Research Article

Received: 31 March 2016 Revised: 8 October 2016 Accepted article published: 24 October 2016 Published online in Wiley Online Library: 29 November 2016

(wileyonlinelibrary.com) DOI 10.1002/jctb.5138

Metabolic engineering and adaptive evolution

of Escherichia coli KO11 for ethanol production
through the Entner–Doudoroff and the
pentose phosphate pathways
Gerardo Huerta-Beristain,a* Rosina Cabrera-Ruiz,b Georgina
Hernandez-Chavez,b Francisco Bolivar,b Guillermo Gossetb and Alfredo
Martinezb*

Abstract
BACKGROUND: Ethanologenic Escherichia coli KO11 was modified to channel carbon flux from glucose through the
Entner–Doudoroff (ED-P) and pentose phosphate (PP-P) pathways by using a phosphoglucose isomerase (pgi) knockout in
the glycolytic pathway. This strain grows very slowly under non-aerated conditions with minimal media supplemented with 4%
glucose. To improve the capacity to grow, KO11 𝚫pgi was evolved for 60 days; the resultant strain was named KO11 E35, which
directs the carbon flux through the PP-P and ED-P to lactate and acetate production.

RESULTS: The activities of glucose-6-phosphate dehydrogenase and the ED-P enzymes increased 17-fold and 2-fold, respectively,
in KO11 E35 in comparison with KO11. A homoethanologenic derivative was constructed from KO11 E35 by deleting the pta,
ack and ldh genes, yielding the KO11 PPAL strain. This strain channels most of the carbon flux from pyruvate to ethanol and
increased expression of heterologous pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis allowed us
to obtain specific ethanol production rates similar to those found in KO11, but with half the cell mass, i.e. larger ethanol/glucose
and ethanol/biomass yields.

CONCLUSIONS: These results suggest that it is possible to obtain the same carbon flux using the PP-P and ED-P as when using
the Embden–Meyerhof–Parnas pathway for glucose catabolism to ethanol.
© 2016 Society of Chemical Industry

Keywords: ethanologenic Escherichia coli; glucose; ethanol; pentose phosphate pathway; Entner–Doudoroff pathway; adaptive
evolution

INTRODUCTION effect was attributed to the limited ability to catabolize glucose

Under aerobic conditions, Escherichia coli has the ability to
metabolize glucose through glycolysis and the pentose phos-
phate pathway (PP-P). These are the main ways it obtains the
intermediates and energy for biosynthesis. However, mixtures
of glucose plus gluconate or glucoronate are metabolized by
the Embden–Meyerhof–Parnas (EMP-P) and Entner–Doudoroff
pathways (ED-P), respectively.1 In E. coli, the ED-P is inducible by
extracellular gluconate.2 The characterization of sugar phospho-
transferase system (PTS) and phosphoglucose isomerase (pgi)
mutants suggest that the ED-P pathway has an important role in
the metabolism of glucose.1,3
Zymomonas mobilis is a unique microorganism that uses the E-D
pathway for glucose catabolism to ethanol and CO2 . This pathway
produces one mole of ATP for each mole of metabolized glucose
under anaerobic conditions.2,4 This strain lacks phosphofructoki-
nase and gluconate-6-phosphate dehydrogenase activities.5
Flores et al.6 showed that an E. coli pgi mutant growing under
990
through the PPP due to blocked glycolysis. The increased flux of
carbon through the oxidative branch of the PPP in the strain with
blocked glycolysis increases the level of NADPH. Under aerobic
conditions, the regeneration of NADPH occurs in the tricarboxylic
∗ Correspondence to: G Huerta-Beristain, Unidad Académica de Ciencias
Químico Biológicas, Universidad Autónoma de Guerrero, Av. Lázaro Cárdenas
S/N. Ciudad Universitaria, CP.39090, Chilpancingo, Guerrero, México. Email:
hbgerardo@gmail.com; or A Martinez, Departamento de Ingeniería Celular
y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma
de México. Apdo. Postal 510-3, Cuernavaca, Morelos 62250, México. Email:
alfredo@ibt.unam.mx
a Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma
de Guerrero, Av. Lázaro Cárdenas S/N. Ciudad Universitaria, CP.39090,
Chilpancingo, Guerrero, México
b Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología,
Universidad Nacional Autónoma de México. Apdo. Postal 510-3, Cuernavaca,

aerobic conditions had an 86% lower specific growth rate. This Morelos 62250, México

