Bioengineered

ISSN: 2165-5979 (Print) 2165-5987 (Online) Journal homepage: http://www.tandfonline.com/loi/kbie20

Engineering the glycolytic pathway: A potential

approach for improvement of biocatalyst
performance

Toru Jojima & Masayuki Inui

To cite this article: Toru Jojima & Masayuki Inui (2015) Engineering the glycolytic pathway: A
potential approach for improvement of biocatalyst performance, Bioengineered, 6:6, 328-334,
DOI: 10.1080/21655979.2015.1111493

To link to this article: http://dx.doi.org/10.1080/21655979.2015.1111493

Accepted author version posted online: 29

Oct 2015.
Published online: 29 Oct 2015.

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REVIEW
Bioengineered 6:6, 328--334; November/December 2015; © 2015 Taylor & Francis Group, LLC

Engineering the glycolytic pathway: A potential

approach for improvement of biocatalyst
performance
Toru Jojima and Masayuki Inui*
Research Institute of Innovative Technology for the Earth; Kizugawa, Kyoto, Japan

Keywords: biochemicals, biofuels, corynebacterium glutamicum, fermentation, glycolytic pathway, metabolic engineering
Abbreviations: ADH, alcohol dehydrogenase; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; ED, Entner-Doudor-
off; EMP, Embden-Meyerhof-Parnas; ENO, enolase; FBP, fructose 1,6-bisphosphate aldolase; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; GLK, glucokinase; GPI, glucose 6-phosphate isomerase; PDC, pyruvate decarboxylase; PFK, 6-phosphofructokinase;
PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; TPI, triosephosphate isomerase.

The glycolytic pathway is a main driving force in the

fermentation process as it produces energy, cell component
precursors, and fermentation products. Given its importance,
the glycolytic pathway can be considered as an attractive
target for the metabolic engineering of industrial
microorganisms. However, many attempts to enhance
glycolytic flux, by overexpressing homologous or
heterologous genes encoding glycolytic enzymes, have been
unsuccessful. In contrast, significant enhancement in
glycolytic flux has been observed in studies with bacteria,
specifically, Corynebacterium glutamicum. Although there
has been a recent increase in the number of successful
applications of this technology, little is known about the
mechanisms leading to the enhancement of glycolytic flux.
To explore the rational applications of glycolytic pathway
engineering in biocatalyst development, this review
summarizes recent successful studies as well as past attempts.
engineering. This is likely because of the many unsuccessful
attempts at enhancing the rate of sugar uptake.6-9 In one such
study aimed toward improving ethanol fermentation in Saccharo-
myces cerevisiae, it was demonstrated that overexpression of the
genes encoding for glycolytic enzymes resulted in no positive
effects on ethanol productivity.7,10 In contrast, recent studies in
glycolytic pathway engineering have demonstrated significant
improvements in sugar consumption. While the current strategy
in metabolic engineering is aimed at redirecting carbon flow
toward a desired compound, the strategy in glycolytic pathway
engineering is to enhance the rate of sugar uptake, which directly
improves productivity of microbial catalysts.
In this review, we describe past and recent attempts in meta-
bolic engineering, showing contrasting results regarding sugar
metabolism. Subsequently, we discuss the potential factors con-
tributing to the different results. However, the regulation mecha-
nism of sugar metabolism is beyond the scope of this review.

Introduction
Over the past decades, the development of various metabolic
engineering technologies has facilitated the expansion of the rep-
ertoire of chemical compounds produced by microbial fermenta-
tion.1,2 The basic strategy of metabolic engineering focuses
largely on engineering the latter part of the metabolic pathway,
specifically, the key enzymes that are directly involved in its regu-
lation. These undesirable regulatory mechanisms, including feed-
back inhibition and transcriptional repression, can be
circumvented by screening for feedback-resistant enzymes and
simply gene overexpression. In some cases, such attempts have
successfully redirected the carbon flux toward the chemicals of
interest.3-5 Despite its central role in sugar metabolism, the glyco-
lytic pathway is hardly recognized as a target in metabolic
*Correspondence to: Masayuki Inui; Email: mmg-lab@rite.or.jp
Submitted: 07/29/2015; Revised: 10/15/2015; Accepted: 10/16/2015
http://dx.doi.org/10.1080/21655979.2015.1111493
Successful Studies to Enhance the Rate of Sugar
Uptake by Overexpression of Glycolytic Genes
Engineering of the glycolytic pathway has been performed
mainly in industrial microorganisms that have established techni-
ques for genetic modification, including Escherichia coli, S. cerevi-
siae, Zymomonas mobilis, and Corynebacterium glutamicum. S.
cerevisiae and C. glutamicum possess the Embden-Meyerhof-Par-
nas (EMP) pathway for sugar metabolism, whereas Z. mobilis
lacks the gene encoding 6-phosphofructokinase (PFK), an essen-
tial enzyme in the EMP pathway, and therefore instead metabo-
lizes sugars via the Entner-Doudoroff (ED) pathway (Fig. 1). E.
coli possesses both pathways. The EMP pathway is the main
route for sugar metabolism in E. coli, whereas sugar acids are
metabolized via the ED pathway. In textbooks, 6-phosphofructo-
kinase (PFK) and pyruvate kinase (PYK) are often described as
“key regulatory enzymes” in glycolysis since, under physiological
conditions, they catalyze irreversible reactions and their activities

