460 http:/ / bioinfo.aizeonpublishers.net/ content/ 2014/ 4/ bioinfo460-465.

pdf

International Journal of Computational Bioinformatics
and In Silico Modeling
Vol. 3, No. 4 (2014): 460-465
Research Article
Open Access
I IS SS SN N: : 2 23 32 20 0- -0 06 63 34 4

In silico metabolic engineering prediction of
Escherichia coli genome model for production of D-
lactic acid from glycerol using the OptFlux software
platform


Bashir Sajo Mienda, Mohd Shahir Shamsir* and Faezah Mohd Salleh


Bioinformatics Research Group (BIRG), Biosciences & Health Sciences Department, Faculty of Biosciences and Medical Engineering,
Universiti Teknologi Malaysia, Skudai 81310 Johor Bahru. Malaysia.

*Corresponding author: Mohd Shahir Shamsir; e-mail: shahir@fbb.utm.my




ABSTRACT
The advent of genome scale metabolic models of Escherichia coli coupled with limited successes in computational
advancement could facilitate rapid advancement in the field of metabolic engineering and synthetic biology. E.
coli has been subjected to various metabolic engineering approaches using established experimental methods to
produce D-lactate under micro-aerobic conditions using glycerol as substrate. However, investigation on the in
silico prediction and/ or deletion of competing pathway genes on glycerol for the production of D-lactate by E.
coli genome scale model using regulatory on or off minimization (ROOM) under the OptFlux software platform is
yet to be elucidated. Here, we show that in silico metabolic engineering using this software platform by
simulating the knocking out of pyruvate formate lyase (pflB/ b0903), fumarate reductase (frdA/ b4154),
phosphoacetyltransferase (pta/ b2297) and alcohol/ acetaldehyde dehydrogenase (adhE/ b1241) have been
predicted to increase D-lactate production in E. coli. The mutant models constructed in this study exhibited
growth rate that is 96 % of the wild-type model, and hence maintaining a significant flux for D-lactate
production. The results reported herein, were found to be in conformity with previously established
experimental studies. These findings indicates that the OptFlux software platform using ROOM as simulation
algorithm hold great promise as potential software platform that can accurately predict metabolic engineering
targets to guide future experimental studies not only for D-lactate production in E. coli but also for other
microbial chemical compounds of interests.

Keywords: Escherichia coli genome model, metabolic engineering, D-lactate, glycerol, OptFlux software,
gene knockout simulation


INTRODUCTION
Lactic acid has been produced by Escherichia coli under
certain environmental condition such as anaerobic and
micro-aerobic conditions after knocking out certain
competing pathway genes. Lactate and its derivatives
have a broad range of application in food,
pharmaceuticals, leather, textile and polymer
industries [1]. E. coli has been considered as a
significant chassis host for the production of D-lactate,
although other microorganisms can produce lactate by
fermentation of glucose and other alternatives
substrates such as xylose and glycerol [2]. Glycerol is
currently advantageous over its carbohydrate
counterparts, as it is being generated in bulk quantities
as by-product of biofuel Industries [3, 4]. The
advantage of using E. coli as chassis host is conspicuous
by its rapid growth rate and simple nutritional
requirements as opposed to other chassis hosts. In
addition, the ease of genetic modification of E. coli
makes possible metabolic engineering strategies for
improving lactate accumulation in E. coli [2, 5].

Received: 07 July 2014 Accepted: 10 August 2014 Online: 12 August 2014

461 http:/ / bioinfo.aizeonpublishers.net/ content/ 2014/ 4/ bioinfo460-465.pdf

Accordingly, there have been several reports on the use
of E. coli systems metabolic engineering for biofuels
and other specialty chemical productions [6, 7].
Therefore systems metabolic engineering, combining
advanced computational tool such as OptFlux and
synthetic micro-biology can provide novel routes and
metabolic engineering targets for enhanced D-lactate
production in E. coli. Genome scale metabolic models of
E. coli have been published [8, 9]. They are considered
as productive tools for their ability to integrate
genomic information to predict desired phenotype and
simulate whole cell physiology in an interconnected
system [10].