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Engineering of E-D and PP pathways in KO11 pgi- knockout www.soci.org

Table 1. Strains, plasmid and primers used in this work

Relevant genotype Source

Strains
DH5𝛼 F− 𝜑80dlacZΔM15 Δ(lacZYA-argF)U169deoR recA1 endA1 Laboratory stock
hsdR17(rk− mk+ ) phoA supE44 𝜆− thi-1 gyrA96 relA1
E. coli KO11 E. coli C Δfrd, pfl::pdc adhB cat Ohta et al.12
KO11 Δpgi KO11, Δpgi This work
KO11 E35 KO11, Δpgi evolved This work
KO11 PPAL KO11 E35, Δack-pta, Δldh This work
Plasmids
pLOI510 PDC and ADH from Z. mobilis Martinez et al.20
Primers Sequence Use
pgiH1P1 5 ́ ATC AGA AGA GTA TTG CTA ATG AAA AAC ATC AAT CCA ACG Deletion of pgi gene in E. coli by substitution with the
CAG TGT GTA GGC TGG AGC TGC TTC G 3 ́ Kanamycin cassette. Datsenko and Wanner14
pgiH2P2 5 ́ CCT ACA TAT CGA CGA TGA TTA ACC GCG CCA CGC TTT ATA
GCG CAT ATG AAT ATC CTC CTT AG 3 ́
ackptaH1P1 5 ́ GTA TCA ATT ATA GGT ACT TCC ATG TCG AGT AAG TTA GTA Deletion of ack and pta genes in E. coli by substitution
CTG GTT GTG TAG GCT GGA GCT GCT TCG 3 ́ with the Kanamycin cassette. Datsenko and
Wanner14
ackptaH2P2 5 ́ CTG CGG ATG ATG ACG AGA TTA CTG CTG CTG TGC AGA CTG
AAT CGC CAT ATG AAT ATC CTC CTT AG 3 ́
ldhH1P1 5’ ATC ACT GGA GAA AGT CTT ATG AAA CTC GCC GTT TAT AGC Deletion of ldh gene in E. coli by substitution with the
ACA GTG TAG GCT GGA GCT GCT TC 3’ Kanamycin cassette. Datsenko and Wanner14
ldhH2P2 5’ TGC AGG GGA GCG GCA AGA TTA AAA CCA GTT GGT TCG GGC
AGG TCA TAT GAA TAT CCT CCT TTA G 3’

