A synthetic glycerol assimilation pathway demonstrates biochemical constraints of cellular metabolism

• Published in: The FEBS journal Vol. 287; no. 1; pp. 160 - 172
• Main Authors: Lindner, Steffen N., Aslan, Selçuk, Müller, Alexandra, Hoffart, Eugenia, Behrens, Patrick, Edlich‐Muth, Christian, Blombach, Bastian, Bar‐Even, Arren
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.01.2020
• Subjects: Assimilation; Dihydroxyacetone; E coli; Escherichia coli; evolution; feedstocks; Glycerol; Metabolic engineering; Metabolism; metabolite toxicity; Metabolites; pathway design; pathway kinetics; pathway thermodynamics; Reaction kinetics; Substrates; synthetic biology; thermodynamics; Toxicity; pathway design; pathway thermodynamics; metabolite toxicity; metabolic engineering; pathway kinetics
• ISSN: 1742-464X; 1742-4658; 1742-4658
• DOI: 10.1111/febs.15048

The engineering of synthetic metabolic routes can provide valuable lessons on the roles of different biochemical constraints in shaping pathway activity. In this study, we designed and engineered a novel glycerol assimilation pathway in Escherichia coli. While the synthetic pathway was based only on well‐characterized endogenous reactions, we were not able to establish robust growth using standard concentrations of glycerol. Long‐term evolution failed to improve growth via the pathway, indicating that this limitation was not regulatory but rather relates to fundamental aspects of cellular metabolism. We show that the activity of the synthetic pathway is fully controlled by three key physicochemical constraints: thermodynamics, kinetics and metabolite toxicity. Overcoming a thermodynamic barrier at the beginning of the pathway requires high glycerol concentrations. A kinetic barrier leads to a Monod‐like growth dependency on substrate concentration, but with a very high substrate saturation constant. Finally, the flat thermodynamic profile of the pathway enforces a pseudoequilibrium between glycerol and the reactive intermediate dihydroxyacetone, which inhibits growth when the feedstock concentration surpasses 1000 mm. Overall, this study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism. We designed and engineered a novel glycerol assimilation pathway in Escherichia coli. We show that the activity of the synthetic pathway is fully controlled by three key physicochemical constraints: thermodynamics, kinetics, and metabolite toxicity. Growth is characterized with a Monod curve with a very high substrate saturation constant. Our study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism.