(614f) Prediction of Double Substrate Microbial Growth Kinetics through Transcriptional Regulation: An Integrated Experimental/Modelling Approach on Pseudomonas Putida Mt-2 | AIChE

(614f) Prediction of Double Substrate Microbial Growth Kinetics through Transcriptional Regulation: An Integrated Experimental/Modelling Approach on Pseudomonas Putida Mt-2


Tsipa, A. - Presenter, Imperial College London
Koutinas, M. - Presenter, Cyprus University of technology
Pistikopoulos, E. N. - Presenter, Texas A&M Energy Institute, Texas A&M University
Mantalaris, A. - Presenter, Imperial College London

of double substrate microbial growth kinetics through transcriptional
regulation: an integrated experimental/modelling approach on Pseudomonas putida

Tsipaa, Michalis Koutinasb, Efstratios N. Pistikopoulosc
and Athanasios Mantalarisa*

a Centre for Process Systems Engineering,
Department of Chemical Engineering, South Kensington Campus, Imperial College
London, SW7 2AZ,
London, UK

b Department of Environmental Science and
Technology, Cyprus University of Technology, 30 Archbishop Kyprianou Str.,
3036, Limassol, Cyprus

cArtie McFerrin Department of Chemical
Engineering, University of Texas, Austin, TX 78712, United States

Pseudomonas putida
is a metabolically versatile soil bacterium and an industrially significant
strain producing a series of fine and bulk chemicals, which has resulted in growing
interest to understand specific metabolic pathways (Ballerstedt et al., 2007). Among the several P. putida strains, mt-2 contains the TOL
plasmid (pWW0), which specifies metabolic pathways for toluene and m-xylene degradation into Krebs cycle
intermediates (Timmis, 2002). Successful activation of TOL relies mainly on the
catabolic enzymes catalysing the degradation of the compound, and the promoters
of genes subject to regulation by specific transcription factors (de Lorenzo
and Perez-Martin, 1996). Therefore, transcriptional
regulation is a key step in the biodegradation process
acting as a
controller regulating the appropriate metabolic cascades in response to the
availability of specific substrate(s) (Diaz and Pieto, 2000).

The TOL plasmid is considered as a paradigm of global and specific gene regulation (Aranda-Olmedo et
al., 2006). The transcriptional regulatory network of TOL constitutes four
transcriptional units (xylR, xylS, upper and meta-operon)
controlled by four promoters (Pr, Ps, Pu
and Pm), respectively (Ramos et al.,
1997). Entry of an effector in TOL provokes a cascade of regulatory events (Figure
1) initiated by Pr expression, which
is followed by an increase in Pu
expression. The latter controls the upper-operon
that encodes for the enzymes transforming the inducer into an intermediate acting
as environmental stimulus for XylS overexpression. XylS protein is produced
constitutively by Ps and serves as
the transcription factor for Pm
activation. Pm controls transcription
from the meta-operon, which encodes
for the enzymes that catalyse the inducer further into Krebs cycle metabolites
required for biomass growth.

Figure 1: The regulatory network of TOL and ortho-cleavage pathways represented as
logic gates of a genetic circuit  (Weiss
et al., 2003)   SHAPE  \* MERGEFORMAT

 : input;  SHAPE  \* MERGEFORMAT

 : output;  SHAPE  \* MERGEFORMAT



 : NOT, i:inactive form of protein, a: active
form of protein

Activation of Pu
leads to the production of upper-operon
enzymes catalysing the transformation of
m-xylene and toluene into methyl
benzoate and benzoate
, respectively. The latter serves as environmental stimulus of the ortho-cleavage pathway of the chromosome
activating PbenR and thus driving expression
of the benR gene, which encodes for
the BenR protein (Figure 1). This
protein acts as transcription factor for
meta-operon activation in TOL,
demonstrating a cross-talk between the two pathways
(Cowles et al., 2000). However, induction with m-xylene leads into a metabolic conflict
(Perez-Pantoja et al., 2014) resulting
in the formation of a dead-end product (Schmidt et al., 1985). Therefore, the benR gene of the chromosome is considered as an essential element
of the catabolic process of toluene (Cuskey and Sprenkle, 1988). The
presence of benzoate leads to a transcriptionally competent form of BenR regulating
expression from PbenA and thus,
transcription from the ben-operon of
the ortho-cleavage pathway (Figure 1).
Therefore, ben- and meta-operons are simultaneously activated
by benzoate (Perez-Pantoja et al.,
2014) triggering the transformation of benzoate into catechol which is further catabolised
into Krebs cycle intermediates (Burlage et
., 1989).

The transcriptional kinetics of the TOL plasmid has been previously
evaluated using m-xylene as the
inducer of the pathway (Koutinas et al., (2010, 2011)). Furthermore, the
specific transcriptional kinetics has been recently monitored upon toluene
introduction in order to demonstrate the interplay between the TOL and the chromosomal
ortho-cleavage pathways (Tsipa et
al., 2015), suggesting interdependence of the two pathways through potential
up-regulation of PbenA by the XylS
protein. Nevertheless, the
present study is the first to our knowledge to evaluate the transcriptional kinetics of P. putida upon simultaneous induction with m-xylene and toluene, although the specific pollutant mixture is
commonly met in petrochemical waste streams. Both substrates are considered as
predominant effectors of the TOL plasmid where m-xylene is strongly preferred by cells as compared to toluene (Duetz
et al., 1998). At the genetic level the four promoters of TOL were stimulated
first by m-xylene, and when the
concentration was reduced below 0.5 mM the activity of the promoters was boosted
due to toluene induction (Figure 2A, B, C, D). The
expression of PbenR was maintained at
a basal level and slightly decreased towards the complete depletion of both
substrates (Figure 2E), while expression of PbenA
was remarkably boosted only when toluene existed as the sole effector remaining
in the culture (Figure 2F).

Taking into account the crucial role of promoters as controllers of
biodegradation, a mechanistic approach
to describe m-xylene and toluene utilization
as well as biomass formation in conjunction to the response of the TOL and
chromosomal regulatory networks
is becoming a necessity. The
performance of biodegradation in similar systems is typically monitored through
Monod-type models. However, these traditional and widely used models are based
on bulk measurements, ignoring the molecular interactions and gene regulation of
the strain employed, resulting in limited predictive capability. Furthermore, prediction
of double substrate microbial growth kinetics still remains a topic which has not
been successfully realised in a wide range of conditions, constituting a major
challenge in biochemical engineering. Herein, we present an experimentally validated mathematical model (using qPCR measurements), which is capable
of capturing the dynamic behaviour of
the key promoters in the TOL and ortho-cleavage
. The model's prediction is used to describe the activity of the
enzymes responsible for substrates catabolism and biomass growth, followed by prediction of specific growth, biomass
formation and substrates utilization rates
. The application of the model in double
substrate cultures exemplifies the importance of using transcriptional regulatory
information for bioprocess prediction linking directly genetic and molecular
mechanisms to macroscopic events. The developed model constitutes a novel
mechanistic approach establishing an integrated framework for the prediction of
microbial growth kinetics in environmental bioprocesses.



Figure 2: Relative
mRNA expression of (A) Pr, (B) Ps, (C) Pu, (D) Pm, (E) PbenR, (F) PbenA promoters upon induction with m-xylene and toluene.


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