acid cycle.6,7 Nevertheless, under anaerobic conditions glucose is
mostly catabolized trough the EMP-P. In addition, Kimata et al.8
reported that a mutation in the pgi gene favored the degradation
of the ptsG mRNA in an RNAse E-dependent manner. This effect is
activated by the accumulation of glucose-6-phosphate.9,10
The ethanologenic strain KO11 is a prototrophic E. coli C deriva-
tive that has the Z. mobilis ethanol pathway integrated into its chro-
mosome. Pyruvate decarboxylase is encoded by pdc, and alcohol
dehydrogenase is encoded by adhB. These genes are under the
control of the pyruvate formate lyase (pfl) promoter.11,12 The strain
also has a deletion of the anaerobic fumarate reductase gene,
which eliminates succinate production.12,13
In this study, we analyzed the effect of the inactivation of the pgi
gene in the ethanologenic E. coli strain KO11 cultivated under fer-
mentative conditions. We found that the KO11 pgi- strain is unable
to grow under anaerobic conditions. To enable growth under fer-
mentative conditions, KO11 Δpgi was subjected to an adaptive
evolution process in minimal medium supplemented with glu-
cose under non-aerated conditions. Furthermore, other fermenta-
tive pathways were inactivated, and the pyruvate decarboxylase
and alcohol dehydrogenase activities were increased to obtain
a homoethanologenic strain that catabolizes glucose to ethanol
through the PP-P and ED-P.
MATERIALS AND METHODS
Bacterial strains and plasmids
Strains, plasmid and primers used in this study are shown in
Table 1. Derivatives of ethanologenic E. coli KO11 were developed
by a combination of gene deletions and metabolic evolution to
optimize the strains for ethanol production through the PP-P and
ED-P. Escherichia coli KO11 was used as the progenitor strain for
construction of the KO11 Δpgi and KO11 PPAL. KO11 PPAL is a
pgi, ack-pta and ldhA were made through a chromosomal gene
inactivation method using PCR products reported by Datsenko
and Wanner,14 using the primers pgiH1P1 and pgiH2P2 to delete
the pgi gene; ackptaH1P1 and ackptaH2P2 to delete ack-pta, and
ldhH1P1 and ldhH2P2 to delete ldh (Table 1). Each knockout was
confirmed by PCR with genomic DNA.
Growth medium and fermentation
Rich medium (Luria Bertani broth) was used for strain construc-
tion and selection and contained the following per liter: 10 g
tryptone, 5 g yeast extract and 5 g sodium chloride. Antibi-
otics were included in appropriate concentrations (40 mg L−1 of
chloramphenicol, 100 and 50 mg L−1 of ampicillin, 40 mg L−1 of
kanamycin).
Batch fermentations were performed with minimal M9
medium15 supplemented with 40 g L−1 glucose (pH adjusted
to 7.0 with 2 N KOH). It contained the following per liter: 6 g
Na2 HPO4 , 3 g KH2 PO4, 1 g NH4 Cl and 0.5 g NaCl. The following
components were sterilized by filtration before being added (per
liter of final medium): 2 mL of 1 mol L−1 MgSO4 . 7H2 O, 1 mL of
0.1 mol L−1 CaCl2 , 1 mL of 1 mg mL−1 thiamin. HCl. Cultures were
grown in 350 mL fleaker mini-fermenters16 containing 200 mL
medium and 40 mg L−1 of chloramphenicol, under non-aerated
conditions, at 35 ∘ C, pH 7 (with 2 N KOH automatic additions) and
100 rpm. All fermentations were carried out in duplicate; averages
and standard errors are shown in plots and tables.
Adaptive evolution
Evolution of KO11 Δpgi was conducted in 200 mL of M9
minimal medium supplemented with 40 g L−1 of glucose in
mini-fermenters under non-aerated conditions as the batch cul-
tures. At the start of evolution, KO11 Δpgi was grown overnight
in 200 mL of M9 medium with 40 g L−1 of glucose in 500 mL Erlen-
meyer flasks at 35 ∘ C and 120 rpm, and the cultures were then
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homoethanologenic mutant derived from KO11 Δpgi. Deletions of

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 www.soci.org G Huerta-Beristain et al.

