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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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Simple mathematical model predicts trade-offs when altering thepathway structure. (A) The model assumes a base pathway comprisinga high-affinity/low-capacity transporter (TH) and a low-affinity/high-capacitytransporter (TL) that import extracellular sugar (S0) into the cell, a catabolic enzyme (E) that degrades thesugar, and a constitutively expressed regulator that upregulates theexpression of the transporters and the enzymes when bound to the sugar.The steady-state expression levels of the enzyme are reported as afunction of extracellular sugar concentration. Note that all variableswere nondimensionalized as part of the model derivation. Dashed linesindicate bifurcation regions. To alter the pathway, TH wasconstitutively expressed (C,D) and the activity of TL wasfurther eliminated (E,F), TL was constitutively expressed(G,H) and the activity of TH was further eliminated (I,J),and the activity of the catabolic enzymes was eliminated (B,D,F,H,J).Three strengths of constitutive expression were selected for TH and TL (low, light blue; medium, blue; high, darkblue). See Supporting Information (SI) formore details.
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fig2: Simple mathematical model predicts trade-offs when altering thepathway structure. (A) The model assumes a base pathway comprisinga high-affinity/low-capacity transporter (TH) and a low-affinity/high-capacitytransporter (TL) that import extracellular sugar (S0) into the cell, a catabolic enzyme (E) that degrades thesugar, and a constitutively expressed regulator that upregulates theexpression of the transporters and the enzymes when bound to the sugar.The steady-state expression levels of the enzyme are reported as afunction of extracellular sugar concentration. Note that all variableswere nondimensionalized as part of the model derivation. Dashed linesindicate bifurcation regions. To alter the pathway, TH wasconstitutively expressed (C,D) and the activity of TL wasfurther eliminated (E,F), TL was constitutively expressed(G,H) and the activity of TH was further eliminated (I,J),and the activity of the catabolic enzymes was eliminated (B,D,F,H,J).Three strengths of constitutive expression were selected for TH and TL (low, light blue; medium, blue; high, darkblue). See Supporting Information (SI) formore details.

Mentions: We builta simple mathematical model of sugar utilization to qualitativelyexplore how different alterations to the pathway influence the responseproperties (see Supporting Information).This model was composed of two transporters that import sugar intothe cell, one enzyme that breaks down the sugar, and one regulatorthat binds the sugar and subsequently activates the expression ofthe transporters and the enzyme. This general configuration capturesthe l-arabinose utilization pathway and many other pathwaysfound in microorganisms.16−25 Parameter values were selected for one high-affinity/low-capacitytransporter and one low-affinity/high-capacity transporter at biologicallyrelevant expression strengths and activities to yield a bistable response(Figure 2, see SupportingInformation). This configuration parallels the l-arabinoseutilization pathway and creates an undesired behavior for titratablecontrol.


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

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

Simple mathematical model predicts trade-offs when altering thepathway structure. (A) The model assumes a base pathway comprisinga high-affinity/low-capacity transporter (TH) and a low-affinity/high-capacitytransporter (TL) that import extracellular sugar (S0) into the cell, a catabolic enzyme (E) that degrades thesugar, and a constitutively expressed regulator that upregulates theexpression of the transporters and the enzymes when bound to the sugar.The steady-state expression levels of the enzyme are reported as afunction of extracellular sugar concentration. Note that all variableswere nondimensionalized as part of the model derivation. Dashed linesindicate bifurcation regions. To alter the pathway, TH wasconstitutively expressed (C,D) and the activity of TL wasfurther eliminated (E,F), TL was constitutively expressed(G,H) and the activity of TH was further eliminated (I,J),and the activity of the catabolic enzymes was eliminated (B,D,F,H,J).Three strengths of constitutive expression were selected for TH and TL (low, light blue; medium, blue; high, darkblue). See Supporting Information (SI) formore details.
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Related In: Results  -  Collection

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

fig2: Simple mathematical model predicts trade-offs when altering thepathway structure. (A) The model assumes a base pathway comprisinga high-affinity/low-capacity transporter (TH) and a low-affinity/high-capacitytransporter (TL) that import extracellular sugar (S0) into the cell, a catabolic enzyme (E) that degrades thesugar, and a constitutively expressed regulator that upregulates theexpression of the transporters and the enzymes when bound to the sugar.The steady-state expression levels of the enzyme are reported as afunction of extracellular sugar concentration. Note that all variableswere nondimensionalized as part of the model derivation. Dashed linesindicate bifurcation regions. To alter the pathway, TH wasconstitutively expressed (C,D) and the activity of TL wasfurther eliminated (E,F), TL was constitutively expressed(G,H) and the activity of TH was further eliminated (I,J),and the activity of the catabolic enzymes was eliminated (B,D,F,H,J).Three strengths of constitutive expression were selected for TH and TL (low, light blue; medium, blue; high, darkblue). See Supporting Information (SI) formore details.
Mentions: We builta simple mathematical model of sugar utilization to qualitativelyexplore how different alterations to the pathway influence the responseproperties (see Supporting Information).This model was composed of two transporters that import sugar intothe cell, one enzyme that breaks down the sugar, and one regulatorthat binds the sugar and subsequently activates the expression ofthe transporters and the enzyme. This general configuration capturesthe l-arabinose utilization pathway and many other pathwaysfound in microorganisms.16−25 Parameter values were selected for one high-affinity/low-capacitytransporter and one low-affinity/high-capacity transporter at biologicallyrelevant expression strengths and activities to yield a bistable response(Figure 2, see SupportingInformation). This configuration parallels the l-arabinoseutilization pathway and creates an undesired behavior for titratablecontrol.

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