Part:BBa_K4324001

lacPO-RBS-XDH-T1

This part is the composite part of the XYL2 gene from S. cerevisiae that induces xylitol dehydrogenase, and has been codon-optimised for expression in E. coli. It has a lac promoter (BBa_K4324201), RBS (BBa_K4324200), and T1 terminator from E. coli's rrnB gene (BBa_B0010).

Figure 1: Protein structure of xylitol dehydrogenase from AlphaFold

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 700
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 357
  

Usage and Biology

Our project focused on the improvement of xylose utilisation in E. coli. One part of this process was to incorporate a yeast-derived XR-XDH pathway

Figure 2: Xylose metabolism pathways of various microorganisms, from Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al.

Xylitol dehydrogenase (EC 1.1.1.9), an oxidoreductase, is an enzyme that serves as a catalyst for the conversion of xylitol into xylulose, and vice versa, according to the following chemical equation:

xylitol + NAD ⇌ D-xylulose + NADH + H⁺

In S. cerevisiae yeast cells, xylitol dehydrogenase forms the second process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.

E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway that directly converts xylose into xylulose. Hence, together with xylose reductase (BBa_K4324100) which can convert xylose to xylitol, xylitol dehydrogenase presents an alternate xylose metabolism pathway for E. coli.

Furthermore, xylitol dehydrogenase enables E. coli to utilise xylitol as an energy source through its direct conversion to xylulose, which then follows the pentose phosphate pathway.

References

1. https://www.uniprot.org/uniprotkb/Q07993/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x