Difference between revisions of "Part:BBa K4324100"

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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
 
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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[[Image:Xylose_metabolism_pathways.jpeg|600px|thumb|center|'''Figure 1''' 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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[[Image:Xylose_metabolism_pathways.jpeg|600px|thumb|center|'''Figure 1:''' 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.]]
  
 
Xylose reductase ([https://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.307 EC 1.1.1.307]), also known as aldose reductase, is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:
 
Xylose reductase ([https://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.307 EC 1.1.1.307]), also known as aldose reductase, is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:
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Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.
 
Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase 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_reductase_kinetic_parameters.png|450px|thumb|left|'''Figure 2:''' Xylose reductase kinetic parameters, from [https://pubmed.ncbi.nlm.nih.gov/3921014/ Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis] by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers]]
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Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a K<sub>m</sub> value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher V<sub>max</sub> values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.
  
 
==Characterisation==
 
==Characterisation==

Revision as of 10:00, 2 October 2022


NAD(P)H-dependent D-xylose reductase from S. stipitis

This part is the CDS of the XYL1 gene from S. stipitis that induces xylose reductase, and has been codon-optimised for expression in E. coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 747

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 1: 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.

Xylose reductase (EC 1.1.1.307), also known as aldose reductase, is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:

D-xylose + NAD(P)H + H+ ⇌ xylitol + NAD(P)+

In S. stipitis yeast cells, XR forms the first 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.

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

Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.

Figure 2: Xylose reductase kinetic parameters, from Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers

Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a Km value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher Vmax values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.

Characterisation

Proof of Expression

Proof of Function

References

1. https://www.uniprot.org/uniprotkb/P31867/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x 3. https://pubmed.ncbi.nlm.nih.gov/3921014/