Difference between revisions of "Part:BBa K4324001"
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<partinfo>BBa_K4324001 short</partinfo>
 
<partinfo>BBa_K4324001 short</partinfo>
  
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This part allows E. coli to express xylitol dehydrogenase (XDH).
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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 ([https://parts.igem.org/Part:BBa_K4324201 BBa_K4324201]), RBS ([https://parts.igem.org/Part:BBa_K4324200 BBa_K4324200]), and T1 terminator from E. coli's rrnB gene ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]).
  
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XDH is an enzyme that allows the conversion of xylitol to xylulose, and vice versa, according to the following chemical equation:
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[[Image: Xylitol_dehydrogenase_structure.png|200px|thumb|right|'''Figure 1:''' Protein structure of xylitol dehydrogenase from [https://alphafold.ebi.ac.uk/entry/Q07993 AlphaFold]]]
  
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xylitol + NAD+ &#8652; D-xylulose + NADH + H+
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4324001 SequenceAndFeatures</partinfo>
  
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In S. cerevisiae yeast cells, XDH forms the second process in the XR-XDH pathway, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.
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===Usage and Biology===
  
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E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway which directly converts xylose into xylulose. Hence, together with xylose reductase (XR) which can convert xylitol to xylulose, XDH presents an alternate xylose metabolism pathway for E. coli.
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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
  
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Furthermore, this part enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.
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[[Image:Xylose_metabolism_pathways.jpeg|600px|thumb|center|'''Figure 2:''' Xylose metabolism pathways of various microorganisms, from [https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x Biochemical routes for uptake and conversion of xylose by microorganisms] by Zhao, Z., Xian, M., Liu, M. et al.]]
  
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<!-- Add more about the biology of this part here
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Xylitol dehydrogenase ([https://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.9 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:
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===Usage and Biology===
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<!-- -->
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<center>'''xylitol + NAD ⇌ D-xylulose + NADH + H<sup>+</sup>'''</center>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4324001 SequenceAndFeatures</partinfo>
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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.
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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 ([https://parts.igem.org/Part:BBa_K4324100 '''BBa_K4324100''']) which can convert xylose to xylitol, xylitol dehydrogenase presents an alternate xylose metabolism pathway for E. coli.
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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.
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==Characterisation==
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===Proof of Expression===
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===Proof of Function===
  
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==References==
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1. https://www.uniprot.org/uniprotkb/Q07993/entry<br>
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2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 06:22, 11 October 2022


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.

Characterisation

Proof of Expression

Proof of Function

References

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