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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


engineering. This is likely because of the many unsuccessful
fermentation process as it produces energy, cell component
precursors, and fermentation products. Given its importance, attempts at enhancing the rate of sugar uptake.6-9 In one such
the glycolytic pathway can be considered as an attractive study aimed toward improving ethanol fermentation in Saccharo-
target for the metabolic engineering of industrial myces cerevisiae, it was demonstrated that overexpression of the
microorganisms. However, many attempts to enhance genes encoding for glycolytic enzymes resulted in no positive
glycolytic flux, by overexpressing homologous or effects on ethanol productivity.7,10 In contrast, recent studies in
heterologous genes encoding glycolytic enzymes, have been glycolytic pathway engineering have demonstrated significant
unsuccessful. In contrast, significant enhancement in improvements in sugar consumption. While the current strategy
glycolytic flux has been observed in studies with bacteria, in metabolic engineering is aimed at redirecting carbon flow
specifically, Corynebacterium glutamicum. Although there toward a desired compound, the strategy in glycolytic pathway
has been a recent increase in the number of successful
engineering is to enhance the rate of sugar uptake, which directly
applications of this technology, little is known about the
improves productivity of microbial catalysts.
mechanisms leading to the enhancement of glycolytic flux.
To explore the rational applications of glycolytic pathway In this review, we describe past and recent attempts in meta-
engineering in biocatalyst development, this review bolic engineering, showing contrasting results regarding sugar
summarizes recent successful studies as well as past attempts. 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
Successful Studies to Enhance the Rate of Sugar
Over the past decades, the development of various metabolic Uptake by Overexpression of Glycolytic Genes
engineering technologies has facilitated the expansion of the rep-
ertoire of chemical compounds produced by microbial fermenta- Engineering of the glycolytic pathway has been performed
tion.1,2 The basic strategy of metabolic engineering focuses mainly in industrial microorganisms that have established techni-
largely on engineering the latter part of the metabolic pathway, ques for genetic modification, including Escherichia coli, S. cerevi-
specifically, the key enzymes that are directly involved in its regu- siae, Zymomonas mobilis, and Corynebacterium glutamicum. S.
lation. These undesirable regulatory mechanisms, including feed- cerevisiae and C. glutamicum possess the Embden-Meyerhof-Par-
back inhibition and transcriptional repression, can be nas (EMP) pathway for sugar metabolism, whereas Z. mobilis
circumvented by screening for feedback-resistant enzymes and lacks the gene encoding 6-phosphofructokinase (PFK), an essen-
simply gene overexpression. In some cases, such attempts have tial enzyme in the EMP pathway, and therefore instead metabo-
successfully redirected the carbon flux toward the chemicals of lizes sugars via the Entner-Doudoroff (ED) pathway (Fig. 1). E.
interest.3-5 Despite its central role in sugar metabolism, the glyco- coli possesses both pathways. The EMP pathway is the main
lytic pathway is hardly recognized as a target in metabolic route for sugar metabolism in E. coli, whereas sugar acids are
metabolized via the ED pathway. In textbooks, 6-phosphofructo-
*Correspondence to: Masayuki Inui; Email: mmg-lab@rite.or.jp kinase (PFK) and pyruvate kinase (PYK) are often described as
Submitted: 07/29/2015; Revised: 10/15/2015; Accepted: 10/16/2015
http://dx.doi.org/10.1080/21655979.2015.1111493 “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 Productivity improvement (fold)* Reference

C. glutamicum GAPDH Alanine 2.7 16


GPI, PFK, GAPDH, PYK Alanine 6.4 17
GPI, PFK, TPI, GAPDH, PYK L-Valine 9 19
GPI, PFK, GAPDH, PYK Isobutanol 1.8 22
GLK D-Lactate 1.8 24
GAPDH D-Lactate 1.5 24
GLK, GAPDH, PFK, FBA, TPI D-Lactate 2.5 25
GPI, PFK, TPI, GAPDH, PYK Ethanol 2.1 21
E. coli GAPDH 1-Butanol 1.5 45
PFK, PYK Ethanol 1.4 31
L. lactis PFK Lactate 2.2 29
Z. mobilis G6PDH Ethanol 1.1 34
GLK Ethanol 1.1 34
S. cerevisiae PFK Ethanol 1.3 32

*Relative productivity compared to that of control strain.


