Part:BBa_K4324300

XR-XDH-XK-PK for E. coli

This part is a collection of all composite parts for xylose reductase (BBa_K4324000), xylitol dehydrogenase (BBa_K4324001), xylulose kinase (BBa_K4324002), and phosphoketolase (BBa_K4324003). This part is a collection of all composite parts within TheKingsSchool_AU_HS's project.

This part enables E. coli to express the XR-XDH pathway, further express XK, and express phosphoketolase (links xylulose-5-phosphate to glycolysis).

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 1649
  Illegal BglII site found at 4065
  Illegal BamHI site found at 4362
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5767
  Illegal AgeI site found at 2929
  Illegal AgeI site found at 3217
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1306
  Illegal SapI.rc site found at 747
  

Xylose Reductase (BBa_K4324000)

This part is the composite part of the XFP gene from B. lactis that induces phosphoketolase, 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).

Our project focused on the improvement of xylose utilisation in E. coli, such that it is able to grow more efficiently on organic bio-waste matter. One part of this process was to incorporate phosphoketolase to induce a part of the PK pathway.

A significant portion of organic biomass contains plant dry matter, or lignocellulose, which is comprised of three substances: cellulose, hemicellulose, and lignin.

Figure 2: Composition of various lignocellulosic biomass, from Production of Bioethanol from Waste Newspaper by Byadgi et al.

Cellulose ([1] KEGG C00760) is a chain of many β-1,4-linked glucose units with a chemical formula of (C₆H₁₀O₅)_n, usually found in plant cell walls. Lignin is comprised of various oxygenated phenylpropane units, usually found between cell walls, such as plant tissues. Hemicellulose is primarily comprised of D-xylose, which is the second most abundant sugar in lignocellulosic biomass, after glucose.

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, but we have implemented it into our project 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.