Part:BBa_K4324002

lacPO-RBS-XK-T1

This part is the composite part of the xylB gene from E. coli that induces xylulose kinase, 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 xylulose kinase from AlphaFold

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 719
  Illegal AgeI site found at 1007
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

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 induce an over-expression of xylulose kinase in E. coli.

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.

In E. coli, D-xylose is directly isomerised by xylose isomerase into D-xylulose.

D-xylulose is a sugar with a chemical formula of C₅H₁₀O₅. E. coli has two transporter systems for xylose - XylE and XylFGH - both of which are inhibited by catabolite repression which is in favour of glucose.

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.

Xylulose kinase (EC 2.7.1.17) is an enzyme that serves as a catalyst for the phosphorylation of xylulose into xylulose-5-phosphate, according to the following chemical equation:

D-xylulose + ATP ⇌ D-xylulose-5-phosphate + ADP + H⁺

E. coli natively expresses xylulose kinase through its xylB gene. In both yeast and E. coli cells, xylulose kinase forms a process that converts xylulose into X5P, for it to then be processed through the pentose phosphate pathway, as shown in Figure 3. Xylulose kinase also serves as a catalyst for the phosphorylation of 1-deoxy-D-xylulose to 1-deoxy-D-xylulose 5-phosphate, albeit with a lower efficiency (Wungsintaweekul et al.).

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

Figure 4: Xylulose kinase kinetic parameters, from Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli by Di Luccio E, Petschacher B, Voegtli J, Chou HT, Stahlberg H, Nidetzky B, Wilson DK.

Xylulose kinase can also utilise D-ribulose, xylitol and D-arabitol as substrates. However, analysing the kinetic parameters of xylulose kinase in Figure 4, we see that it has a K_m value of 0.29mM for D-xylulose, whilst the K_m values for the other substrates are comparatively high, from 14mM (D-ribulose) to 127mM (xylitol) and 141 (D-arabitol). This demonstrates that xylulose kinase in E. coli has a significantly higher affinity for xylulose of any other substrates. This is further confirmed through comparing the k_cat values of each substrate, with D-xylulose inducing the highest turnover.

References

1. https://www.uniprot.org/uniprotkb/P09099/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x
3. https://pubmed.ncbi.nlm.nih.gov/17123542/
4. https://pubmed.ncbi.nlm.nih.gov/11168365/