Abbreviations are listed in Fig. 1.

consumption in C. glutamicum? To answer this question, Tsuge and glycerol should increase with increasing concentrations of
et al.24 examined the effects of overexpressing each of genes cod- the precursors DHAP and DHA, the substrates of HdpA and
ing for the glycolytic enzymes on D-lactate productivity. A ButA, respectively, because the Michaelis constant Km of HdpA
D-lactate-producing strain was developed by overexpression of and ButA for their substrates are very high.27,28 Consequently,
D-lactate dehydrogenase from Lactobacillus delbrueckii and inacti- the production of DHA and glycerol are increased when overex-
vation of endogenous L-lactate dehydrogenase. The study showed pression of glycolytic enzymes enhances the intracellular concen-
that increases in the activities of glucokinase (GLK), GAPDH, trations of DHAP.
PFK, fructose 1,6-bisphosphate aldolase (FBP), and triose phos-
phate isomerase (TPI) enhanced glucose consumption and Studies with other microorganisms
D-lactate productivity when a sufficient amount of D-lactate Overexpression of the PFK gene in Lactococcus lactis LM0230
dehydrogenase activity was present. Interestingly, all these enhances the rates of glucose consumption and lactate produc-
enzymes catalyze the reactions upstream of the EMP pathway. tion in proportion to PFK activity.29 A recombinant L. lactis
Overexpression of the glycolytic genes also affects end-product strain showing a 2-fold increase in PFK activity by overexpression
yields. The yield of alanine was shown to increase with increasing of pfkA and its truncated gene fragment (pfk13) from Aspergillus
volumetric productivity by overexpression of the glycolytic niger exhibited a 1.5-fold increase in the maximum rate of glu-
genes.17 Metabolic flux via the EMP pathway increases by overex- cose consumption under glucostat culture conditions. Interest-
pression of glycolytic genes, whereas metabolic flux to the by- ingly, the results of Koebmann et al.30 were in stark contrast to
products is unchanged. The net result is that the metabolic flux those of this aforementioned study. They constructed recombi-
toward alanine is relatively increased and the alanine yield is nant L. lactis strains with various levels of PFK activities, showing
improved.17 While this model explains the decreased yields of 1.4- to 11-fold differences compared to the wild-type level, and
acetate and succinate, the major by-products of the growth- demonstrated that increased PFK activity has no effect on glucose
arrested bioprocess, it cannot be applied to the production of consumption in L. lactis. These controversial results will be fur-
dihydroxyacetone (DHA) and glycerol. In the D-lactate-produc- ther discussed in the next section, though the results obtained in
ing C. glutamicum, overexpression of glk significantly enhances the former study clearly indicate that improvement of glucose
glucose consumption; however, glycerol and DHA production uptake rate by engineering of the glycolytic pathway is not lim-
increases at least 5-fold.25 This observation suggests that the over- ited in C. glutamicum.
flow in metabolism is caused by differences in the metabolic flux Increased enzyme activities of PFK and PYK in ethanoL-pro-
upstream and downstream of the EMP pathway.26-28 Co-overex- ducing E. coli KO20 moderately enhances glucose consumption
pression of glk and gapA reverts the DHA and glycerol yields under specific conditions.31 Resting cells of the recombinant strain
back to levels of the non-glk-overexpressing strain. Under oxygen with simultaneous increased PFK and PYK activities exhibit 25%
deprivation, glycerol is produced in C. glutamicum via DHA, higher glucose consumption and 35% higher ethanol production
from the glycolytic intermediate dihydroxyacetone phosphate compared to the parental strain when the cells are harvested from
(DHAP), by DHAP phosphatase HdpA and butanediol dehy- aerobic cultures. However, glycolytic flux is not enhanced when
drogenase.27,28 The model mentioned above assumes that the the recombinant strain is cultured anaerobically. Because the glu-
metabolic flux toward the by-products remains unchanged even cose consumption of E. coli KO20 is maximized under anaerobic
when the concentrations of the intracellular precursors of succi- conditions, the authors concluded that glucose flux under submaxi-
nate and acetate increase. However, the metabolic fluxes to DHA mal (aerobic) conditions can be enhanced by overexpression of