328 Bioengineered Volume 6 Issue 6

 acid, but is unable to grow dur-
ing these conversion reac-
tion.12,13 The inherent abilities
of C. glutamicum has enabled the
development of a novel biopro-
cess which decouples microbial
growth-phase from production-
phase. A wide range of commod-
ity chemicals including organic
acids (D-lactate,14 succinate,15)
amino acids (alanine.16,17 and L-
valine,18,19) and fuels (etha-
nol.20,21 and isobutanol.22) can
be produced by this growth-
arrested bioprocess.23
Successful engineering of the
glycolytic pathway was achieved
in the studies with C. glutami-
cum. Overexpression of some
genes encoding glycolytic
enzymes in oxygen-deprived C.
glutamicum enhances conversion
reaction rates (Table 1). In the
first attempt aimed at improving
alanine productivity, the gapA
gene encoding glyceraldehyde 3-
phosphate dehydrogenase
(GAPDH) was selected as a tar-
get. GAPDH was chosen as it
was speculated that under oxygen
deprivation inhibition of
GAPDH by high intracellular
NADH/NADC ratio suppresses
glucose consumption.16 The
wild-type strain of C. glutamicum
produces small amounts of ala-
nine from sugar under oxygen
deprivation, but overexpression
Figure 1. Glycolysis via the EMP and ED pathways. Names of enzymes are underlined. Abbreviations: 6PGD,
gluconate 6-phosphate dehydratase; 6PGL, 6-phosphogluconolactonase; ENO, enolase; FBA, fructose 1,6- of alanine dehydrogenase from
bisphosphate aldolase; G6PDH, glucose 6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate Lysinibacillus sphaericus results in
dehydrogenase; GLK, glucokinase; GPI, glucose 6-phosphate isomerase; KDPGA, 2-keto-3-deoxy-6-phospho- hyperproduction of alanine by C.
gluconate aldolase; PFK, 6-phosphofructokinase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate glutamicum.16 When GAPDH
mutase; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system; PYK, pyruvate kinase; TPI,
activity was increased in the ala-
triosephosphate isomerase; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; KDPG,
2-keto-3-deoxy-6-phosphogluconate. nine-producing strain of C. glu-
tamicum, alanine productivity
was enhanced by 2.7-fold com-
pared to that of the parental
are controlled allosterically by ATP and ADP. As a result, PFK strain. Further investigations demonstrated that stepwise overex-
and PYK become primary targets for engineering. pression of the genes encoding glucose 6-phosphate isomerase
(GPI), PFK, and PYK successively improved alanine productivity
Studies with C glutamicum and enhanced glucose consumption.17 This metabolic engineer-
C. glutamicum is a gram-positive bacterium used for industrial ing strategy also improved productivity of other engineered
production of amino acids such as L-glutamic acid and L-lysine.11 strains to produce ethanol,21 L-valine,19 and isobutanol.22
Normally, this microbe is cultivated aerobically for amino acid (Table 1)
production. Under anaerobic conditions, C. glutamicum con- These investigations prompted the question: which enzymes
sumes sugars to produce lactic acid, succinic acid, and acetic in the glycolytic pathway are able to enhance glucose