The use of in silico metabolic engineering software
platform to predict metabolic engineering targets have
received remarkable attention in recent years, an
example is seen in the use of the OptFlux software to
predict metabolic engineering interventions using E.
coli genome scale as described previously [11].
Although other software platforms such as OptKnock
has been used to predict metabolic engineering for
lactic acid production in E. coli [10]. In addition,
OptGene, is also another alternative method that is
applied for gene knockout in genome scale metabolic
models. It has been clearly established that limitation
to the use of Opt knock and Opt Gene still exists, which
include the fact that they only use metabolic
information, determining sets of reactions to be
eliminated from the metabolic models, instead of sets
of genes to knockout, which is the real purpose [12].
Therefore, in order to have a desired and/ or
appropriate mutant in the lab, it is important to
determine which sets of genes can lead to the
elimination of a given set of reactions [12] This is
because the rule 1 gene: 1 enzyme: 1 reaction was
not universal as reported elsewhere [12]. However,
there are many exceptions, such as isoenzymes, protein
complexes, or enzymes that catalyze several reactions
[12]. In contrast, OptFlux software platform uses the E.
coli genome scale model and the method of Flux
balance analysis (FBA) to predict the phenotype
simulation of both wild-type and mutant models. The
software is characterized with plug-in architecture,
where an algorithm called Regulatory on or off
minimization of metabolic flux changes after genetic
perturbations (ROOM) was plugged-in to simulate
mutant whole cell behavior after genetic perturbation
or gene knockout [11, 13].

Regulatory on or off minimization (ROOM) is a
constraint-based algorithm for predicting the post
perturbation metabolic steady state. It aims to
minimize the number of significant flux changes (hence
on or off) with respect to the wild type, and it was
shown to accurately predict steady-state metabolic
fluxes that maintain flux linearity in agreement with
experimental flux measurements, and to correctly
identify short alternative pathways used for rerouting
metabolic flux in response to gene knockouts [13].

However, few studies have reported on the use of
computational tools such as Opt Knock [14] for
studying a number of gene knock out to increase the
production of lactic acid using E. col. We have reported
elsewhere that combining computational
breakthroughs with synthetic microbiology would
increase the prospects of metabolic engineering for
increased microbial chemical synthesis [15]. In
addition, Gonzalez and co-workers have reported the
experimental deletion of some competing pathway
genes in E. coli using glycerol to increase D-lactate
under anaerobic and micro-aerobic conditions [1]. The
engineered E. coli strain obtained was capable of D-
lactate production. Other researchers also reported
experimentally engineered E. coli strain for the
production of D-lactate from carbohydrates rich
feedstock. The choice of substrate is critical to the
production of microbial chemicals, although currently
glycerol has become an inexpensive and abundant
substrate due to its generation in large quantities as by-
products of biodiesel and bioethanol industries [3, 4]. It
was previously proposed that conversion of glycerol to
high value products can serve as a path to economic
viability for the biofuels industry [16].

Although this is not the first approach to use glycerol as
a substrate in metabolic engineered E. coli to produced
D-lactate, because the pathways involved in anaerobic
or micro-aerobic utilization of glycerol were
established and elucidated elsewhere [1, 3, 4, 16-18]. In
the work presented here, the knowledge base created
by the studies mentioned above was used to investigate
whether the in silico prediction of metabolic
engineering targets under the OptFlux software
platform using ROOM as the simulation algorithm can
tally with previously established experimental
metabolic engineering approaches. This study inform
other studies that the OptFlux software platform using
ROOM algorithm can prospectively and effectively
predict in silico knocking out of competing pathway
genes, namely: pyruvate formate lyase B
(pflB/b0903), fumarate reductase A (frdA/b4154),
phosphoacetyltransferase (pta/ b2297) and
alcohol/ acetaldehyde dehydrogenase (adhE/ b1241)
that can direct carbon flux to D-lactate production
using the most recent metabolic reconstruction of E.
coli iJ01366 model. These findings clearly
demonstrates that the OptFlux software platform using
ROOM can accurately guide future experimental
metabolic engineering strategies for increased D-
lactate production in E. coli or any other established
selected chassis host.