330 Bioengineered Volume 6 Issue 6


glycolytic enzymes to the level observed under maximum (anaero- observed when glycolytic genes were overexpressed, was also
bic) conditions. Similarly, immobilized S. cerevisiae cells with demonstrated in the study with Z. mobilis.8 This negative effect
increased PFK activity show 1.3-fold higher rate of glucose con- on glucose metabolism could be caused by a protein burden
sumption under aerobic conditions, but not under anaerobic con- effect that is often observed during overproduction of proteins in
ditions.32 Both studies imply that the increased rate of glucose recombinant cells.
consumption, induced by the overproduction of glycolytic By using metabolic control analysis, Jensen and co-workers
enzymes, does not exceed the maximum glucose consumption rate investigated the role of glycolytic enzymes on the control of gly-
of the organisms. This idea emphasizes the importance of the gly- colytic flux.30,36-39 They developed a series of synthetic pro-
colytic pathway engineering for improving fermentation perfor- moters showing various expression levels and used those
mance of newly isolated microorganisms because optimal culture promoters to construct L. lactis strains with multilevel activities
conditions of those microorganisms are hardly established espe- of glycolytic enzymes (PFK, PYK, TPI, GAPDH, ENO, PGM).
cially in the early phase of strain development. These studies showed that recombinant strains with higher activi-
Z. mobilis is well known as an efficient ethanol producer.33 ties of glycolytic enzymes exhibited similar levels of glucose con-
This microorganism possesses the ED pathway for sugar metabo- sumption to wild-type L. lactis. The authors concluded that none
lism. The recombinant strain of Z. mobilis, displaying 20% of these enzymes influenced the glycolytic flux, and these
increase in glucose 6-phosphate dehydrogenase activity, shows enzymes were therefore present in excess in L. lactis.
10% higher glycolytic flux. However, further increase in enzyme There has also been report of an unsuccessful study in oxygen
activity shows no effect on glycolytic flux.34 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
Difference Between Success and Failure in equivalents. To achieve this, the fdh gene encoding formate dehy-
Glycolytic Pathway Engineering drogenase from Mycobacterium vaccae was introduced into the C.
glutamicum genome and formate was added as a co-substrate to
In the last section, we described studies that were successful at provide additional NADH for succinate production. The authors
enhancing the rate of glucose consumption by overexpression of examined the effect of gapA overexpression in the fdh-harboring
the glycolytic gene(s). However, historically, there have been strain. The succinate yield was improved in the fdh-harboring
many studies where overexpression of the glycolytic genes strain in the presence of formate, but the strain enters a dormancy
resulted in no positive effects on glucose consumption. In the fol- phase for approximately 10h after the 3h reaction. The dormancy
lowing sections, we summarize those studies and discuss the fac- phase disappears by overexpression of gapA. However, the glu-
tors determining success and failure in glycolysis engineering. cose consumption rate of the gapA-overexpressing strain was not
enhanced.
Past studies showing no improvement in glucose uptake
Schaaff et al. constructed recombinant S. cerevisiae strains Is overexpression of glycolytic genes really effective?
overexpressing each of the genes encoding for the following 6 gly- Contrasting results described in the last sections make us hesi-
colytic enzymes: hexokinase, GPI, PFK, phosphoglycerate tate to conclude that overexpression of the glycolytic genes is an
mutase (PGM) or PYK, and an enzyme for ethanol fermentation, effective means to enhance glycolytic flux. What is different
pyruvate decarboxylase (PDC) or alcohol dehydrogenase between successful and failed attempts? Two studies with PFK-
(ADH).10 Although enzyme activities were increased 3.7- to overproducing L. lactis conducted by different research groups
13.9-fold compared to wild-type levels, overexpression of the will be used as examples. Papagianni et al.29 demonstrated a 2-
genes had no effect on the rate of ethanol production. The fold increase in glucose consumption by overproduction of PFK,
authors speculated that simultaneous increase in several enzyme whereas Jensen and coworkers showed that PFK has no control
activities would be required to enhance the glycolytic flux. The on glycolytic flux,30 but they also found that a mutant strain hav-
study by Hauf et al. tested this hypothesis by simultaneously ing 40% reduced PFK activity showed 34% decrease in glycolytic
overexpressing the genes encoding for the following 7 enzymes: flux compared to the wild-type strain.41 This suggests that the
GAPDH, phosphoglycerate kinase (PGK), PGM, enolase glycolytic flux of L. lactis is sensitive to PFK activity. Moreover,
(ENO), PYK, PDC, and ADH. However, the results showed the strains and culture conditions were different between the
that ethanol fermentation was almost equal in both recombinant studies showing enhanced.29 and unchanged.30 glycolytic flux. If
and wild-type strains and only a slight increase in initial ethanol these differences affect the protein level of PFK in the each strain,
production rate was observed in the recombinant strain.7 it might provide an explanation for the contrasting results.
The effects of overproduction of PFK, PGK, PYK, and sugar From the studies on S. cerevisiae described above, it appears that
transporter GalP in ethanoL-producing E. coli KO11 were inves- the glycolytic enzyme level is high enough to drive glucose metabo-
tigated.35 GalP is a galactose:HC symporter that also shows glu- lism in the wild-type strain. A recent kinetic model of S. cerevisiae
cose uptake activity. Most of the recombinant strains showed glycolysis demonstrated that control of the glycolytic pathway is
similar levels of glucose flux, but the strains overexpressing the distributed among many enzyme reactions.42 There is a paradoxi-
genes for PFK and PYK exhibited a 60% lower rate in glucose cal example in S. cerevisiae where overexpression of PGM2, which
consumption. This decrease in glucose consumption rate, is involved in galactose metabolism, actually enhances galactose

