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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
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.
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
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
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