Difference between revisions of "Part:BBa K4324102"
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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<sub>m</sub> value of 0.29mM for D-xylulose, whilst the K<sub>m</sub> 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<sub>cat</sub> values of each substrate, with D-xylulose inducing the highest turnover. | 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<sub>m</sub> value of 0.29mM for D-xylulose, whilst the K<sub>m</sub> 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<sub>cat</sub> values of each substrate, with D-xylulose inducing the highest turnover. | ||
<br><br> | <br><br> | ||
+ | |||
+ | ==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. | ||
+ | |||
+ | [[Image:Xk kan growth curve.png|500px|thumb|center|'''Figure A:''' Growth rate of XK on M9 media with KAN (glucose, xylose, xylitol)]] | ||
+ | |||
+ | E. coli containing XK were grown in the M9 media with KAN 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, IPTG helped double the growth rate in glucose and significantly increase growth rate in xylose. They grew well in these two carbon sources, with glucose still the most preferable, due to E. coli's inherent characteristics. Interestingly, this engineered strain of E. coli grew well in xylitol, with the growth rate as fast as xylose. More tests and analysis needs to be done to understand these unusual characteristics. | ||
+ | |||
+ | ===Spot Growth=== | ||
+ | Plasmids for XDH, XK, and phosphoketolase were transformed into E. coli K12, which were grown on M9 media (with KAN) with their respective parts induced for a few days to check growth on glycerol as a sole carbon source. | ||
+ | |||
+ | [[Image:M9 glycerol kan.png|200px|thumb|center|'''Figure B:''' Spot growth on M9 media of glycerol (with KAN) of control, XR, XDH and phosphoketolase]] | ||
+ | |||
+ | On the M9 media with glycerol and KAN, we observed minimal growth of the controls, no growth of XDH and XK, but a large growth of XK. Through literature research, we discovered that xylulose kinase could actually phosphorylate glucose to some extent, which we believed was causing it to display prominent growth on the glycerol medium (Luccio et al., Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli, Journal of Molecular Biology, Volume 365, Issue 3, 2007, Pages 783-798, https://doi.org/10.1016/j.jmb.2006.10.068). | ||
==References== | ==References== |
Latest revision as of 15:03, 12 October 2022
Xylulose Kinase from E. coli
This part is the CDS of the xylB gene from E. coli that induces xylulose kinase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 747
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.
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.
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:
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.).
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 Km value of 0.29mM for D-xylulose, whilst the Km 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 kcat values of each substrate, with D-xylulose inducing the highest turnover.
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.
E. coli containing XK were grown in the M9 media with KAN 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, IPTG helped double the growth rate in glucose and significantly increase growth rate in xylose. They grew well in these two carbon sources, with glucose still the most preferable, due to E. coli's inherent characteristics. Interestingly, this engineered strain of E. coli grew well in xylitol, with the growth rate as fast as xylose. More tests and analysis needs to be done to understand these unusual characteristics.
Spot Growth
Plasmids for XDH, XK, and phosphoketolase were transformed into E. coli K12, which were grown on M9 media (with KAN) with their respective parts induced for a few days to check growth on glycerol as a sole carbon source.
On the M9 media with glycerol and KAN, we observed minimal growth of the controls, no growth of XDH and XK, but a large growth of XK. Through literature research, we discovered that xylulose kinase could actually phosphorylate glucose to some extent, which we believed was causing it to display prominent growth on the glycerol medium (Luccio et al., Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli, Journal of Molecular Biology, Volume 365, Issue 3, 2007, Pages 783-798, https://doi.org/10.1016/j.jmb.2006.10.068).
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/