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ACS Synth Biol. 2017 May 19;6(5):793-805. doi: 10.1021/acssynbio.6b00230. Epub 2017 Feb 9.

Refactoring the Embden-Meyerhof-Parnas Pathway as a Whole of Portable GlucoBricks for Implantation of Glycolytic Modules in Gram-Negative Bacteria.

Author information

1
Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco , 28049 Madrid, Spain.

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.

KEYWORDS:

Escherichia coli; PHB; Pseudomonas putida; glycolysis; metabolic engineering; standardization

PMID:
28121421
PMCID:
PMC5440799
DOI:
10.1021/acssynbio.6b00230
[Indexed for MEDLINE]
Free PMC Article

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