Analytical methods
Table 2. Enzyme specific activities (IU per mgPROT ) for strains KO11,
KO11 Δpgi and KO11 E35. The cell extracts were obtained from Growth was spectrophotometrically determined as optical density
cultures under non-aerated conditions at 600 nm (DU-70, Beckman Instruments, Inc. Fullerton, CA, USA)
and converted to dry cell weight (DCW) per liter using a calibra-
KO11 KO11 Δpgi KO11 E35 tion curve (1 OD600nm = 0.37 gDCW L−1 ). Samples were centrifuged,
ZWF 0.17 ± 0.03 0.32 ± 0.02 2.92 ± 0.01 and cell-free culture broth was frozen until analysis. Acetic, formic,
PGI 3.6 ± 0.3 0.014 ± 0.002 0.009 ± 0.001 lactic, and pyruvic acids were measured by HPLC analysis (Waters,
ED-P 0.08 ± 001 0.021 ± 0.01 0.26 ± 0.01 Millipore Co., Milford, MA, USA), using an Aminex HPX-87H ion
PFL 0.06 ± 0.01 0.05 ± 0.003 0.08 ± 0.003 exclusion column (300 × 7.8 mm; Bio-Rad Laboratories, Hercules,
LDH 0.68 ± 0.01 0.06 ± 0.002 0.51 ± 0.006 CA, USA), with 5.0 mmol L−1 H2 SO4 aqueous solution as the mobile
PDC 0.43 ± 0.03 0.27 ± 0.01 0.65 ± 0.03 phase (0.5 mL min−1 ) at 50 ∘ C and a photodiode array detector at
ADH 0.13 ± 0.03 0.11 ± 0.01 0.22 ± 0.04 210 nm (Model 996, Waters, Millipore Co., Milford, MA, USA). Glu-
cose was measured with an enzymatic analyzer (Model 2700, Yel-
low Spring Instruments, Co. Inc. Ohio, USA). Ethanol was analyzed
by gas chromatography using n-butanol as an internal standard
transferred to mini-fermenters for adaptive evolution at an initial (6850 Series GC System, Agilent Wilmington, DE, USA).
optical density at 600 nm (OD600nm ) of 0.01. In the mini-fermenters,
cells were grown until reaching an OD600nm of 1.0, then diluted by
serial transfer into fresh medium. Each transfer into fresh medium Preparation of cells extracts and measurement of enzymatic
was started at an OD600nm of 0.01. This process, in batch growth activities
and serial dilution, was conducted for 60 days for a total of 35 For the enzymatic assays, E. coli cells were cultivated in M9 mini-
transfers to fresh medium, accounting a total of 240 generations. mal medium supplemented with glucose in 200 mL batch cultures
The strain from the 35th transfer, at 60 days of evolution, was in mini-fermenters under non-aerated conditions. Cells from the
cultured until a stable growth rate was achieved for 10 additional exponential growth phase (6 h of fermentation for KO11 or 12 h
days, accounting for a total 1680 h of evolution). The selected of fermentation for other strains) were harvested by centrifuga-
evolved mutant was called KO11 E35. tion (10 000 × g, 10 min, 4 ∘ C), washed twice with 100 mmol L−1

Figure 1. Carbon metabolism central pathways in KO11 Δpgi and KO11 PPAL (Δpgi, Δpta, Δack and Δldh). Abbreviations: G-6-P, glucose-6-phosphate;
6-PGL, 6-phosphogluconatelactone; CO2, carbon dioxide; NADPH/NADP+ , reduced/oxidized nicotinamide adenine dinucleotide phosphate; NADH/NAD+ ,
reduced/oxidized nicotinamide adenine dinucleotide; ADP, adenosine diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate;
RYBU-5-P, rybulose-5-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; F-6-P, fructose-6-phosphate; F-1,6-DP, fructose-1-6-diphosphate; G-3-P,
glyceraldehyde-3-phosphate; ACALD, acetaldehyde; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; PTS, phosphoenolpyruvate carbohy-
drate phosphotransferase system. Enzymes: GLK, glucokinase; PGI, phosphoglucose isomerase; ZWF, glucose-6-phosphate dehydrogenase; GDH, glu-
conate dehydrogenase; EDD, Entner–Doudoroff dehydratase; EDA, Entner–Doudoroff aldolase; PFL, pyruvate formate lyase; PTA, phosphotransacetylase;
ACK, acetate kinase; LDH, lactate dehydrogenase; PDCZm and ADHBZm , pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis.
992

Dotted line indicates several biochemical reactions. Blocked pathways are indicated with an ‘X’.