www.tandfonline.com Bioengineered 331


uptake by 70%.43 Thus, overexpression of a glycolytic gene will testing, since the degree of improvement of glucose uptake seems
enhance metabolic flux when a cell faces a deficiency in an to vary depending on the host species considered. For rational
enzyme. There is another possible factor leading to disappointed modification of the glycolytic pathway, a remaining challenge is
results on strain development. The effect of protein burden might to gain a detailed understanding of the mechanism of the tech-
be underestimated because of the difficulty to quantitatively evalu- nology in a model organism. An ideal model for further studies
ate its effects. It is possible that the negative effects of the protein would be with C. glutamicum under oxygen deprivation condi-
burden, caused by overproduction of the glycolytic enzymes, will tions. Under these conditions, its growth is arrested and the
mask positive effects on glycolytic flux. In the study by Hauf et al., number of active pathways is limited, which simplifies the choice
activities of PYK and PGM were increased 10- to 20-fold in S. cer- of targets in future research. In contrast, since microorganisms
evisiae. The expression levels of glycolytic enzymes are generally grow in normal fermentation processes, glucose consumption
high, and according to published data,42 PYK and PGM in S. cere- must be influenced by complex mechanisms associating the regu-
visiae were estimated to account for approximately 25 wt% among lation of cell growth. Another reason to select C. glutamicum is
the glycolytic enzymes. Increasing protein levels of PGM and PYK that this microbe is currently used in the biotechnology industry.
by 10-fold, assuming that the protein levels of other glycolytic Consequently, knowledge from basic research can be directly
enzymes are unchanged, results in an increase in the total amount transferred to industrial applications.
of glycolytic enzymes by approximately 3-fold compared to the In E. coli, ATP demand is known to control glycolytic flux.44
level of the wild-type strain. Such a large increase in protein con- Significant increase in glycolytic flux is observed when the genes
centrations might cause a protein burden. encoding the F1 portion of (F1F0) HC-ATP synthase are overex-
Although the most successful results from glycolytic pathway pressed to deplete ATP. Since growth of C. glutamicum is
engineering have been observed from the studies in C. glutami- arrested under oxygen deprivation, under these conditions, ATP
cum under oxygen deprivation, under other conditions, no posi- demand is supposed to be low. Therefore, the combined effects
tive effects were found. For example, under oxygen-deprived of increasing ATP demand and overexpression of the glycolytic
conditions, gapA overexpression in alanine-producing C. glutami- genes in C. glutamicum are of great interest. Takeno et al.46
cum enhanced the rate of glucose consumption. However, under developed a recombinant C. glutamicum strain to enhance L-
aerobic conditions, glucose consumption was almost the same as lysine production by replacing wild-type NAD-dependent
that of the reference strain.16 GAPDH is not inhibited under aer- GAPDH with NADP-dependent GAPDH (GapN) from Strepto-
obic conditions because in the presence of oxygen, the intracellu- coccus mutans, because the supply of NADPH is important for
lar NADH/NADC ratio does not increase to levels that can efficient L-lysine production. In this strain, NADPH is synthe-
inhibit GAPDH. Given this, it can be understood that no sized through this modified EMP pathway and L-lysine produc-
enhancement was observed in the study with succinate-producing tion was largely improved. There is another feature in the
C. glutamicum.40 Due to redox imbalance (i.e., 2 mols of succi- modified EMP pathway where no ATP is formed in the engi-
