Biology Reference
In-Depth Information
Chapter 10
putida
KT2440 Metabolic Network
Jacek Pucha
ł
ka, Matthew A. Oberhardt, Miguel Godinho,
Agata Bielecka, Daniela Regenhardt, Kenneth N. Timmis,
Jason A. Papin, and Vítor A. P. Martins dos Santos
INTRODUCTION
A cornerstone of biotechnology is the use of microorganisms for the efficient produc-
tion of chemicals and the elimination of harmful waste.
Pseudomonas putida
is an ar-
chetype of such microbes due to its metabolic versatility, stress resistance, amenability
to genetic modifications, and vast potential for environmental and industrial applica-
tions. 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 (CB) 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. The FBA and flux variability analysis (FVA) were
used to analyze the properties, potential, and limits of the model. These analyses al-
lowed 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 pheno-
typing data,
13
C-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 (PHAs), a class of biotechnologi-
cally 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.
Pseudomonas putida
is one of the best studied species of the metabolically versa-
tile and ubiquitous genus of the Pseudomonads [1-3]. As a species, it exhibits a wide
biotechnological potential, with numerous strains (some of which solvent-tolerant [4,
5]) able to effi ciently produce a range of bulk and fi ne chemicals. These features, along
with their renowned stress resistance, amenability for genetic manipulation and suit-
ability as a host for heterologous expression, make
Pseudomonas putida
particularly
