Figure 7C). The high reproducibility of such measurements was sufficient to draw conclusions on the effect of providing the substrates for malonyl-CoA production (see below).

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.


Related in: MedlinePlus