Based on the parts (BBa_K2314913 & BBa_K2314324) from OUC_China in 2017 iGEM, we built a more efficient xylose utilization part BBa_K3803016. Apart from the original xylose reductase (XR) and xylitol dehydrogenase (XDH) genes, considering the low copy number of native Xylulose kinase (XK) gene in S. cerevisiae, we introduced an extra XK gene to improve the xylose utilization ability.
Fig.1 Improvement of Existing Part
In the XR/XDH pathway, XR first transforms xylose to xylitol, and XDH further converts xylitol to xylulose. Xylulose will be converted to xylulose 5-phosphate (X5P) by the native XK from S. cerevisiae.
The xylulose has some cytotoxicity effects on yeast cells so that the extra XK can not only improve the xylose utilization ability but also can avoid negative influence by the accumulation of intracellular xylulose.
Fig.2 Xylose Utilization Pathway
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.3 The OD600 Value with Different Xylose Utilization Genes
For our product yield, our production also improved after substituting our three-gene cassette for OUC_China's two-gene one.
Fig.4 The HPLC Results with Different Xylose Utilization Genes