Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture.
This study focused on elucidating metabolism of xylose in a Saccharomyces cerevisiae strain that overexpresses xylose reductase and xylitol dehydrogenase from Pichia stipitis, as well as the endogenous xylulokinase. The influence of xylose on overall metabolism was examined supplemented with low glucose levels with emphasis on two potential bottlenecks; cofactor requirements and xylose uptake. Results of metabolic flux analysis in continuous cultivations show changes in central metabolism due to the cofactor imbalance imposed by the two-step oxidoreductase reaction of xylose to xylulose. A comparison between cultivations on 27:3g/L xylose-glucose mixture and 10g/L glucose revealed that the NADPH-generating flux from glucose-6-phosphate to ribulose-5-phosphate was almost tenfold higher on xylose-glucose mixture and due to the loss of carbon in that pathway the total flux to pyruvate was only around 60% of that on glucose. As a consequence also the fluxes in the citric acid cycle were reduced to around 60%. As the glucose level was decreased to 0.1g/L the fluxes to pyruvate and in the citric acid cycle were further reduced to 30% and 20%, respectively. The results from in vitro and in vivo xylose uptake measurements showed that the specific xylose uptake rate was highest at the lowest glucose level, 0.1g/L.
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland.
Juha-Pekka
JP
Pitkänen
Aristos
A
Aristidou
Laura
L
Salusjärvi
Laura
L
Ruohonen
Merja
M
Penttilä
D000332:Aerobiosis / Q000502:physiology; D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism*; D000693:Anaerobiosis / Q000502:physiology; D019149:Bioreactors / Q000382:microbiology; D018929:Cell Culture Techniques / Q000379:methods*; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D050542:D-Xylulose Reductase; D004734:Energy Metabolism / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D003067:Coenzymes; D011994:Recombinant Proteins; D014994:Xylose; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; D050542:D-Xylulose Reductase; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D005947:Glucose; D010100:Oxygen