Premium
A synthetic glycerol assimilation pathway demonstrates biochemical constraints of cellular metabolism
Author(s) -
Lindner Steffen N.,
Aslan Selçuk,
Müller Alexandra,
Hoffart Eugenia,
Behrens Patrick,
EdlichMuth Christian,
Blombach Bastian,
BarEven Arren
Publication year - 2020
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15048
Subject(s) - dihydroxyacetone , assimilation (phonology) , glycerol , synthetic biology , metabolic engineering , metabolic pathway , metabolism , biochemistry , chemistry , cellular metabolism , substrate (aquarium) , metabolite , enzyme , biophysics , biology , computational biology , ecology , linguistics , philosophy
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 m m . Overall, this study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism.