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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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Linearizing the response to d-xylose. The wild type E. coli strain (A), the strain constitutively expressingthe high-affinity transporter xylFGH (B), and thestrain constitutively expressing the high-affinity transporter xylFGH and lacking the catabolic operon xylAB (C) each harbored the reporter plasmid pUA66-pxylA. The designatedstrain was back-diluted into M9 minimal medium supplemented with theindicated concentration of d-xylose and grown for 6 h toABS600 ∼ 0.4 prior to flow cytometry analysis. SeeFigure 3 for more information on the dot plots.Each dot plot is representative of at least three experiments conductedfrom independent colonies.
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fig5: Linearizing the response to d-xylose. The wild type E. coli strain (A), the strain constitutively expressingthe high-affinity transporter xylFGH (B), and thestrain constitutively expressing the high-affinity transporter xylFGH and lacking the catabolic operon xylAB (C) each harbored the reporter plasmid pUA66-pxylA. The designatedstrain was back-diluted into M9 minimal medium supplemented with theindicated concentration of d-xylose and grown for 6 h toABS600 ∼ 0.4 prior to flow cytometry analysis. SeeFigure 3 for more information on the dot plots.Each dot plot is representative of at least three experiments conductedfrom independent colonies.

Mentions: To extend the generality of our insights, we focused on the d-xylose utilization pathway in E. coli. Similarto the l-arabinose utilization pathway, the d-xyloseutilization pathway encodes a high-affinity transporter and a low-affinitytransporter, a transcriptional activator that recognizes d-xylose, and enzymes that shunt d-xylose into the pentosephosphate pathway (Figure S1B). Also parallelingthe l-arabinose utilization pathway, the d-xyloseutilization pathway exhibits a bimodal response when tracking theactivity of the xylA promoter (Figure 5A).


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

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

Linearizing the response to d-xylose. The wild type E. coli strain (A), the strain constitutively expressingthe high-affinity transporter xylFGH (B), and thestrain constitutively expressing the high-affinity transporter xylFGH and lacking the catabolic operon xylAB (C) each harbored the reporter plasmid pUA66-pxylA. The designatedstrain was back-diluted into M9 minimal medium supplemented with theindicated concentration of d-xylose and grown for 6 h toABS600 ∼ 0.4 prior to flow cytometry analysis. SeeFigure 3 for more information on the dot plots.Each dot plot is representative of at least three experiments conductedfrom independent colonies.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Linearizing the response to d-xylose. The wild type E. coli strain (A), the strain constitutively expressingthe high-affinity transporter xylFGH (B), and thestrain constitutively expressing the high-affinity transporter xylFGH and lacking the catabolic operon xylAB (C) each harbored the reporter plasmid pUA66-pxylA. The designatedstrain was back-diluted into M9 minimal medium supplemented with theindicated concentration of d-xylose and grown for 6 h toABS600 ∼ 0.4 prior to flow cytometry analysis. SeeFigure 3 for more information on the dot plots.Each dot plot is representative of at least three experiments conductedfrom independent colonies.
Mentions: To extend the generality of our insights, we focused on the d-xylose utilization pathway in E. coli. Similarto the l-arabinose utilization pathway, the d-xyloseutilization pathway encodes a high-affinity transporter and a low-affinitytransporter, a transcriptional activator that recognizes d-xylose, and enzymes that shunt d-xylose into the pentosephosphate pathway (Figure S1B). Also parallelingthe l-arabinose utilization pathway, the d-xyloseutilization pathway exhibits a bimodal response when tracking theactivity of the xylA promoter (Figure 5A).

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