www.tandfonline.com Bioengineered 329

Table 1. Improvement of productivity by engineering of the glycolytic pathway.
Organism Overproduced enzyme Product
C. glutamicum GAPDH Alanine
GPI, PFK, GAPDH, PYK Alanine
GPI, PFK, TPI, GAPDH, PYK L-Valine
GPI, PFK, GAPDH, PYK Isobutanol
GLK D-Lactate
GAPDH D-Lactate
GLK, GAPDH, PFK, FBA, TPI D-Lactate
GPI, PFK, TPI, GAPDH, PYK Ethanol
E. coli GAPDH 1-Butanol
PFK, PYK Ethanol
L. lactis PFK Lactate
Z. mobilis G6PDH Ethanol
GLK Ethanol
S. cerevisiae PFK Ethanol
*Relative productivity compared to that of control strain.
Abbreviations are listed in Fig. 1.
consumption in C. glutamicum? To answer this question, Tsuge
et al.24 examined the effects of overexpressing each of genes cod-
ing for the glycolytic enzymes on D-lactate productivity. A
D-lactate-producing strain was developed by overexpression of
D-lactate dehydrogenase from Lactobacillus delbrueckii and inacti-
vation of endogenous L-lactate dehydrogenase. The study showed
that increases in the activities of glucokinase (GLK), GAPDH,
PFK, fructose 1,6-bisphosphate aldolase (FBP), and triose phos-
phate isomerase (TPI) enhanced glucose consumption and
D-lactate productivity when a sufficient amount of D-lactate
dehydrogenase activity was present. Interestingly, all these
enzymes catalyze the reactions upstream of the EMP pathway.
Overexpression of the glycolytic genes also affects end-product
yields. The yield of alanine was shown to increase with increasing
volumetric productivity by overexpression of the glycolytic
genes.17 Metabolic flux via the EMP pathway increases by overex-
pression of glycolytic genes, whereas metabolic flux to the by-
products is unchanged. The net result is that the metabolic flux
toward alanine is relatively increased and the alanine yield is
improved.17 While this model explains the decreased yields of
acetate and succinate, the major by-products of the growth-
arrested bioprocess, it cannot be applied to the production of
dihydroxyacetone (DHA) and glycerol. In the D-lactate-produc-
ing C. glutamicum, overexpression of glk significantly enhances
glucose consumption; however, glycerol and DHA production
increases at least 5-fold.25 This observation suggests that the over-
flow in metabolism is caused by differences in the metabolic flux
upstream and downstream of the EMP pathway.26-28 Co-overex-
pression of glk and gapA reverts the DHA and glycerol yields
back to levels of the non-glk-overexpressing strain. Under oxygen
deprivation, glycerol is produced in C. glutamicum via DHA,
from the glycolytic intermediate dihydroxyacetone phosphate
(DHAP), by DHAP phosphatase HdpA and butanediol dehy-
drogenase.27,28 The model mentioned above assumes that the
metabolic flux toward the by-products remains unchanged even
when the concentrations of the intracellular precursors of succi-
nate and acetate increase. However, the metabolic fluxes to DHA
330 Bioengineered
Productivity improvement (fold)* Reference
2.7 16
6.4 17
9 19
1.8 22
1.8 24
1.5 24
2.5 25
2.1 21
1.5 45
1.4 31
2.2 29
1.1 34
1.1 34
1.3 32
and glycerol should increase with increasing concentrations of
the precursors DHAP and DHA, the substrates of HdpA and
ButA, respectively, because the Michaelis constant Km of HdpA
and ButA for their substrates are very high.27,28 Consequently,
the production of DHA and glycerol are increased when overex-
pression of glycolytic enzymes enhances the intracellular concen-
trations of DHAP.
Studies with other microorganisms
Overexpression of the PFK gene in Lactococcus lactis LM0230
enhances the rates of glucose consumption and lactate produc-
tion in proportion to PFK activity.29 A recombinant L. lactis
strain showing a 2-fold increase in PFK activity by overexpression
of pfkA and its truncated gene fragment (pfk13) from Aspergillus
niger exhibited a 1.5-fold increase in the maximum rate of glu-
cose consumption under glucostat culture conditions. Interest-
ingly, the results of Koebmann et al.30 were in stark contrast to
those of this aforementioned study. They constructed recombi-
nant L. lactis strains with various levels of PFK activities, showing
1.4- to 11-fold differences compared to the wild-type level, and
demonstrated that increased PFK activity has no effect on glucose
consumption in L. lactis. These controversial results will be fur-
ther discussed in the next section, though the results obtained in
the former study clearly indicate that improvement of glucose
uptake rate by engineering of the glycolytic pathway is not lim-
ited in C. glutamicum.
Increased enzyme activities of PFK and PYK in ethanoL-pro-
ducing E. coli KO20 moderately enhances glucose consumption
under specific conditions.31 Resting cells of the recombinant strain
with simultaneous increased PFK and PYK activities exhibit 25%
higher glucose consumption and 35% higher ethanol production
compared to the parental strain when the cells are harvested from
