Refactoring the Embden-Meyerhof-Parnas pathway as a whole of portable GlucoBricks for implantation of glycolytic modules in Gram-negative bacteria

Alberto Sanchez-Pascuala, Victor de Lorenzo, Pablo Ivan Nikel

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Abstract

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 pyruvate in E. coli recombinants, thereby resulting in a 3-fold increase of poly(3-hydroxybutyrate) synthesis from glucose. Refactoring whole metabolic blocks in the fashion described in this work thus eases the engineering of biochemical processes where the optimization of carbon traffic is facilitated by the operation of the EMP pathway—which yields more ATP than other glycolytic routes such as the Entner–Doudoroff pathway.
Original languageEnglish
JournalA C S Synthetic Biology
Volume6
Issue number5
Pages (from-to)793-805
ISSN2161-5063
DOIs
Publication statusPublished - 2017
Externally publishedYes

Bibliographical note

ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Keywords

  • Escherichia coli
  • Pseudomonas putida
  • Standardization
  • Glycolysis
  • Metabolic engineering
  • PHB

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