Difference between revisions of "Part:BBa K4324002"

Latest revision as of 15:04, 12 October 2022

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

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:

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.

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 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.

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

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/
5. https://doi.org/10.1016/j.jmb.2006.10.068