aerobic cultures. However, glycolytic flux is not enhanced when
the recombinant strain is cultured anaerobically. Because the glu-
cose consumption of E. coli KO20 is maximized under anaerobic
conditions, the authors concluded that glucose flux under submaxi-
mal (aerobic) conditions can be enhanced by overexpression of
Volume 6 Issue 6
glycolytic enzymes to the level observed under maximum (anaero-
bic) conditions. Similarly, immobilized S. cerevisiae cells with
increased PFK activity show 1.3-fold higher rate of glucose con-
sumption under aerobic conditions, but not under anaerobic con-
ditions.32 Both studies imply that the increased rate of glucose
consumption, induced by the overproduction of glycolytic
enzymes, does not exceed the maximum glucose consumption rate
of the organisms. This idea emphasizes the importance of the gly-
colytic pathway engineering for improving fermentation perfor-
mance of newly isolated microorganisms because optimal culture
conditions of those microorganisms are hardly established espe-
cially in the early phase of strain development.
Z. mobilis is well known as an efficient ethanol producer.33
This microorganism possesses the ED pathway for sugar metabo-
lism. The recombinant strain of Z. mobilis, displaying 20%
increase in glucose 6-phosphate dehydrogenase activity, shows
10% higher glycolytic flux. However, further increase in enzyme
activity shows no effect on glycolytic flux.34
Difference Between Success and Failure in
Glycolytic Pathway Engineering
In the last section, we described studies that were successful at
enhancing the rate of glucose consumption by overexpression of
the glycolytic gene(s). However, historically, there have been
many studies where overexpression of the glycolytic genes
resulted in no positive effects on glucose consumption. In the fol-
lowing sections, we summarize those studies and discuss the fac-
tors determining success and failure in glycolysis engineering.
Past studies showing no improvement in glucose uptake
Schaaff et al. constructed recombinant S. cerevisiae strains
overexpressing each of the genes encoding for the following 6 gly-
colytic enzymes: hexokinase, GPI, PFK, phosphoglycerate
mutase (PGM) or PYK, and an enzyme for ethanol fermentation,
pyruvate decarboxylase (PDC) or alcohol dehydrogenase
(ADH).10 Although enzyme activities were increased 3.7- to
13.9-fold compared to wild-type levels, overexpression of the
genes had no effect on the rate of ethanol production. The
authors speculated that simultaneous increase in several enzyme
activities would be required to enhance the glycolytic flux. The
study by Hauf et al. tested this hypothesis by simultaneously
overexpressing the genes encoding for the following 7 enzymes:
GAPDH, phosphoglycerate kinase (PGK), PGM, enolase
(ENO), PYK, PDC, and ADH. However, the results showed
that ethanol fermentation was almost equal in both recombinant
and wild-type strains and only a slight increase in initial ethanol
production rate was observed in the recombinant strain.7
The effects of overproduction of PFK, PGK, PYK, and sugar
transporter GalP in ethanoL-producing E. coli KO11 were inves-
tigated.35 GalP is a galactose:HC symporter that also shows glu-
cose uptake activity. Most of the recombinant strains showed
similar levels of glucose flux, but the strains overexpressing the
genes for PFK and PYK exhibited a 60% lower rate in glucose
consumption. This decrease in glucose consumption rate,
www.tandfonline.com Bioengineered
observed when glycolytic genes were overexpressed, was also
demonstrated in the study with Z. mobilis.8 This negative effect
on glucose metabolism could be caused by a protein burden
effect that is often observed during overproduction of proteins in
recombinant cells.
By using metabolic control analysis, Jensen and co-workers
investigated the role of glycolytic enzymes on the control of gly-
colytic flux.30,36-39 They developed a series of synthetic pro-
moters showing various expression levels and used those
promoters to construct L. lactis strains with multilevel activities
of glycolytic enzymes (PFK, PYK, TPI, GAPDH, ENO, PGM).
These studies showed that recombinant strains with higher activi-
ties of glycolytic enzymes exhibited similar levels of glucose con-
sumption to wild-type L. lactis. The authors concluded that none
of these enzymes influenced the glycolytic flux, and these
enzymes were therefore present in excess in L. lactis.
There has also been report of an unsuccessful study in oxygen
deprived C. glutamicum where the gapA gene was overexpressed
in succinate-producing strain.40 This study aimed at improving
succinate yield, which is normally limited by shortage of reducing
equivalents. To achieve this, the fdh gene encoding formate dehy-
drogenase from Mycobacterium vaccae was introduced into the C.
glutamicum genome and formate was added as a co-substrate to
provide additional NADH for succinate production. The authors
