Its all bout pentose phosphate pathway and how one can manipulate it.

5. The practice of optimizing genetic and regulatory processes within cells to
increase the cells' production of a certain substance
Chemical networks-a series of biochemical reactions and enzymes that allow
cells to convert raw materials into molecules necessary for the cell’s survival
Mathematically model these networks, calculate a yield of useful products,
and pin point parts of the network that constrain the production of these
products.
Existing metabolic engineering methodologies include
– pathway deletion
– pathway addition
– pathway modification: amplification, modulation or use of isozymes
(or enzyme from directed evolution study) with different enzymatic
properties
– Cofactors play an essential role in a large number of
biochemical reactions

6. Pichia stipitis genes XLY1 and XLY2 encoding xylose reductase and
xylitol dehydrogenase were cloned in S. cerevisiae.
To increase the flux through PPP, genes TKL1 and TAL1 encoding
transketolase and transaldolase were overexpressed.
Strains expressing all the genes together, showed considerable growth
on xylose.
The results indicate that the transaldolase level in S. cerevisiae is
insufficient for the efficient utilization of pentose phosphate pathway
metabolites.
1995 http://journals.asm.org

7. FIG. S104-TKL-TAL cultivated under three different levels of oxygenation
in SC medium-Leu-uracil containing 20 g of xylose per liter.
(A) Aerobic cultivation;
(B) oxygen-limited cultivation;
(C) anaerobic cultivation.
Symbols: ◊, xylose; , ○xylitol; , ●optical density at 600 nm (OD 600).
With decreasing oxygenation the biomass formation was reduced and
the xylitol production was increased.
http://journals.asm.org

8. FIG. Proposed metabolic pathways for xylose and glucose utilization in
yeasts. Abbreviations: XK, xylulokinase; Ribulose 5P, ribulose-5-phosphate;
Ribose 5P,ribose-5-phosphate; Xylulose 5P, xylulose-5-phosphate; S7P,
sedoheptulose-7-phosphate; G3P, glyceraldehyde-3-phosphate; E4P, erythrose-
4-phosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; FDP,
fructose diphosphate.
http://journals.asm.org

9. A classically derived tryptophan-producing Corynebacterium glutamicum was
significantly improved both by plasmid-mediated amplification of the genes for the
rate-limiting enzymes in the terminal pathways and by construction of a plasmid
stabilization system so that it produced more tryptophan.
At the late stage of the fermentation, tryptophan yield decreased with a
concomitant increase in CO₂ yield, suggesting a transition in the distribution of carbon
flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis.
To circumvent this transition by increasing the supply of erythrose 4-phosphate, a
direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum
was coamplified in the engineered strain by using low-copy-number plasmids which
were compatible with the resident plasmid.
This engineered strain, KY9218 carrying pIK9960, produced 58g of tryptophan per
liter from sucrose after 80h in fed-batch cultivation without antibiotic pressure
1999 http://journals.asm.org

10. FIG. Construction of low-copy-number
plasmid pIK9960
containing the transketolase gene
as well as the DS gene, the PGD
gene, and the tryptophan
biosynthetic gene cluster.
Stippled bars, C. glutamicum
KY10694 chromosomal DNA
fragment containing the DS gene (3-
deoxy-D-arabinoheptulosonic acid
7-phosphate);
Solid bars, C. glutamicum KY10894
chromosomal DNA fragment
containing the tryptophan-biosynthetic
gene cluster (trp genes)
Hatched bars, C. glutamicum ATCC
31833 chromosomal DNA fragment
containing the PGD gene(3-
phosphoglycerate dehydrogenase)
Cross-hatched bar, C. glutamicum
ATCC 31833 chromosomal DNA
fragment containing the
transketolase (TK) gene
Open bars, vector

11. FIG. Tryptophan fermentation by strain KY9218 carrying pSW9911 or pIK9960 in fed-batch
jar-fermentor cultivation.
Symbols: ●, tryptophan; ○, biomass; X, sugar.
For comparison, the profiles of tryptophan production by strain KY9218 carrying
pKW9901 are shown as controls. Arrows indicate the points at which feeding with a
60% sucrose solution began. Data represent mean values from three independent
cultures. OD660, optical density at 660 nm

12. Enhanced tryptophan production occurred because
increased activity of transketolase directed more carbon to
E4P formation through the nonoxidative pentose phosphate
pathway and contributed to increased availability of E4P.
This is one of few examples of successful metabolic
engineering with practical significance and thus should
provide valuable insight into the construction of industrially
useful production strains.
http://journals.asm.org

13. Hydrogen is widely recognized as an alternative, renewable energy source because
it is nonpolluting, producing only water as a byproduct, and it has a high energy
density.
Biological hydrogen production processes are known to be less energy intensive and
more environmental friendly than physico-chemical processes.
Among various routes for the biological hydrogen production, the NAD(P)H-dependent
pentose phosphate (PP) pathway is the most efficient for the dark
fermentation.
The co-overexpression of glpX with zwf genes encoding glucose-6-phosphate-1-
dehydrogenase and FBPase II increased the hydrogen yield to 2.32-fold.
These results indicate that activation of the PP pathway by glpX overexpression-enhanced
gluconeogenic flux is crucial for the increase of NAD(P)Hdependent
hydrogen production in E. coli BL21(DE3).
2011 Wiley Periodicals, Inc.

14. FIG.Simplified glycolysis/gluconeogenesis pathway in E. coli
Activation of the PP pathway, however, should be accompanied by activation of the
gluconeogenic pathway because it is necessary to recover 5 of 6 moles of G-6-P to
maximize the PP pathway
Fructose 1,6-bisphosphatase (FBPase), which converts fructose 1,6-bisphosphate to
fructose 6-phosphate is a key enzyme in the gluconeogenic pathway

15. FIG. Hydrogen production yield by
recombinant E. coli BL21(DE).
The potential of zwf and glpX overexpression was investigated to improve hydrogen
production in recombinant E. coli BL21(DE3) containingthe ferredoxin-dependent
hydrogenase system by activating the PP pathway.
Furthermore, co-overexpression of zwf improved the hydrogen yield to 2.32-fold
that of the HFdY strain.

16. Engineering the pentose phosphate pathway of
Saccharomyces cerevisiae for production of ethanol and
xylitol
Mervi Toivari