Coding

Part:BBa_K2314913

Designed by: Yuqing Chen   Group: iGEM17_OUC-China   (2017-10-22)

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, 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: http://parts.igem.org/wiki/images/2/2a/T--OUC-China--xylose content.png the growth curve of xylose strain: http://parts.igem.org/wiki/images/2/2a/T--OUC-China--xylose OD.png

Improved by Jiangnan_China 2021

We built a more efficient composite part BBa_K3803016 to achieve a functional improvement based on the parts BBa_K2314913 & BBa_K2314324 (Although they were uploaded as basic parts separately, they worked together as a composite part) from OUC_China in 2017 iGEM.

We express this composite part on pY15TEF1 vector and then introduced it into S. cerevisiae to test its function.

Design

Xylose reductase (XR) will first transform xylose to xylitol, and xylitol dehydrogenase (XDH) can further convert xylitol to xylulose. Then, xylulose will be converted to xylulose 5-phosphate (X5P) by the native xylulose kinase (XK). Apart from the original XR and XDH genes, considering the low copy number of native XK gene in S. cerevisiae, we introduced an extra XK gene to improve the xylose utilization ability.


Fig.1 Xylose Utilization Pathway


Fig.2 The Existing & Improved Parts

Xylose Utilization Ability: For Cell Growth

For cell growth, OUC_China achieved a final OD600 around 2.35 in YNB-based xylose media. After our improvement on their parts, we could get a final OD600 around 20.


Fig.2 The OD600 Value with Different Xylose Utilization Genes

Xylose Utilization Ability: For Product Yield

For our own product yield, our production also improved after substituting our three-gene cassette for OUC_China's two-gene one. The improved production was very obvious after the parts improvement based on our HPLC results.


Fig.3 The HPLC Results with Different Xylose Utilization Genes

For more information about our parts and improvement, you can see details here

Reference

[1] Construction of efficient xylose-fermenting Saccharomyces cerevisiae through a synthetic isozyme system of xylose reductase from Scheffersomyces stipitis. Bioresource Technology, Jung-Hyun Jo, Yong-Cheol Park, Yong-Su Jin, Jin-Ho Seo, Bioresource Technology, 241 (2017) 88–94.

[2] Metabolic engineering of Saccharomyces cerevisiae for production of Shinorine, a sunscreen material, from xylose. Seong-Hee Park, Kyusung Lee, Jae Woo Jang and Ji-Sook Hahn, ACS Synthetic Biology, 2019 (8), 346−357.

Sequence and Features BBa_K2314913 SequenceAndFeatures __NOTOC__ BBa_K1583002 short

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