examined the effect of gapA overexpression in the fdh-harboring
strain. The succinate yield was improved in the fdh-harboring
strain in the presence of formate, but the strain enters a dormancy
phase for approximately 10h after the 3h reaction. The dormancy
phase disappears by overexpression of gapA. However, the glu-
cose consumption rate of the gapA-overexpressing strain was not
enhanced.
Is overexpression of glycolytic genes really effective?
Contrasting results described in the last sections make us hesi-
tate to conclude that overexpression of the glycolytic genes is an
effective means to enhance glycolytic flux. What is different
between successful and failed attempts? Two studies with PFK-
overproducing L. lactis conducted by different research groups
will be used as examples. Papagianni et al.29 demonstrated a 2-
fold increase in glucose consumption by overproduction of PFK,
whereas Jensen and coworkers showed that PFK has no control
on glycolytic flux,30 but they also found that a mutant strain hav-
ing 40% reduced PFK activity showed 34% decrease in glycolytic
flux compared to the wild-type strain.41 This suggests that the
glycolytic flux of L. lactis is sensitive to PFK activity. Moreover,
the strains and culture conditions were different between the
studies showing enhanced.29 and unchanged.30 glycolytic flux. If
these differences affect the protein level of PFK in the each strain,
it might provide an explanation for the contrasting results.
From the studies on S. cerevisiae described above, it appears that
the glycolytic enzyme level is high enough to drive glucose metabo-
lism in the wild-type strain. A recent kinetic model of S. cerevisiae
glycolysis demonstrated that control of the glycolytic pathway is
distributed among many enzyme reactions.42 There is a paradoxi-
cal example in S. cerevisiae where overexpression of PGM2, which
is involved in galactose metabolism, actually enhances galactose
331
uptake by 70%.43 Thus, overexpression of a glycolytic gene will
enhance metabolic flux when a cell faces a deficiency in an
enzyme. There is another possible factor leading to disappointed
results on strain development. The effect of protein burden might
be underestimated because of the difficulty to quantitatively evalu-
ate its effects. It is possible that the negative effects of the protein
burden, caused by overproduction of the glycolytic enzymes, will
mask positive effects on glycolytic flux. In the study by Hauf et al.,
activities of PYK and PGM were increased 10- to 20-fold in S. cer-
evisiae. The expression levels of glycolytic enzymes are generally
high, and according to published data,42 PYK and PGM in S. cere-
visiae were estimated to account for approximately 25 wt% among
the glycolytic enzymes. Increasing protein levels of PGM and PYK
by 10-fold, assuming that the protein levels of other glycolytic
enzymes are unchanged, results in an increase in the total amount
of glycolytic enzymes by approximately 3-fold compared to the
level of the wild-type strain. Such a large increase in protein con-
centrations might cause a protein burden.
Although the most successful results from glycolytic pathway
engineering have been observed from the studies in C. glutami-
cum under oxygen deprivation, under other conditions, no posi-
tive effects were found. For example, under oxygen-deprived
conditions, gapA overexpression in alanine-producing C. glutami-
cum enhanced the rate of glucose consumption. However, under
aerobic conditions, glucose consumption was almost the same as
that of the reference strain.16 GAPDH is not inhibited under aer-
obic conditions because in the presence of oxygen, the intracellu-
lar NADH/NADC ratio does not increase to levels that can
inhibit GAPDH. Given this, it can be understood that no
enhancement was observed in the study with succinate-producing
C. glutamicum.40 Due to redox imbalance (i.e., 2 mols of succi-
nate produced requires more reducing equivalents that are pro-
duced from one mole of glucose), the NADH/NADC ratio is
expected to remain low. Although no positive effects of gapA
overexpression on succinate production by C. glutamicum have
been reported, this does not necessarily mean that glycolytic engi-
neering is a futile approach for improving succinate productivity.
For the development of D-lactate-producing C. glutamicum
strains, all the genes encoding for glycolytic enzymes were exam-
ined to find one that had positive effects on glycolytic flux.24,25
This approach can be similarly applied in developing an
improved strain for succinate production.
On the basis of the above discussions, the effects of glycolytic
pathway engineering vary depending on the microbial species,
culture conditions, and concentrations of key intracellular com-
pounds, including NAD(P)H. To apply this metabolic engineer-
ing strategy, all the glycolytic enzymes should be explored to
improve the fermentation performance of the production strain
