A synthetic glycerol assimilation pathway demonstrates biochemical constraints of cellular metabolism

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 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.

Complementation of an Escherichia coli K-12 Mutant Strain Deficient in KDO Synthesis by Forming D-Arabinose 5-Phosphate from Glycolaldehyde with Fructose 6-Phosphate Aldolase (FSA)

“…This opened a pathway for the production of DHA and glycerol from glucose [23]. The opposite reaction, e.g., growth on glycerol via DHA to F6P in specific mutants, was achieved as well by the group of the late Arren Bar-Even [31]. While the present manuscript was in preparation, the Walther's group proved that FSAA can be used to implement a novel in vivo metabolic pathway to yield the product acetyl-CoA.…”

Evaluation of Cellular Responses of Heterotrophic Escherichia coli Cultured with Autotrophic Chlamydomonas reinhardtii as a Nutrient Source by Analyses Based on Microbiology and Transcriptome

“…The central metabolism from glucose to pyruvate is deeply involved in the metabolism (catabolism and assimilation) of carbon sources, and it is known that the metabolic system is activated during glucose catabolism [ 25 ] and glycerol assimilation [ 26 ]. The gene transcription levels in glycolysis of E. coli were comprehensively evaluated ( Figure 5 a-1,a-2).…”

Engineering an efficient whole‐cell catalyst for d‐allulose production from glycerol

“…dehydrogenase (GlpD); (2) glycerol is oxidized to dihydroxyacetone (DHA) by glycerol dehydrogenase (GldA), and subsequently phosphorylated to DHAP by dihydroxyacetone kinase (DAK1, DKA2, or DhaMLK). [31,32] Alditol oxidase from Streptomyces coelicolor A3 and NADP + -dependent alcohol dehydrogenases have been reported to be capable of oxidizing glycerol to D-GA. [30,33,34] This study will systematically explore the glycerol assimilation pathways, alditol oxidase, and NADP + -dependent alcohol dehydrogenases from different organisms (Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, Gluconobacter oxydans, and S. coelicolor), and find efficient pathways to convert glycerol to DHAP and D-GA. Assembling these pathways with D-allulose producing module (composed of DHAP-dependent aldolases and phosphatase) will finally achieve D-allulose production utilizing cheap glycerol feedstock from biodiesel industry (Figure 1B).…”

“…Glycerol assimilation involves two routes to make DHAP: (1) glycerol is first phosphorylated to glycerol 3‐phosphate by glycerol kinase (GlpK), and subsequently oxidized to DHAP by glycerol 3‐phosphate dehydrogenase (GlpD); (2) glycerol is oxidized to dihydroxyacetone (DHA) by glycerol dehydrogenase (GldA), and subsequently phosphorylated to DHAP by dihydroxyacetone kinase (DAK1, DKA2, or DhaMLK). [ 31,32 ] Alditol oxidase from Streptomyces coelicolor A3 and NADP + ‐dependent alcohol dehydrogenases have been reported to be capable of oxidizing glycerol to d ‐GA. [ 30,33,34 ] This study will systematically explore the glycerol assimilation pathways, alditol oxidase, and NADP + ‐dependent alcohol dehydrogenases from different organisms ( Escherichia coli , Saccharomyces cerevisiae , Bacillus subtilis , Gluconobacter oxydans , and S. coelicolor ), and find efficient pathways to convert glycerol to DHAP and d ‐GA.…”