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Engineering of E-D and PP pathways in KO11 pgi- knockout
0.7
0.6
0.5
Biomass (g/L)
0.4
0.3
0.2
0.1
0.0
0 240 480 720
Time (h)
Figure 2. Comparison of growth during 35 serial transfers of KO11 Δpgi to obtain KO11 E35. The strain KO11 Δpgi was sequentially transferred in M9
minimal medium supplemented with glucose (40 g L−1 ).
Tris-HCl (pH 7.0) containing 20 mmol L−1 KCl, 5 mmol L−1 MnSO4 ,
2 mmol L−1 DTT and 0.1 mmol L−1 EDTA, resuspended in the same
buffer and disrupted by sonication: four pulses of 15 s in a cold bath
(Ultrasonic Disrupter, Soniprep 150).17 The cell debris was removed
by centrifugation (10 000 × g, 10 min, 4 ∘ C), and the resulting crude
cell extracts were immediately used for measurement of enzymatic
activities or stored at –20 ∘ C.
Enzyme activities were determined spectrophotometrically
(BioMate 5, Thermo Spectronic, NY, USA) by coupling reac-
tions to NADH+ reduction (extinction coefficient of 6.22 cm−1
mM−1 ) at 340 nm and 30 ∘ C. One international unit (IU) of spe-
cific enzyme activity was defined as the amount of enzyme
required to convert 1 mmol of substrate into the specific prod-
uct per minute per milligram of protein. Protein concentrations
were measured using the Bradford assay. All measurements
were performed in triplicate. Enzyme activities were determined
by methods previously reported17,18 for phosphoglucose iso-
merase (PGI), glucose-6-phosphate dehydrogenase (ZWF), lactate
dehydrogenase (LDH), pyruvate formate-lyase (PFL), and by the
methods reported by Hoppner and Doelle19 for enzymes of the
Entner–Doudoroff pathway (ED-P). Methods described by Mar-
tinez et al.20 were used for pyruvate decarboxylase (PDC) and
alcohol dehydrogenase (ADH). All enzymatic activity and protein
measurements were performed in triplicate.
Kinetic parameter calculations
Specific growth rates were calculated as the slopes of linear
regression equations describing the logarithm of gDCW L−1 vs time
during the exponential growth phase. Specific rates of glucose
consumption (qS ), ethanol production (qEt-OH ), and organic acid
formation (acetate, formate and lactate) were estimated during
the exponential phase. These rates were converted to product flux
as reported elsewhere.21
RESULTS
Construction of the strain with blocked glycolysis, E. coli
KO11 𝚫pgi, and cell growth characterization
In this study, the pgi gene encoding phosphoglucose isomerase
in KO11 was deleted using methods reported by Datsenko and
Wanner,14 using the primers pgiH1P1 and pgiH2P2 (Table 1). The
pgi knockout strain was confirmed by PCR analysis. The phenotype
J Chem Technol Biotechnol 2017; 92: 990–996 © 2016 Society of Chemical Industry
www.soci.org
960 1200 1440 1680
was verified by measuring PGI specific activity (Table 2). In this
strain, KO11 Δpgi, the glycolytic pathway was blocked, so all of
the carbon flux from glucose-6-P was channeled through the PP-P
and ED-P (Fig. 1). Because the strain had limited ability to channel
carbon flux through the pentose phosphate pathway, KO11 Δpgi
grew slowly in glucose–mineral medium under non-aerated con-
ditions. Glucose-6-phosphate (G6P) could probably accumulate in
this strain, which would impair glucose consumption, perhaps due
to ptsG degradation by RNAse E, as reported by Morita et al.9
Adaptive evolution of KO11 𝚫pgi and characterization of the
evolved mutant
The adaptive evolution of KO11 Δpgi was carried out by sequen-
tial transfer into M9 minimal medium with 4% glucose, using
pH-controlled mini-fermenters under non-aerated conditions. The
slight increases in growth rate and cell yield, after the initial trans-
fers, allowed us to increase the transfer frequency until 35 transfers
were conducted (Fig. 2). From the pool of strains obtained in trans-
fer number 35, a colony with increased growth on plates under
anaerobic conditions was selected. The isolated strain was named
KO11 E35 (evolved KO11 Δpgi).
When the KO11 strain was grown under non-aerated conditions,
it produced ethanol, lactate, acetate and formate (Fig. 3(A)). As
the KO11 Δpgi evolved, the growth rate gradually increased,
and we assume that the increase can be ascribed to increases
in key carbon metabolic fluxes.22 Therefore, several enzymatic
activities in the PP-P and ED-P were measured on samples of
cell extracts grown under non-aerated conditions. As shown in
Table 2, increased levels of the ZWF made it possible to channel
the G6P through the PP-P and ED-P and to increase the flux to
ethanol production. The specific growth, glucose consumption
and ethanol production rates in KO11 E35 were only 26%, 39%
ans 32%, respectively, relative to the values obtained with KO11
under similar conditions (Table 3).
Comparison of specific activities
The main differences between KO11 E35 and KO11 were that the
PFL, PDC and ADH activities were slightly higher in KO11 E35. In
contrast, the ZWF and E-D enzyme activities were 17 and 2-fold
higher, respectively, in KO11 E35 (Table 2). In accordance with
previous reports,23 as a result of an increase in enzymatic activities,
flux redistribution and/or up regulation in existing pathways was
993
observed in response to the adaptive evolution process.
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 www.soci.org G Huerta-Beristain et al.