MATERIALS AND METHODS
Model
The Metabolic reconstruction of Escherichia coli
iJ01366 developed by Orth and co-workers [8] was
used as a model for all the wild-type and mutant strains
described in this study. The model used herein was
previously tested and validated against experimental
data, and was shown to be capable of predicting
accurate growth rates, metabolite excretion rates, and a
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growth phenotypes on a number of substrates and
genetic conditions [8, 9, 19, 20]. The substrate used in
this study is glycerol unless otherwise stated.

Flux Balance Analysis
The reference computational tool for metabolic
engineering called OptFlux software, considered to be
an open source platform www.optflux.org [11] was
used for the flux balance analysis (FBA). Regulatory
on/ off minimization of metabolic flux changes after
genetic perturbations (ROOM) [13] was used as a
simulation method for gene knockouts, and it was
implemented using the Java programming within the
framework of the OptFlux. All simulation of mutant
strains and wild-type models were performed using the
OptFluxv3.06.

The chosen solitary carbon source is glycerol, and the
uptake rate of the carbon source was constrained to a
maximum of 20.0 mmolgDW
1
h
1.
The oxygen uptake
rate was constrained to be 5.0 mmolgDW
1
h
1
as the
simulation condition was micro-aerobic for
fermentative production of D-lactate. These values
were chosen based on slightly close experimental
observation of micro-aerobic and anaerobic growth of
E. coli [21-23].

Gene knockout under the OptFlux software
platform
Gene knockout simulation was conducted under the
OptFlux software platform using ROOM [13] as
simulation method. Flux balance analysis (FBA) was
used for simulation of the wild-type model, which
predicts metabolic flux distributions at steady state by
using linear programming, while ROOM employs mixed
integer linear programming (MILP) to find flux
distribution that satisfies the same constraint as FBA
while minimizing the number of significant flux
changes [13]. The algorithm accounted only for
significant flux changes (0.001 flux prediction) because
of the inherent noise in biological systems and to
reduce the running time, as described in their original
documentation [13]. The wild-type model obtained
from Biomodels database [24], constructed by Orth [8]
was designated as BSM (WT Orth Model) and the
mutant model/ strain with pyruvate formate lyase B
(pflB/ b0903) and fumarate reductase A (frdA/ b4154)
double genes knockout was designated as BSM01
(pflB frdA), while on the other hand, the mutant
model with phosphoacetyltransferase (pta/ b2297),
alcohol/ acetaldehyde dehydrogenase (adhE/ b1241)
and fumarate reductase (frdA/ b4154) triple genes
knockout was designated as BSM02 (pta adhE
frdA). The in silico gene knockouts simulation were
run to completion using ROOM, as previously
established and described [13].

RESULTS AND DISCUSSION
Escherichia coli exhibited a heterofermentative
behavior that resulted in the synthesis of significant
amounts of ethanol, acetate succinate and formate but
only a negligible production of D-lactate is achieved
when glycerol is metabolized under anaerobic and
micro-aerobic conditions [1, 4, 18].

Identification of metabolic engineering targets to
improve D-lactate production in engineered E. coli
strains using established experimental approach have
been reported previously [1, 25]. The target pathway
genes for engineering to increase D-lactate production
are pflB, frdA, pta and adhE. This is because the D-
lactate is synthesized from pyruvate using D-lactate
dehydrogenase (ldhA) see figure 1. The blocking of the
competing pathway genes that lead to the syntheses of
succinate (frdA), ethanol (adhE) formate (pflB) and
acetate (pta) have been the established metabolic
engineering strategies to increase D-lactate production
in E. coli.

