Difference between revisions of "Part:BBa K4324002"

 
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<partinfo>BBa_K4324002 short</partinfo>
 
<partinfo>BBa_K4324002 short</partinfo>
  
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This part allows E. coli to express xylulose kinase (XK).
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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 ([https://parts.igem.org/Part:BBa_K4324201 BBa_K4324201]), RBS ([https://parts.igem.org/Part:BBa_K4324200 BBa_K4324200]), and T1 terminator from E. coli's rrnB gene ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]).
  
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XK is an enzyme that allows the conversion of xylulose to xylulose-5-phosphate (X5P), and vice versa, according to the following chemical equation:
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[[Image:Xylulose kinase structure.png|200px|thumb|right|'''Figure 1:''' Protein structure of xylulose kinase from [https://alphafold.ebi.ac.uk/entry/P09099 AlphaFold]]]
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D-xylulose + ATP &#8652; D-xylulose 5-phosphate + ADP + H+
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In E. coli, XK forms part of the xylose metabolism pathway by converting xylulose to X5P, which is then further metabolised in the pentose phosphate pathway.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4324002 SequenceAndFeatures</partinfo>
  
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E. coli already exhibit XK genes.
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===Usage and Biology===
  
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Ultimately, this part serves to consolidate the efficiency of conversion from xylulose to X5P in E. coli.
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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.
  
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<!-- Add more about the biology of this part here
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A significant portion of organic biomass contains plant dry matter, or lignocellulose, which is comprised of three substances: cellulose, hemicellulose, and lignin.
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===Usage and Biology===
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[[Image:Composition-of-Cellulose-Hemicellulose-and-Lignin-for-different-Lignocellulosic.png|500px|thumb|center|'''Figure 2:''' Composition of various lignocellulosic biomass, from [https://www.researchgate.net/publication/305892656_Production_of_Bioethanol_from_Waste_Newspaper Production of Bioethanol from Waste Newspaper] by Byadgi et al.]]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4324002 SequenceAndFeatures</partinfo>
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Cellulose ([https://www.kegg.jp/entry/C00760] KEGG C00760) is a chain of many β-1,4-linked glucose units with a chemical formula of '''(C<sub>6</sub>H<sub>10</sub>O<sub>5</sub>)<sub>n</sub>''', 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.
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In E. coli, D-xylose is directly isomerised by xylose isomerase into D-xylulose.
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D-xylulose is a sugar with a chemical formula of '''C<sub>5</sub>H<sub>10</sub>O<sub>5</sub>'''. 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.
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[[Image:Xylose_metabolism_pathways.jpeg|600px|thumb|center|'''Figure 2:''' Xylose metabolism pathways of various microorganisms, from [https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x Biochemical routes for uptake and conversion of xylose by microorganisms] by Zhao, Z., Xian, M., Liu, M. et al.]]
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Xylulose kinase ([https://www.genome.jp/dbget-bin/www_bget?ec:2.7.1.17 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:
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<center>'''D-xylulose + ATP ⇌ D-xylulose-5-phosphate + ADP + H<sup>+</sup>'''</center>
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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.).
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[[Image:X5P_in_pentose_phosphate_pathway.png|300px|thumb|center|'''Figure 3:''' Xylulose-5-phosphate within the pentose phosphate pathway, from [https://scholar.uwindsor.ca/cgi/viewcontent.cgi?article=1091&context=etd Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume]]]
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[[Image:Xylulose kinase kinetic parameters.png|400px|thumb|left|'''Figure 4:''' Xylulose kinase kinetic parameters, from [https://pubmed.ncbi.nlm.nih.gov/17123542/ 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.]]
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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.
  
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==References==
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1. https://www.uniprot.org/uniprotkb/P09099/entry<br>
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2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x<br>
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3. https://pubmed.ncbi.nlm.nih.gov/17123542/<br>
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4. https://pubmed.ncbi.nlm.nih.gov/11168365/
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 06:24, 11 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


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 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.

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/