(A) increased, and the lactate flux increased 3-fold, probably due to
the increase in pyruvate availability as demonstrated by Tarmy and

Glucose, Ethanol, Acetate,

Kaplan.24 Furthermore, the drive to lactate flux is also due to the

Formate, Lactate (g/L)

need to resolve the NADH redox balance.22
Biomass (g/L)

Construction of a homoethanologenic strain and increase

PDC and ADH activities in KO11 PPAL
When the KO11 E35 strain was grown in minimal medium under
non-aerated conditions, it produced ethanol, lactate, acetate and
formate (Fig. 3C). The genes encoding, acetate kinase, phos-
photransacetylase and lactate dehydrogenase were deleted from
KO11 E35 to eliminate these losses of carbon. The ack, pta and
ldh genes were deleted by the method reported by Datsenko
(B) and Wanner,14 using the primers ackptaH1P1 and ackptaH2P2 to
delete ack and pta genes, and the primers ldhH1P1 and ldhH2P2 to
delete ldh gene (Table 1): the resultant strain was called KO11 PPAL.
When the KO11 PPAL strain was grown under non-aerated con-
ditions, it produced ethanol as the only fermentation product, but
the productivity was low compared with KO11 (Fig. 4 and Table 3).
To determine the feasibility of increasing the ethanol yield and
specific rate of production in KO11 PPAL growing in minimal media
supplemented with glucose, we increased the PDC and ADH activ-
ities, which could potentially be limiting the ethanol flux. KO11
PPAL was transformed with the plasmid pLOI510 containing genes
encoding pyruvate decarboxylase (PDCZm ) and alcohol dehydro-
(C)
C)
Glucose, Ethanol, Acetate,

genase (ADHZm ) from Z. mobilis. These enzymes channel pyruvate

Formate, Lactate (g/L)

to ethanol and CO2 .

PDCZm and ADHZm amplification increased the glucose and
Biomass (g/L)

ethanol fluxes during the exponential phase of the glucose cul-

tures relative to KO11 PPAL (Table 4 and Fig. 4). It is worth men-
tioning that relative to the parental strain KO11, the specific rates
of growth and glucose consumption were 85% and 22% lower
in KO11 PPAL/pLOI510, but the specific ethanol production rate
was similar (only 5.1% lower in KO11 PPAL/pLOI510) (Table 3).
The ethanol yield on biomass (YEtOH/X in gEtOH /gDCW ) was 4.44 for
KO11 and 24.38 for KO11 PPAL / pLOI510, indicating a 5.5-fold
better efficiency of KO11 PPAL / pLOI510 to channel glucose
to ethanol.

Figure 3. Kinetics of growth, glucose consumption and production of

ethanol and organic acids in minimal media (4% glucose) by strains (A)
KO11; (B) KO11 Δpgi; and (C) KO11 E35. Symbols: , Biomass; , Glucose;
, Ethanol; , Acetate; , Lactate; , Formate.
Metabolic flux in KO11 E35 and KO11 𝚫pgi in minimal
medium with glucose under non-aerated conditions
Specific growth, glucose consumption and ethanol production
rates in KO11 E35 were low compared with KO11 (Table 3). E35 also
showed decreased glucose consumption and ethanol production
(Fig. 3C). However, the fluxes to acetate and formate were slightly
DISCUSSION
Under non-aerated conditions the ethanologenic E. coli strain
KO11 produces ethanol efficiently by using glucose as carbon
source through the glycolytic pathway with a yield of 2 mol of
ethanol per mol of glucose.12,23 It also produces acetate (with
concomitant production of ATP), formate and lactate.21 In this
study, we found that strain KO11 Δpgi is not able to grow under
non-aerated conditions in minimal medium with glucose (Fig. 3B),
due to the lack of the enzyme phosphoglucose isomerase, which
channels glucose-6-phosphate to the EMP-P. The absence of

