Coding

Part:BBa_K2314913

Designed by: Yuqing Chen   Group: iGEM17_OUC-China   (2017-10-22)
Revision as of 16:46, 1 November 2017 by Capreolus (Talk | contribs)

Genes XYL1 encoding xylose reductase (XR) from Pichia stipitis

This part is Gene XYL1, which encoding NAD(P)H-dependent D-xylose reductase, it's short name is xylose reductase (XR) .It from Pichia stipitis (taxonomic classification has been changed to Scheffersomyces stipitis). The function of the protein is reduces D-xylose into xylitol, has a preference for NADPH, but can also utilize NADH as cosubstrate, facilitated xylose assimilation in yeast.

This protein is involved in the pathway D-xylose degradation, which is part of Carbohydrate metabolism. Xylose reductase (XR) from Pichia stipitis is one of the enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. Catalytic activity i Xylitol + NAD(P)+ = D-xylose + NAD(P)H. Kinetics i KM=142 mM for xylose KM=38 µM for NADH KM=3 µM for NADPH In our project, we expressed the XYL1 and XYL2 genes, which are associated with xylose metabolism in Saccharomyces cerevisiae EBY100. The whole process of xylose metabolism is as follows:
Xylose is reduced to xylulose under the action of NADPH-dependent xylose reductase (xylosereductase XR). Xylitol is then oxidized to xylulose under the action of xylitol dehydrogenase, which relies on NAD. Xylulose is then phosphorylated by xylulokinase to form 5-phosphate xylose and then into the pentose phosphate pathway, PPP. The intermediate product of PPP pathway 6-phosphate glucose and 3-phosphoric acid glycerol aldehyde through glycolysis pathway to form pyruvate. L-lactate dehydrogenase and coenzyme NADH reduced pyruvate to L-lactic acid. Saccharomyces cerevisiae is not able to use xylose as a result of the lack of enzymes that convert xylose into xylose, but can use the isomers of xylose-xylulose. In Saccharomyces cerevisiae, Xylulose is also first phosphorylated to form 5-phosphate sugar, which enters the PPP pathway and forms lactic acid by glycolysis.

We have constructed engineered bacteria that can use xylose as a carbon source, which can be used as xylose and fermented xylose to produce ethanol, which is the result of our experiment.

[1]Kötter, Peter, and M. Ciriacy. "Xylose fermentation by Saccharomyces cerevisiae." Applied Microbiology & Biotechnology 38.6(1993):776-783. [2]Yu, S., H. Jeppsson, and Barbel Hahnhagerdal. "Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains.." Applied Microbiology and Biotechnology (1995): 314-320.


In short，Genes XYL1 encoding xylose reductase (XR) from Pichia stipitis (taxonomic classification has been changed to Scheffersomyces stipitis). Reduces D-xylose into xylitol, facilitated xylose assimilation in yeast


The content of xylose in the medium:


https://static.igem.org/mediawiki/parts/2/2a/T--OUC-China--xylose content.png

the growth curve of xylose strain:

https://static.igem.org/mediawiki/parts/2/2a/T--OUC-China--xylose OD.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 89
    Illegal BglII site found at 478
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 109
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 433


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