Composite
PK

Part:BBa_K4324003

Designed by: Chris Yoo   Group: iGEM22_TheKingsSchool_AU_HS   (2022-10-02)
Revision as of 06:27, 11 October 2022 by PenTest-duck (Talk | contribs)


lacPO-RBS-PK-T1

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).

Figure 1: Protein structure of phosphoketolase from AlphaFold

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 210
    Illegal BamHI site found at 507
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1912
  • 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 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 Km value for F6P is lower (10mM) than it is for X5P (45mM), meaning it has a higher affinity for F6P.

Characterisation

Proof of Function

References

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


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