Table 3. Kinetic and stoichiometric parameters obtained with the E. coli strains KO11 E35, KO11 PPAL, KO11 PPAL/pLOI510 and the parental strain
KO11 growing on glucose

KO11 KO11 E35 KO11 PPAL KO11 PPAL/pLOI510

𝜇 (h−1 ) 0.42 ± 0.0007 0.11 ± 0.01 0.11 ± 0.001 0.065 ± 0.005

YX/Glc (gDCW /gGlc ) 0.081 ± 0.01 0.022 ± 0.001 0.066 ± 0.007 0.016 ± 0.005
YEtOH/Glc (gEtOH /gGlc ) 0.36 ± 0.02 0.44 ± 0.05 0.37 ± 0.009 0.39 ± 0.02
qGlc (gGlc /gDCW .h) 5.2 ± 0.6 2.05 ± 0.07 1.54 ± 0.2 4.04 ± 0.03
qEtOH (gEtOH /gDCW .h)) 1.17 ± 0.1 0.38 ± 0.03 0.53 ± 0.01 1.11 ± 0.01
994

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Engineering of E-D and PP pathways in KO11 pgi- knockout www.soci.org

(A) the cell. After adaptive processes other Δpgi mutants resolve

Glucose, Ethanol, Acetate,

0.8 40 this issue through mutations in key genes, such as rpoS and the
transhydrogenases udhA and pntAB, that suppress the bacte-
rial stress response and probably reduce the redox imbalance,

Formate (g/L)
0.6 30
respectively.22
Biomass (g/L)

Previous works involving adaptive evolution of E. coli pgi mutants

0.4 20 under aerobic conditions reported changes occurring in 7 and 35
genes in two different strains.23 Thus, the pgi mutants obtained
under different evolution strategies have evolved to increase
0.2 10 the growth rate using different strategies. KO11 Δpgi regained
the ability to grow in glucose-supplemented minimal medium
0.0 0
under non-aerated conditions by using the PP-P and ED-P for

Glucose, Ethanol, Acetate,

(B) 0.8 0 24 48 72 96 12 040 glucose catabolism to ethanol and organic acids. We designated
the evolved mutant strain KO11 E35. Our results showed that
the activities of glucose-6-phosphate dehydrogenase (ZWF) and

Formate (g/L)
0.6 30
the two enzymes from the Entner–Doudoroff pathway increased
Biomass (g/L)

17-fold and 2-fold, respectively, in KO11 E35 compared with KO11.

