Brief Guides
A Brief Guide to Pseudomonas putida as a microbial cell factory
Pablo I. Nikel
Systems and Synthetic Biology Program, Centro Nacional de
Biotecnología (CNB-CSIC), Madrid, Spain
Corresponding author:
Pablo I. Nikel
E-mail: pablo.nikel@cnb.csic.es
Under the general premise of environmental
strains (many of them solvent-tolerant), able to
protection and biosustainability, the quest for
efficiently produce several bulk and fine chemicals
efficient and reliable microbial cell factories
(Fig. 1).
(MCFs) is constantly underway. Resurging interest
in the exploration of microbes as industrial
biocatalysts leads to a shift of strategies away from
traditional one-gene-at-a-time biotechnology approaches to new strategies that yield MCFs deeply
engineered to perform highly specific tasks à la
carte. Although Escherichia coli continues to be a
relevant MCF in a variety of industrial applications
(e.g.
recombinant
protein
production),
other
microorganisms are rapidly gaining attention as
attractive alternatives to this biotechnological
workhorse, which is not devoid of limitations in
both industrial and environmental settings. The soil
bacterium Pseudomonas putida thus emerges
primus inter pares as an attractive candidate. One
of the best studied species of the metabolically
Figure 1. A simplified roadmap sketching basic, omic-based
knowledge and industrial applications of a P. putida-based microbial
cell factory. A non-exhaustive list of bioproducts with
biotechnological interest (corresponding to both bulk biochemicals
and fine chemistry specialties) is given. For other relevant examples,
see Poblete-Castro et al. [10]. Note that, besides biotechnological
synthesis of relevant biochemicals, P. putida is a promising biological
agent of in situ and ex situ bioremediation processes. PHAs,
polyhydroxyalkanoates.
versatile and ubiquitous genus of the pseudomonads [1], this species exhibits huge biotechnological potential, comprising a number of
It should also not be forgotten that one of the first
phenotypic
Keywords: biodegradation; microbial cell factory; oxidative
stress; Pseudomonas putida; redox and energy homeostasis
Abbreviations:
MCF,
microbial
cell
factory;
GRAS,
traits
of
biotechnological
interest
associated with P. putida was its remarkable ability
to degrade recalcitrant xenobiotic compounds,
generally recognized as safe; ED pathway, Entner-Doudoroff
such as toluene and xylenes [2] (later demon-
pathway; EMP pathway, Embden-Meyerhof-Parnas pathway.
strated to be encoded in the archetypal TOL
Please note: this contribution was not peer-reviewed.
1
plasmid pWW0). These interesting features, along
putida KT2440 has been elegantly exposed by
with their entire lack of pathogenesis determinants
Ebert et al. [4] by introducing systematic per-
(P. putida KT2440, a plasmid-less derivative of the
turbations in NADH and ATP demand.
original P. putida isolate termed mt-2, has been
Another hurdle that calls for attention is the
given the GRAS status), high stress resistance,
metabolic architecture of sugar catabolism in P.
and amenability to profound genetic refactoring
putida. Glucose is one of the most attractive
and heterologous gene expression, make P. putida
substrates to fuel the metabolism of micro-
an ideal starting point in the search for superior
organisms used in energy-dependent biopro-
MCFs. Then, the relevant question that needs to
cesses. The biochemical network of P. putida
be addressed is: what are the current limits for the
seems geared for the catabolism of a large variety
wide biotechnological application of P. putida? And
of substrates (e.g. organic acids) with a clear focus
even more relevant, what needs to be done to
on metabolic diversity rather than efficiency. P.
overcome these limits?
putida KT2440, like many other Pseudomonas
Many of the potential P. putida-based applications
species and rhizosymbionts, has an incomplete
are still at an early stage of progress due to the
Embden-Meyerhof-Parnas (EMP) pathway be-
lack of knowledge on the genotype/phenotype
cause of a missing 6-phospho-fructokinase [5].
relationships under conditions relevant for both
The initial steps of glucose metabolism in this
industrial and environmental endeavors. Recent
bacterium occur through a set of three convergent
advances in molecular biology tools, genome-
pathways
scale models, theoretical understanding, and
gluconate, adding a further level of complexity to
kinetic modeling of P. putida have increased the
hexose catabolism. As glucose is catabolized
interest in using targeted metabolic engineering
almost entirely through the Entner-Doudoroff (ED)
strategies to program this bacterium for bio-
pathway, utilization of hexoses is not optimal from
technological purposes. In particular, the complete
the biotechnological point of view since it yields
genomic sequencing of strain KT2440 [3] has
half of the ATP obtained through a bona fide EMP
provided
its
pathway. Most surprisingly, knocking out eda
metabolic potential, opening avenues for several
(encoding the second step of the ED pathway) and
omics studies. However, the nub of the problem of
expressing pfkA from E. coli (encoding the missing
designing an improved MCF based on this
bioreaction needed to sustain a complete EMP
microorganism mostly resides in the difficulty of
pathway) not only failed to activate an EMP
partially or totally dissociating catabolism from
metabolism, but also turned out to be detrimental
anabolism, as needed in a suite of applications.
to growth on different carbon sources, interfering
When the aim is shifting its metabolic network
with both the redox and energy balance [6]. This
entirely towards the formation of a heterologous
points to an evolutionary advantage of an ED-
product, one should minimize by-product synthesis
based metabolism, and also demonstrates the
and cell growth. But, as it happens, the extant
central role of catabolic pathways in P. putida
metabolic properties of the cells, including redox
(while posing quite a challenge to metabolic
and energy homeostasis, often act in the opposite
engineers!). As a corollary, it seems likely that
way. The surprising – and somewhat annoying –
environmental bacteria hosting an ED pathway
robustness of the central metabolic pathways in P.
over an EMP pathway do so to gear their aerobic
diverse
means
of
investigating
Please note: this contribution was not peer-reviewed.
that
eventually
yield
6-phospho-
2
metabolism for enduring oxidation-related insults
nutritional resources (and starving the biocatalyst
[7]. And, on this very subject, moving the window
is not the brightest idea to harness its full
of metabolic operativity of P. putida in terms of
potential). With the continuous advent of new and
oxygen availability towards more reduced environ-
more sophisticated tools for genome manipulation,
ments would also be a welcome industrial trait [8].
the design of a device that efficiently switches the
cell state from
growing conditions into the
Finally, as reproduction can be regarded as a
production status can be envisaged in the near
seriously distractive (yet unavoidable) process
future.
from other cellular activities, it would be desirable
From its humble beginning in the soil towards its
to synthesize heterologous products in non-
promising future as a fully programmable MCF, the
growing cells. This strategy would allow efficient
exciting trajectory of P. putida keeps offering room
channeling of metabolic resources into the desired
for improvement. The burgeoning field of systems
product rather than into biomass (in this context,
metabolic engineering, guided by recent advances
an unwanted by-product). Despite many efforts, it
in Synthetic Biology [9], will surely help to exploit
is still enormously difficult to stop bacterial growth
the maximum industrial potential of P. putida and
without stressing the cells
put it in the middle of the biotechnological arena.
by limiting their
Acknowledgements
I am indebted to Prof. Víctor de Lorenzo for guiding me into the amazing world of metabolic engineering of Pseudomonas,
and for inspiring discussions on the contents of this article. I would like to thank Esteban Martínez-García for sharing the
P. putida micrograph used in the figure. The author is a researcher from the Consejo Nacional de Investigaciones
Científicas y Tecnológicas (Argentina), and holds a Long-Term Fellowship from the European Molecular Biology
Organization (EMBO ALTF 13-2010).
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