Difference between revisions of "Part:BBa K4324100"

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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.  
 
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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[[Image:Composition-of-Cellulose-Hemicellulose-and-Lignin-for-different-Lignocellulosic.png|300px|thumb|right|'''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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[[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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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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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.  
  
 
D-xylose is a aldopentose sugar with a chemical formula of '''C<sub>5</sub>H<sub>10</sub>O<sub>5</sub>'''. It can serve as a sole carbon source for E. coli with its XI pathway and pentose phosphate pathway that is explained further below. 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.
 
D-xylose is a aldopentose sugar with a chemical formula of '''C<sub>5</sub>H<sub>10</sub>O<sub>5</sub>'''. It can serve as a sole carbon source for E. coli with its XI pathway and pentose phosphate pathway that is explained further below. 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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In S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.
 
In S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.
  
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[[Image:X5P_in_pentose_phosphate_pathway.png|300px|thumb|right|'''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: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]]]
  
 
E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway that directly isomerises xylose into xylulose using the xylose isomerase enzyme. Hence, together with xylitol dehydrogenase ([https://parts.igem.org/Part:BBa_K4324101 '''BBa_K4324101''']) which can convert xylitol to xylulose, xylose reductase presents an alternate xylose metabolism pathway for E. coli.
 
E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway that directly isomerises xylose into xylulose using the xylose isomerase enzyme. Hence, together with xylitol dehydrogenase ([https://parts.igem.org/Part:BBa_K4324101 '''BBa_K4324101''']) which can convert xylitol to xylulose, xylose reductase presents an alternate xylose metabolism pathway for E. coli.
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Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.
 
Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.
  
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[[Image:Xylose_reductase_kinetic_parameters.png|350px|thumb|left|'''Figure 3:''' Xylose reductase kinetic parameters, from [https://pubmed.ncbi.nlm.nih.gov/3921014/ Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis] by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers]]
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[[Image:Xylose_reductase_kinetic_parameters.png|400px|thumb|center|'''Figure 3:''' Xylose reductase kinetic parameters, from [https://pubmed.ncbi.nlm.nih.gov/3921014/ Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis] by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers]]
  
 
Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a K<sub>m</sub> value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher V<sub>max</sub> values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.
 
Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a K<sub>m</sub> value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher V<sub>max</sub> values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.

Revision as of 04:28, 3 October 2022


NAD(P)H-dependent D-xylose reductase from S. stipitis

This part is the CDS of the XYL1 gene from S. stipitis that induces xylose reductase, and has been codon-optimised for expression in E. coli.

Figure 1: Protein structure of xylose reductase 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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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 incorporate a yeast-derived XR-XDH 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-xylose is a aldopentose sugar with a chemical formula of C5H10O5. It can serve as a sole carbon source for E. coli with its XI pathway and pentose phosphate pathway that is explained further below. 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.

Xylose reductase (EC 1.1.1.307), an aldose reductase, is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:

D-xylose + NAD(P)H + H+ ⇌ xylitol + NAD(P)+

In S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.

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 the XR-XDH pathway, instead having an XI pathway that directly isomerises xylose into xylulose using the xylose isomerase enzyme. Hence, together with xylitol dehydrogenase (BBa_K4324101) which can convert xylitol to xylulose, xylose reductase presents an alternate xylose metabolism pathway for E. coli.

Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.

Figure 3: Xylose reductase kinetic parameters, from Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers

Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a Km value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher Vmax values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.

The reaction is reversible, from xylitol to D-xylose, and on expression within E.coli, would allow utilization of xylitol as the sole carbon source. This will occur first by this reverse reaction to xylose, then by direct isomerisation through xylose isomerase (XI pathway) which exists natively within E. coli. However, it must be noted that the reverse reaction incurs a reaction rate which is 4-5% that of the forward reaction, and so it is hardly useful.

Characterisation

Proof of Expression

Proof of Function

References

1. https://www.uniprot.org/uniprotkb/P31867/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x
3. https://pubmed.ncbi.nlm.nih.gov/3921014/