0.4
0.2
0.0
0 24 48 72 96
Time (h)
Figure 4. Kinetics of growth, glucose consumption and production of
ethanol, acetate and formate in minimal media (4% glucose) by strains: (A)
KO11 PPAL; and (B) KO11 PPAL/pLOI510. Symbols: , Biomass; , Glucose;
, Ethanol; , Acetate; , Lactate; , Formate.
PGI blocks the glycolytic pathway, redirecting all carbon flux
through the PP-P. After the evolution process of KO11 Δpgi, in
the resulting strain the specific growth rate was only 26% of
that of KO11. Also this strain has a 39% drop in specific glucose
consumption rate, compared with KO11; hence, overall glucose
consumption was restricting growth rate as well in KO11 E35.
This strategy can increase the flux of enzymes that catalyze alter-
native reactions for the metabolite, which can be accumulated
after gene deletion or either contributing to the activation of
silent genes or activating otherwise down-regulated pathways.23
The improved growth rate obtained in KO11 E35 is probably a
consequence of increases in the carbon flux for metabolic inter-
mediates synthesis, which are necessary for cellular biosynthesis
and energy production. Improved growth rate in KO11 E35 could
also be due to improved redox balance, given that fermentative
by-products are increased. Furthermore, it has been reported
that E. coli Δpgi strains force the glycolytic flux through the
pentose phosphate pathway and creates a redox imbalance in
20 Nevertheless, the flux to ethanol production was low in KO11
E35. In contrast, the carbon flux to organic acids was increased,
probably due to the lower levels of PDC activity, which lim-
10 ited the ethanol flux.21 In addition, the accumulation of intra-
cellular pyruvate could induce increases in LDH activity, which
0 would increase carbon flux to lactate.24 The elimination of ack,
120 pta and ldh (encoding acetate kinase, phosphotransacetylase
and lactate dehydrogenase, respectively) in KO11 E35 allowed
us to obtain a homoethanologenic strain named KO11 PPAL.
To overcome the limitation of PDC, the pdc and adhB genes
from Z. mobilis (pLOI510 plasmid) were overexpressed in E. coli
KO11 PPAL. Increased expression of pyruvate decarboxylase and
alcohol dehydrogenase in KO11 PPAL, and the elimination of
other fermentative pathways, made it possible to obtain specific
ethanol formation rates similar to those found in KO11; but with
a lower specific growth rate, half of the cell mass, no carbon
flux to acetate, formate or lactate and a 5.5-fold better YEtOH/X
of KO11 PPAL/pLOI510. Thus, most of the flux was channeled
through the PP-P and ED-P for glucose metabolism and ethanol
production.
CONCLUSION
Adaptive evolution restored the ability of E. coli KO11 Δpgi to
grow in minimal medium supplemented with glucose under
non-aerated conditions. Due to increased ZWF specific activity, the
evolved strain, KO11 E35, had increased levels of PP-P, intermedi-
ates that are necessary for the biosynthesis of plasmid DNA and
encoded proteins for the cogeneration and recycling of NADPH
and NADH. Furthermore, the elimination of fermentative pathways
and the increases of PDC and ADH II from Z. mobilis improved the

Table 4. Carbon flux distribution in E. coli strains KO11 PPAL, KO11 PPAL/pLOI510 and the parental strain KO11 obtained at the exponential phase
of glucose fermentations

Product fluxa

Strain Consumed glucose Ethanol CO2 Acetate Lactate Formate Biomass

KO11 173 ± 22 51 ± 5 25.5 ± 2.5 25 ± 0 10.3 ± 0 18.3 ± 0 16.8 ± 0.03

KO11 PPAL 51 ± 7 23.2 ± 0.7 11.6 ± 0.3 3.8 ± 0 0 0.2 ± 0 4.4 ± 0.04
KO11 PPAL/ pLOI510 135 ± 29 48.4 ± 0.8 24.2 ± 0.4 1.1 ± 0.06 0 0±0 2.6 ± 0.2
a All values are in mmolC /gDCW .h. An approximation for the CO2 flux was estimated from a stoichiometric balance of the ethanol flux.
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 www.soci.org
ethanol flux to levels similar to those obtained with the ethanolo-
genic strain KO11, but using the PP and E-D pathways instead of
the EM-P for glucose catabolism. These results demonstrate that
it is possible to obtain the same carbon flux by using the PP-P and
the ED-P as alternative routes for the EMP-P for glucose catabolism
under non-aerated conditions.
ACKNOWLEDGEMENTS
Strains KO11 and plasmid pLOI510 were kindly provided by Dr
Lonnie O. Ingram (University of Florida, USA). We thank Arturo
Ocádiz Ramírez, Juan Manuel Hurtado Ramírez, Martín Patiño Vera
and Mario Trejo Loyo for technical assistance. This work was sup-
ported by the Mexican Council of Science and Technology (CONA-
CyT) through the Bioenergy Thematic Network (‘Red Mexicana
de Bioenergía’), grant 260457 and FONCICYT ERANet-LAC Grant
C0013-248192. G.H-B. held a scholarship from CONACyT.
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