In this study, we employ in silico metabolic engineering
for the prediction of D-lactate production in E. coli
using genome scale model under the OptFlux software
platform. Regulatory on/ off minimization (ROOM) has
been used as the algorithm for gene knockout
simulation. The competing pathway genes (pflB, adhE,
pta and frdA) used in this study has been previously
reported to increase D-lactate production in E. coli
experimentally. We used these genes knockout to
investigate whether the OptFlux software platform
using ROOM as gene knockout algorithm can accurately
predict D-lactate flux/ production by using in silico E.
coli genome scale metabolic model.

The results obtained in double gene knockout strain
BSM01 (pflB frdA) for D-lactate flux prediction is
0.001 mmolgDW
1
h
1
see table 1 and fig 2. This value
looks negligible among others, but ROOM algorithm
was specifically designed with a small chosen minimal
values in some parameters that influences the running
time of the MILP solver; hence 0.001 was considered
significant for flux predictions, and the choice of these
parameters influences the corresponding flux
distributions of the algorithm by allowing only a small
amount of additive and multiplicative flux to routes via
alternative pathways with no cost, as described in their
original documentation [13]. Similar flux prediction for
D-lactate production was seen in the triple gene
knockout strain BSM02 (pta adhE frdA). Both
mutant strains exhibited a growth rate that is 96.8 % of
their parent strain BSM (WT Orth Model), see table 1
and Figure 2.

Although there have been several reports describing
metabolically engineered E. coli strains for increased D-
lactate production on glycerol using an alternative
approach to divert carbon towards the synthesis of D-
lactate [1]. Eliminating the pathway genes to the
undesired products such as ethanol, acetate and
succinate have been the widely used metabolic
engineering strategies. In this study in silico knockout
of pflB/ b0903 gene which catalyzes the conversion of
pyruvate to acetyl-CoA and formate (Figure 1) and the
knockout of pta/ b2297 gene (see Figure 1), which
converts acetyl-CoA to acetyl-phosphate (considered as
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the first step in acetate production from AcCoA) have
been conducted to see whether D-lactate flux can be
increase. In practice, when the aforementioned
mutations are introduced in to E. coli using glycerol
under micro-aerobic conditions, the pool of pyruvate
increases, as previously reported that the deletion of
pta/ b2297 and pflB/ b0903 do not affect lactate
dehydrogenase A (ldhA) [1, 26] (see Figure 1).

Based on the achieved engineering targets described
above, D-lactate production was engineered in E. coli
using the OptFlux software platform using ROOM
algorithm as mentioned earlier. The deletion of pflB
strain BSM01 is extremely important, since PFL is the
primary routes for pyruvate conversion to AcCoA
during the micro-aerobic utilization of glycerol [1, 17],
therefore pflB mutation has been shown to effectively
minimize both acetate and ethanol production from
AcCoA (see table 1 and Figure 1). The construction of
double mutant model BSM01 (pflB frdA) lead to
reduced succinate production by in silico knocking out
fumarate reductase (frdA) gene. Correspondingly,
Mutant model BSM01 (pflB frdA) produced D-lactate
as predicted (see table 1 and Figure 2) as the primary
product of glycerol metabolism. These findings are in
agreement with the previously reported
experimentally engineered E. coli [1] using glycerol as
substrate, where D-lactate was produced as primary
product of glycerol metabolism at high concentration of
12.g l
1
and a yield of about 0.69 g Lactate g glycerol
1

[1].

On the other hand, the triple mutant model BSM02
(pta adhE frdA) was constructed to minimized the
synthesis of succinate by in silico knocking out frdA and
that of ethanol by deleting adhE. The elimination of pta
lead to reduced acetate flux (see table 1) compared to
the wild-type model (BSM Orth model). The triple
mutant model BSM02 (pta adhE frdA), exhibited a
very similar D-lactate production and other properties
such as 96.8% growth rate and reduced acetate
production with the double mutant model BSM01
(pflB frdA) relative to the wild-type model (see table
1 and Figure 2). The properties displayed by triple
mutant model BSM02 (pta adhE frdA) is in
consistent with the experimentally reported findings
elsewhere [1], where the same genes were deleted in E.
coli using glycerol as substrate, leading to D-lacttate
production of about 10.9 g l
1
[1]. On the bases of these
findings, the production of D-lactate in engineered E.
coli using glycerol as the substrate in both mutant
models could be attributed to an increase in pyruvate
pool, which is known to allosterically activate ldhA (see
Figure 1).

