Refactoring the Embden–Meyerhof–ParnasPathway as a Whole of Portable GlucoBricks for Implantation of GlycolyticModules in Gram-Negative Bacteria

摘要

The Embden–Meyerhof–Parnas (EMP) pathway is generally considered to be the biochemical standard for glucose catabolism. Alas, its native genomic organization and the control of gene expression in Escherichia coli are both very intricate, which limits the portability of the EMP pathway to other biotechnologically important bacterial hosts that lack the route. In this work, the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (GlucoBrick) that enables their grouping in the form of functional modules at the user’s will. After verifying their activity in several glycolytic mutants of E. coli, the versatility of these GlucoBricks was demonstrated in quantitative physiology tests and biochemical assays carried out in Pseudomonas putida KT2440 and P. aeruginosa PAO1 as the heterologous hosts. Specific configurations of GlucoBricks were also adopted to streamline the downward circulation of carbon from hexoses to pyruvatein E. coli recombinants, thereby resulting ina 3-fold increase of poly(3-hydroxybutyrate) synthesis from glucose.Refactoring whole metabolic blocks in the fashion described in thiswork thus eases the engineering of biochemical processes where theoptimization of carbon traffic is facilitated by the operation ofthe EMP pathway—which yields more ATP than other glycolyticroutes such as the Entner–Doudoroff pathway.