Coding
PK

Part:BBa_K4324103

Designed by: Chris Yoo   Group: iGEM22_TheKingsSchool_AU_HS   (2022-09-27)


Phosphoketolase from B. lactis

This part is the CDS of the XFP gene from B. lactis that induces phosphoketolase, and has been codon-optimised for expression in E. coli.

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 116
    Illegal BamHI site found at 413
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1818
  • 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

Optical Density Growth Curve

We measured the growth rate of E. coli on various types of media by measuring the optical density through a biophotometer.

Figure A: Growth rate of PK on M9 media with CAM (glucose, xylose, xylitol)

E. coli containing PK were grown in the M9 media with CAM antibiotics, containing different carbon sources (glucose, xylose and Xylitol) over a period of 26 hours, with and without IPTG induction. OD600 were taken every 3 hours.

Analysing the results, cells grew slightly better without IPTG induction. As expected, Glucose is the most preferred carbon source, with the cell growth rate more than 3 times faster than in xylose. Also as expected, PK cells could not grow in xylitol, as E. coli does not natively have a xylose metabolism pathway. Interestingly, the induction of PK reduced the growth rate slightly on xylose. As the main intention of adding phosphoketolase was to alleviate the flux of X5P, there may not have been enough flux generated by the XI pathway alone for there to be a benefit in adding phosphoketolase, and hence random deviations in growth produced the slightly differing results.

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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