CONCLUSION
In conclusion, the notion for D-lactate production in E.
coli reported in this study is not the first to bridge the
world of metabolic engineering and synthetic biology
[1, 3, 17, 18] but it is the first to use OptFlux software
platform with ROOM algorithm to predict metabolic
engineering targets with reasonable degree of accuracy
consistent with previously established experimental
studies. We previously reiterated the significance of
combining computational pathway prediction tools
with experimental validation to fully achieve the
prospects of metabolic engineering and synthetic
biology [15]. The in silico metabolic engineering using
OptFlux hold great promise as potential software
platform that can predict metabolic engineering
targets to guide future experimental studies not only
for D-lactate production in E. coli but also for other
microbial chemical compounds of interests.


Table 1. E. coli strain design properties on glycerol under the OptFlux software platform
Knockout
genes/
strains
Growth
rates
%
Growth
rate
Lactate
(mmolgDW
1
h
1
)
Acetate
(mmolgDW
1
h
1
)
Ethanol
(mmolgDW
1
h
1
)
Succinate
(mmolgDW
1
h
1
)
Formate
(mmolgDW
1
h
1
)
BSM (WT
Orth
Model)
0.65960226 100 0.000 30.16366 - - 32.70878
BSM01(pflB
frdA)
0.638808 96.8 0.001 29.2699 0.00227 0.00061 31.7361
BSM02 (Pta
adhE frdA)
0.638808 96.8 0.001 29.264 0.00227 - 31.7203

Maximum uptake rates for glycerol were set to be 20 mmolgDW
1
h
1
and the corresponding Oxygen uptake rate was 5 mmolgDW
1
h
1
for
micro-aerobic simulation.

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HO
OH
OH
Glycerol
gldA
NADH
Dihydroxyacetone
Phosphoenolpyruvate
Pyruvate
OH OH
O
Dihydroxyacetone phosphate
NADH ATP
Phosphoenolpyruvate
O
OH
p O
OH
HO
O
CO
2
+NADH
OH
OH
O
O
OH
OH
O
O
NADH/H
2
Fumarate
Succinate
OH
O
O
Pyruvate
H OH
O
Formate O
CoA
Acetyl-Coenzyme A
NADH
H
O
OH
NADH
adhE/b1241 Acetaldehyde
adhE/b1241
O
O
OH
OH
P
O
HO O
P
OH
O
OH
O
OH
O
ackA
ATP
Acetate
Ethanol
ATP
Acetyl-phosphate
pyk
X
OH
O
OH
D-Lactate
NADH
ldhA
CO
2
+H
2
X
CO
2
+ NADH
lpdA
aceEF
X
X
fdhF, hycB-1
0.5 O
2
H
2
O
P/O ATP
NADH/QH
2
/H
2
frdA/b4154
X
pflB/b0903
pta/b2297

Figure 1. Pathways involved in micro-aerobic utilization of glycerol to produce D-lactate, partially adopted from Ref: [1, 17].
Genetic modification supporting the metabolic engineering is indicated in red. The enzymes in red and their corresponding b
numbers were knocked out to direct the carbon flux towards the production of D-lactate indicated in red. Broken lines illustrate
multiple steps. Relevant reactions are represented by the names of the gene(s) coding for the enzymes: lactate dehydrogenase
(ldhA/ b1380), fumarate reductase (frdA/ b4154), pyruvate formate lyase (pflB/ b0903) and acetaldehyde / alcohol
dehydrogenase (adhE/ b1241).


Figure 2. Growth rates and D-lactate production of the E. coli mutants and wild-type models under micro-aerobic condition on
glycerol.
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