Gmppp

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

The practice of optimizing genetic and regulatory processes within cells to increase the cells' production of a certain substanceChemical networks-a series of biochemical reactions and enzymes that allow cells to convert raw materials into molecules necessary for the cell’s survivalMathematically model these networks, calculate a yield of useful products, and pin point parts of the network that constrain the production of these products.

1995 http://journals.asm.org

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.

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.

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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.

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1999 http://journals.asm.org

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. glutamicumwas 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

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. glutamicumKY10694 chromosomal DNA fragment containing the DS gene (3-deoxy-D-arabinoheptulosonic acid 7-phosphate);Solid bars, C. glutamicum KY10894chromosomal DNA fragment containing the tryptophan-biosynthetic gene cluster (trp genes)Hatched bars, C. glutamicum ATCC 31833 chromosomal DNA fragmentcontaining the PGD gene(3-phosphoglycerate dehydrogenase)Cross-hatched bar, C. glutamicumATCC 31833 chromosomal DNA fragment containing the transketolase (TK) geneOpen bars, vector

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

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.

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2011 Wiley Periodicals, Inc.

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)Hdependenthydrogen production in E. coli BL21(DE3).

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 tomaximize the PP pathwayFructose 1,6-bisphosphatase (FBPase), which converts fructose 1,6-bisphosphate to fructose 6-phosphate is a key enzyme in the gluconeogenic pathway

FIG. Hydrogen production yield by recombinant E. coli BL21(DE).

The potential of zwf and glpX overexpression was investigated to improve hydrogenproduction 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.

Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitolMervi Toivari