Abstract
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 Escherichiacoli. 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 profileof the pathway enforces a pseudoequilibrium between glycerol and the reactive intermediate dihydroxyacetone, which inhibits growth when the feedstock concentration surpasses 1000mm. Overall, this study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism.
Highlights
The structure of metabolic pathways reflects a complex interplay between different selection pressures and biochemical constraints
We deleted, within the DglpK DdhaK strain, the two isozymes of fructose bisphosphatase [10], resulting in a DglpK DdhaK Dfbp DglpX strain. This strain was expected to grow on glucose, but growth on pyruvate – or any other ‘lower metabolism’ carbon source – should not be possible, since gluconeogenesis is blocked at the level of fructose 1,6-bisphosphate and the cell cannot synthesize essential sugar phosphates: fructose 6-phosphate (F6P), glucose 6-phosphate, erythrose 4-phosphate and ribose 5-phosphate
To confirm that the growth on pyruvate and glycerol follows the metabolic pattern we predicted, we cultivated the strain on completely 13C-labelled glycerol and unlabelled pyruvate and measured the labelling pattern in different proteinogenic amino acids. Both alanine and serine – which can be derived solely from pyruvate – are negligibly labelled, whereas histidine – two carbons of which are expected to be derived from glycerol (Fig. 3A) – is almost completely doubly labelled. These results demonstrate that the combined activities of GldA and fructose 6-phosphate aldolase (F6PA) can support flux in the desired assimilatory direction. (We note the different labelling of the amino acids within a WT strain, where the labelling of serine and alanine indicates that glycerol is oxidized by glycolysis and the labelling of histidine indicates that it is mainly produced from glycerol.)
Summary
The structure of metabolic pathways reflects a complex interplay between different selection pressures and biochemical constraints. Even the canonical structure of central metabolism shows major variations that reflect changing constraints under different environmental conditions. To give just few examples, nonphosphorylating glycolysis represents an adaptation to very high temperatures, where the instability of phosphorylated sugars becomes a major constraint [1,2,3]; the Entner– Doudoroff pathway is preferred over (Embden–Meyerhof–Parnas) glycolysis for obligatory aerobic metabolism in which the rate of glycolysis is more important than its ATP yield [4]; the methylglyoxal bypass, converting dihydroxyacetone phosphate into the highly reactive compound methylglyoxal, is utilized when phosphate starvation constrains glycolytic flux [5,6]; and the use of pyruvate formate-lyase, supporting 50% increase in ATP yield during sugar fermentation, reflects adaptation to microaerobic and anaerobic conditions [7,8]. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies
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