nate produced requires more reducing equivalents that are pro- neered pathway, whereas 2 mol of ATP is generated in the native
duced from one mole of glucose), the NADH/NADC ratio is EMP pathway from 1 mol of glucose degradation. Since this
expected to remain low. Although no positive effects of gapA recombinant strain dose not grow on glucose as the sole carbon
overexpression on succinate production by C. glutamicum have source because of NADPH accumulation,47 it is not possible to
been reported, this does not necessarily mean that glycolytic engi- investigate the effect of reduced ATP formation on glycolytic
neering is a futile approach for improving succinate productivity. flux under culture conditions. However, the glycolytic flux can
For the development of D-lactate-producing C. glutamicum be determined under growth-arrested conditions by using the
strains, all the genes encoding for glycolytic enzymes were exam- cells cultivated in appropriate conditions, which is yet another
ined to find one that had positive effects on glycolytic flux.24,25 advantage of using C. glutamicum in glycolytic engineering
This approach can be similarly applied in developing an research.
improved strain for succinate production. Another interesting approach for engineering of the glycolytic
On the basis of the above discussions, the effects of glycolytic pathway was explored by Benisch and Boles,48 who investigated
pathway engineering vary depending on the microbial species, the possibility of introducing the ED pathway to S. cerevisiae,
culture conditions, and concentrations of key intracellular com- which normally metabolizes glucose via the EMP pathway. The
pounds, including NAD(P)H. To apply this metabolic engineer- ED pathway generates half the amount of ATP per glucose com-
ing strategy, all the glycolytic enzymes should be explored to pared to the EMP pathway, which results in reduced biomass for-
improve the fermentation performance of the production strain mation. In Z. mobilis, where the ED pathway is responsible for
of interest. In addition, moderate levels of gene overexpression glucose catabolism, the yield of ethanol is high due to low bio-
appear to be sufficient for enhancing glycolytic flux. 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
Outlook growth. Unfortunately, the ED pathway did not function in S.
cerevisiae due to the very low enzyme activity of bacterial 6-phos-
Engineering of the glycolytic pathway is a developing technol- phogluconate dehydratase (PGDH), one of the key enzymes in
ogy, and its outcomes are currently not predictable prior to the ED pathway. The authors speculated that inefficient loading

332 Bioengineered Volume 6 Issue 6


of the iron-sulfur cluster to bacterial PGDH protein might have would help expand the use of biorefineries. Thus, development
caused the low enzyme activity, since expression of the PGDH of technologies to enhance glycolytic flux will be one of key chal-
protein was detected in Western blot analysis after codon optimi- lenges in this research field.
zation. Development of a novel method for functional expression
of the bacterial enzyme is anticipated.
Increasing concerns regarding global climate change necessi- Disclosure of Potential Conflicts of Interest
tates a decrease in consumption of fossil resources. Consequently, No potential conflicts of interest were disclosed.
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 Funding
biomass. Industrial production of cellulosic ethanol from ligno- This work was partially supported by New Energy and Indus-
cellulosic biomass started in 2014 in the United States,49 but fur- trial Technology Development Organization (NEDO) of Japan
ther improvement of biocatalysts in yield, titer and productivity and the Ministry of Economy, Trade and Industry (Japan).

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