Difference between revisions of "Part:BBa K1585213"
 
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<partinfo>BBa_K1585213 short</partinfo>
 
<partinfo>BBa_K1585213 short</partinfo>
  
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This is the translational unit for the D-Xylulose-5-phosphate-phosphoketolase (Xpk). It derives from Bifidobacterium adolescentis and is codon optimized for E. coli.
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This is the translational unit for the D-Xylulose-5-phosphate-phosphoketolase (Xpk). It derives from ''Bifidobacterium adolescentis'' and is codon optimized for ''E. coli''.
  
  
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|<html> <img src="https://static.igem.org/mediawiki/parts/0/05/Aachen_Reaction_xpk.png" width="600px"></html>
 
|<html> <img src="https://static.igem.org/mediawiki/parts/0/05/Aachen_Reaction_xpk.png" width="600px"></html>
 
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|'''{{{title|Catalysed reaction by Xpk}}}'''<br />D-Xylulose-5-phosphate + phosphate → Acetyl-phosphate + D-Glyceraldehyde-3-phosphate.
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|'''{{{title|Catalyzed reaction by Xpk}}}'''<br />D-Xylulose-5-phosphate + phosphate → Acetyl-phosphate + D-Glyceraldehyde-3-phosphate.
 
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[[File:Aachen_Reaction_xpk.png]]
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==Usage and Biology: TheKingsSchool_AU_HS==
  
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D-Xylulose-5-phosphate + phosphate → Acetyl-phosphate + D-Glyceraldehyde-3-phosphate
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D-xylulose-5-phosphate is a phosphorylated sugar with a chemical formula of '''C<sub>5</sub>H<sub>11</sub>O<sub>8</sub>P'''. In xylose metabolism, it generally occurs as a result of the phosphorylation of xylulose by xylulose kinase. 
  
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<!-- Add more about the biology of this part here
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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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===Usage and Biology===
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Phosphoketolase ([https://www.genome.jp/dbget-bin/www_bget?ec:4.1.2.9 EC 4.1.2.9]) is an enzyme that serves as a catalyst for the conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate, according to the following chemical equation:
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<center>'''D-xylulose-5-phosphate + phosphate ⇌ D-glyceraldehyde-3-phosphate + acetyl phosphate + H<sub>2</sub>O'''</center>
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In E. coli cells, xylulose-5-phosphate generally leads into the pentose phosphate pathway, as shown in Figure 3. Phosphoketolase allows X5P to also be broken down through glycolysis through its conversion to G3P. Thiamine diphosphate is a cofactor of phosphoketolase.
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[[Image:X5P_in_pentose_phosphate_pathway.png|300px|thumb|center|'''Figure 3:''' Xylulose-5-phosphate within the pentose phosphate pathway, from [https://scholar.uwindsor.ca/cgi/viewcontent.cgi?article=1091&context=etd Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume]]]
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E. coli do not exhibit phosphoketolase natively, it may help to alleviate the flux of X5P through another method of metabolism.
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Phosphoketolase can also utilise fructose-6-phosphate as a substrate, and in fact, the K<sub>m</sub> value for F6P is lower (10mM) than it is for X5P (45mM), meaning it has a higher affinity for F6P.
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==Characterisation==
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[[File: Aachen 15-08-10 Mdh, Xpk f. Wiki 2.png]]
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Mdh and Xpk in a pSB1A30 backbone on SDS page after IPTG induction with a RFP expression strain as negative control|subtitle1=all genes expressed in BL21 Gold (DE3), samples taken from 50 ml main culture in shake flasks after 6 h and 21 h after IPTG induction|subtitle2=all genes expressed in BL21 Gold (DE3), 2 ml samples taken from 50 ml main culture in shake flasks after 6h and 21 h after IPTG induction.
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==References==
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1. https://www.uniprot.org/uniprotkb/Q9AEM9/entry<br>
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2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x<br>
  
 
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Latest revision as of 15:19, 12 October 2022

B0034.XPK

This is the translational unit for the D-Xylulose-5-phosphate-phosphoketolase (Xpk). It derives from Bifidobacterium adolescentis and is codon optimized for E. coli.


Catalyzed reaction by Xpk
D-Xylulose-5-phosphate + phosphate → Acetyl-phosphate + D-Glyceraldehyde-3-phosphate.



Usage and Biology: TheKingsSchool_AU_HS

D-xylulose-5-phosphate is a phosphorylated sugar with a chemical formula of C5H11O8P. In xylose metabolism, it generally occurs as a result of the phosphorylation of xylulose by xylulose kinase.

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.

Phosphoketolase (EC 4.1.2.9) is an enzyme that serves as a catalyst for the conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate, according to the following chemical equation:

D-xylulose-5-phosphate + phosphate ⇌ D-glyceraldehyde-3-phosphate + acetyl phosphate + H2O

In E. coli cells, xylulose-5-phosphate generally leads into the pentose phosphate pathway, as shown in Figure 3. Phosphoketolase allows X5P to also be broken down through glycolysis through its conversion to G3P. Thiamine diphosphate is a cofactor of phosphoketolase.

Figure 3: Xylulose-5-phosphate within the pentose phosphate pathway, from Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume

E. coli do not exhibit phosphoketolase natively, it may help to alleviate the flux of X5P through another method of metabolism.

Phosphoketolase can also utilise fructose-6-phosphate as a substrate, and in fact, the Km value for F6P is lower (10mM) than it is for X5P (45mM), meaning it has a higher affinity for F6P.

Characterisation

Aachen 15-08-10 Mdh, Xpk f. Wiki 2.png

Mdh and Xpk in a pSB1A30 backbone on SDS page after IPTG induction with a RFP expression strain as negative control|subtitle1=all genes expressed in BL21 Gold (DE3), samples taken from 50 ml main culture in shake flasks after 6 h and 21 h after IPTG induction|subtitle2=all genes expressed in BL21 Gold (DE3), 2 ml samples taken from 50 ml main culture in shake flasks after 6h and 21 h after IPTG induction.

References

1. https://www.uniprot.org/uniprotkb/Q9AEM9/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1426
  • 1000
    COMPATIBLE WITH RFC[1000]