of interest. In addition, moderate levels of gene overexpression
appear to be sufficient for enhancing glycolytic flux.
Outlook
Engineering of the glycolytic pathway is a developing technol-
ogy, and its outcomes are currently not predictable prior to
332 Bioengineered
testing, since the degree of improvement of glucose uptake seems
to vary depending on the host species considered. For rational
modification of the glycolytic pathway, a remaining challenge is
to gain a detailed understanding of the mechanism of the tech-
nology in a model organism. An ideal model for further studies
would be with C. glutamicum under oxygen deprivation condi-
tions. Under these conditions, its growth is arrested and the
number of active pathways is limited, which simplifies the choice
of targets in future research. In contrast, since microorganisms
grow in normal fermentation processes, glucose consumption
must be influenced by complex mechanisms associating the regu-
lation of cell growth. Another reason to select C. glutamicum is
that this microbe is currently used in the biotechnology industry.
Consequently, knowledge from basic research can be directly
transferred to industrial applications.
In E. coli, ATP demand is known to control glycolytic flux.44
Significant increase in glycolytic flux is observed when the genes
encoding the F1 portion of (F1F0) HC-ATP synthase are overex-
pressed to deplete ATP. Since growth of C. glutamicum is
arrested under oxygen deprivation, under these conditions, ATP
demand is supposed to be low. Therefore, the combined effects
of increasing ATP demand and overexpression of the glycolytic
genes in C. glutamicum are of great interest. Takeno et al.46
developed a recombinant C. glutamicum strain to enhance L-
lysine production by replacing wild-type NAD-dependent
GAPDH with NADP-dependent GAPDH (GapN) from Strepto-
coccus mutans, because the supply of NADPH is important for
efficient L-lysine production. In this strain, NADPH is synthe-
sized through this modified EMP pathway and L-lysine produc-
tion was largely improved. There is another feature in the
modified EMP pathway where no ATP is formed in the engi-
neered pathway, whereas 2 mol of ATP is generated in the native
EMP pathway from 1 mol of glucose degradation. Since this
recombinant strain dose not grow on glucose as the sole carbon
source because of NADPH accumulation,47 it is not possible to
investigate the effect of reduced ATP formation on glycolytic
flux under culture conditions. However, the glycolytic flux can
be determined under growth-arrested conditions by using the
cells cultivated in appropriate conditions, which is yet another
advantage of using C. glutamicum in glycolytic engineering
research.
Another interesting approach for engineering of the glycolytic
pathway was explored by Benisch and Boles,48 who investigated
the possibility of introducing the ED pathway to S. cerevisiae,
which normally metabolizes glucose via the EMP pathway. The
ED pathway generates half the amount of ATP per glucose com-
pared to the EMP pathway, which results in reduced biomass for-
mation. In Z. mobilis, where the ED pathway is responsible for
glucose catabolism, the yield of ethanol is high due to low bio-
mass formation.33 The specific productivity of ethanol is also
higher in Z. mobilis than in S. cerevisiae, because Z. mobilis
requires a high flux to obtain sufficient ATP to support its
growth. Unfortunately, the ED pathway did not function in S.
cerevisiae due to the very low enzyme activity of bacterial 6-phos-
phogluconate dehydratase (PGDH), one of the key enzymes in
the ED pathway. The authors speculated that inefficient loading
Volume 6 Issue 6
of the iron-sulfur cluster to bacterial PGDH protein might have
caused the low enzyme activity, since expression of the PGDH
protein was detected in Western blot analysis after codon optimi-
zation. Development of a novel method for functional expression
of the bacterial enzyme is anticipated.
Increasing concerns regarding global climate change necessi-
tates a decrease in consumption of fossil resources. Consequently,
much attention has been focused on using alternative biorefinery
strategies. In this strategy, a range of bulk chemicals and fuels are
produced from renewable resources, especially lignocellulosic
biomass. Industrial production of cellulosic ethanol from ligno-
cellulosic biomass started in 2014 in the United States,49 but fur-
ther improvement of biocatalysts in yield, titer and productivity
would help expand the use of biorefineries. Thus, development
of technologies to enhance glycolytic flux will be one of key chal-
lenges in this research field.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was partially supported by New Energy and Indus-
trial Technology Development Organization (NEDO) of Japan
and the Ministry of Economy, Trade and Industry (Japan).

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