Abstract
A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, 13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype–phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.
Highlights
Pseudomonas putida is one of the best studied species of the metabolically versatile and ubiquitous genus of the Pseudomonads [1,2,3]
The pseudomonads include a diverse set of bacteria whose metabolic versatility and genetic plasticity have enabled their survival in a broad range of environments
To better understand the growth and metabolism of these bacteria, we developed a large-scale mathematical model of the metabolism of Pseudomonas putida, a representative of the industrially relevant pseudomonads
Summary
Pseudomonas putida is one of the best studied species of the metabolically versatile and ubiquitous genus of the Pseudomonads [1,2,3] As a species, it exhibits a wide biotechnological potential, with numerous strains (some of which solvent-tolerant [4,5]) able to efficiently produce a range of bulk and fine chemicals. It exhibits a wide biotechnological potential, with numerous strains (some of which solvent-tolerant [4,5]) able to efficiently produce a range of bulk and fine chemicals These features, along with their renowned stress resistance, amenability for genetic manipulation and suitability as a host for heterologous expression, make Pseudomonas putida attractive for biocatalysis. In an effort towards the generation of critical knowledge, the genomes of several members of the Pseudomonads have been or are currently being sequenced (http://www.genomesonline.org, http://www.pseudomonas.com), and a series of studies are underway to elucidate specific aspects of their genomic programs, physiology and behavior under various stresses (e.g., http://www.psysmo.org, http://www.probactys.org, http://www.kluyvercentre.nl)
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