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Trade-offs in engineering sugar utilization pathways for titratable control.

Afroz T, Biliouris K, Boykin KE, Kaznessis Y, Beisel CL - ACS Synth Biol (2014)

Bottom Line: We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration.For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway.Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, North Carolina 27695, United States.

ABSTRACT
Titratable systems are common tools in metabolic engineering to tune the levels of enzymes and cellular components as part of pathway optimization. For nonmodel microorganisms with limited genetic tools, inducible sugar utilization pathways offer built-in titratable systems. However, these pathways can exhibit undesirable single-cell behaviors that hamper the uniform and tunable control of gene expression. Here, we applied mathematical modeling and single-cell measurements of L-arabinose utilization in Escherichia coli to systematically explore how sugar utilization pathways can be altered to achieve desirable inducible properties. We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration. For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway. Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter. Equivalent modifications to the D-xylose utilization pathway yielded similar responses, demonstrating the applicability of our observations. Overall, our findings indicate that there is no ideal set of typical alterations when co-opting natural utilization pathways for titratable control and suggest design rules for manipulating these pathways to advance basic genetic studies and the metabolic engineering of microorganisms for optimized chemical production.

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Effect of cell density in the presence or absenceof sugar catabolism.(A) Model predictions for the pathway with a constitutively expressedlow-affinity transporter (TL = 0.2) and the deleted high-affinitytransporter (αH = 0) when accounting for depletionof extracellular sugar through catabolism. Each simulation was conductedto τ = 10. The different curves reflect the relative volumeof the cells to the medium (ν). Note that all variables werenondimensionalized as part of the model derivation. See Supporting Information for more details. (B)Growth curves for the Pcon-araE ΔaraFGH strain with or without (ΔaraBAD) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments.The SEM for each measurement was smaller than the symbol. (C) Representativedot plots for both strains in log phase grown to the indicated finalcell densities. See Figure 3 for more informationon the dot plots. Each dot plot is representative of at least threeexperiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicateexperiments.
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fig4: Effect of cell density in the presence or absenceof sugar catabolism.(A) Model predictions for the pathway with a constitutively expressedlow-affinity transporter (TL = 0.2) and the deleted high-affinitytransporter (αH = 0) when accounting for depletionof extracellular sugar through catabolism. Each simulation was conductedto τ = 10. The different curves reflect the relative volumeof the cells to the medium (ν). Note that all variables werenondimensionalized as part of the model derivation. See Supporting Information for more details. (B)Growth curves for the Pcon-araE ΔaraFGH strain with or without (ΔaraBAD) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments.The SEM for each measurement was smaller than the symbol. (C) Representativedot plots for both strains in log phase grown to the indicated finalcell densities. See Figure 3 for more informationon the dot plots. Each dot plot is representative of at least threeexperiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicateexperiments.

Mentions: Catabolism can deplete thereserve of sugar in the medium, whichhas been viewed as a barrier to the generation of titratable systems.37 However, such depletion would form negativefeedback by depleting the available sugar to induce the pathway, potentiallybenefiting the response. To explore these potential effects, we firstmodified the simple model to allow for the depletion of the extracellularsugar through catabolism (see Supporting Information). The resulting model was then employed to predict the responsein the presence of catabolism. As part of the model, we could specifythe relative volume of the cells to the medium (ν), which dictatesin part the rate of depletion. We specifically focused on the pathwaywith the constitutively expressed low-affinity transporter (TL = 0.02) and the deleted high-affinity transporter (αH = 0) that yielded a graded response in the presence or absenceof sugar catabolism. The model predicted that depletion of the extracellularsugar flattened the curve and elevated the EC50 value (Figure 4A). High depletion (ν = 0.01) was detrimentalbased on the sharp response close to saturating sugar concentrations,although intermediate depletion (ν < 0.01) improved the overalllinearity of the response.


Trade-offs in engineering sugar utilization pathways for titratable control.

Afroz T, Biliouris K, Boykin KE, Kaznessis Y, Beisel CL - ACS Synth Biol (2014)

Effect of cell density in the presence or absenceof sugar catabolism.(A) Model predictions for the pathway with a constitutively expressedlow-affinity transporter (TL = 0.2) and the deleted high-affinitytransporter (αH = 0) when accounting for depletionof extracellular sugar through catabolism. Each simulation was conductedto τ = 10. The different curves reflect the relative volumeof the cells to the medium (ν). Note that all variables werenondimensionalized as part of the model derivation. See Supporting Information for more details. (B)Growth curves for the Pcon-araE ΔaraFGH strain with or without (ΔaraBAD) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments.The SEM for each measurement was smaller than the symbol. (C) Representativedot plots for both strains in log phase grown to the indicated finalcell densities. See Figure 3 for more informationon the dot plots. Each dot plot is representative of at least threeexperiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicateexperiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384834&req=5

fig4: Effect of cell density in the presence or absenceof sugar catabolism.(A) Model predictions for the pathway with a constitutively expressedlow-affinity transporter (TL = 0.2) and the deleted high-affinitytransporter (αH = 0) when accounting for depletionof extracellular sugar through catabolism. Each simulation was conductedto τ = 10. The different curves reflect the relative volumeof the cells to the medium (ν). Note that all variables werenondimensionalized as part of the model derivation. See Supporting Information for more details. (B)Growth curves for the Pcon-araE ΔaraFGH strain with or without (ΔaraBAD) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments.The SEM for each measurement was smaller than the symbol. (C) Representativedot plots for both strains in log phase grown to the indicated finalcell densities. See Figure 3 for more informationon the dot plots. Each dot plot is representative of at least threeexperiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicateexperiments.
Mentions: Catabolism can deplete thereserve of sugar in the medium, whichhas been viewed as a barrier to the generation of titratable systems.37 However, such depletion would form negativefeedback by depleting the available sugar to induce the pathway, potentiallybenefiting the response. To explore these potential effects, we firstmodified the simple model to allow for the depletion of the extracellularsugar through catabolism (see Supporting Information). The resulting model was then employed to predict the responsein the presence of catabolism. As part of the model, we could specifythe relative volume of the cells to the medium (ν), which dictatesin part the rate of depletion. We specifically focused on the pathwaywith the constitutively expressed low-affinity transporter (TL = 0.02) and the deleted high-affinity transporter (αH = 0) that yielded a graded response in the presence or absenceof sugar catabolism. The model predicted that depletion of the extracellularsugar flattened the curve and elevated the EC50 value (Figure 4A). High depletion (ν = 0.01) was detrimentalbased on the sharp response close to saturating sugar concentrations,although intermediate depletion (ν < 0.01) improved the overalllinearity of the response.

Bottom Line: We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration.For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway.Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, North Carolina 27695, United States.

ABSTRACT
Titratable systems are common tools in metabolic engineering to tune the levels of enzymes and cellular components as part of pathway optimization. For nonmodel microorganisms with limited genetic tools, inducible sugar utilization pathways offer built-in titratable systems. However, these pathways can exhibit undesirable single-cell behaviors that hamper the uniform and tunable control of gene expression. Here, we applied mathematical modeling and single-cell measurements of L-arabinose utilization in Escherichia coli to systematically explore how sugar utilization pathways can be altered to achieve desirable inducible properties. We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration. For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway. Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter. Equivalent modifications to the D-xylose utilization pathway yielded similar responses, demonstrating the applicability of our observations. Overall, our findings indicate that there is no ideal set of typical alterations when co-opting natural utilization pathways for titratable control and suggest design rules for manipulating these pathways to advance basic genetic studies and the metabolic engineering of microorganisms for optimized chemical production.

Show MeSH
Related in: MedlinePlus