Part:BBa_K1585213

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 K_m value for F6P is lower (10mM) than it is for X5P (45mM), meaning it has a higher affinity for F6P.

Characterisation

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]