Difference between revisions of "Part:BBa K1602004"
m (→‎Improvements: TheKingsSchool_AU_HS)
 
(15 intermediate revisions by 2 users not shown)
Line 14: Line 14:
 
             <tr>
 
             <tr>
 
                 <td>
 
                 <td>
−
                 <b>cadA</b> is a gene, coding for a cis-aconitate decarboxylase in Aspergillus terreus. The dehydrogenase catalyzes the conversion from cis-aconitate to itaconic acid. The reaction takes place in the cytosol of the host.
+
                 <b>GRE3</b> is a gene, coding for an aldose reductase. The reductase catalyzes the conversion from xylose to xylitol in dependance of NADPH.
 
<!--<br>
 
<!--<br>
 
                 FUELLTEXT
 
                 FUELLTEXT
Line 21: Line 21:
 
-->
 
-->
 
                 </td>
 
                 </td>
−
                 <td>
+
                 <td align="center" valign="middle">
−
<div style="width: 300px; height: 300px; align: center; margin-left: 100px; margin-right:100px">
+
<video width="300" height="300" controls loop autoplay>
−
<video controls loop autoplay>
+
   <source src="https://static.igem.org/mediawiki/2015/e/e0/GRE3_low_hq.mp4" type="video/mp4">
−
   <source src="https://static.igem.org/mediawiki/2015/b/b0/GRE3_low_animated.mp4" type="video/mp4">
+
 
</video>
 
</video>
−
</div>
 
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 33: Line 31:
 
                     <!-- BLANK -->
 
                     <!-- BLANK -->
 
                 </td>
 
                 </td>
−
                 <td>
+
                 <td align="center" valign="middle">
−
                     <b>Figure 1</b> cis-aconitate decarboxylase (coded by cadA)
+
                     <b>Figure 1</b> aldose reductase (coded by GRE3)
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 48: Line 46:
 
         <br>
 
         <br>
 
         <table>
 
         <table>
−
             <colgroup width=50% span="4">
+
             <colgroup width=55% span="4">
 
             <tr>
 
             <tr>
 
                 <td>
 
                 <td>
Line 54: Line 52:
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
−
                     52754.05
+
                     37118.78
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 62: Line 60:
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
−
                     490
+
                     327
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 70: Line 68:
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
−
                     -10.0
+
                     3.5
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 78: Line 76:
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
−
                     5.1050
+
                     7.0925
 
                 </td>
 
                 </td>
 
             </tr>
 
             </tr>
Line 86: Line 84:
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
−
                     59360 (reduced)  60110 (cystine bridges)
+
                     45380 (reduced)  45755 (cystine bridges)
 
                 </td>
 
                 </td>
 +
            </tr>
 +
<tr>
 +
                <td>
 +
                    Improbability of expression in inclusion bodies
 +
                </td>
 +
<td>
 +
0.605
 +
</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
Line 98: Line 104:
 
         <br>
 
         <br>
 
     </body>
 
     </body>
−
</html>  
+
</html>
  
 
===<h2>Sequence and Features</h2>===
 
===<h2>Sequence and Features</h2>===
 
<partinfo>BBa_K1602004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1602004 SequenceAndFeatures</partinfo>
 +
 +
===<h2>Usage and Biology: TheKingsSchool_AU_HS</h2>===
 +
Aldose reductase is also notated as '''NAD(P)H-dependent D-xylose reductase''', or '''xylose reductase''' (XR).
 +
 +
[[Image:Xylose_metabolism_pathways.jpeg|600px|thumb|center|'''Figure 1:''' 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.]]
 +
 +
Xylose reductase ([https://www.genome.jp/dbget-bin/www_bget?ec:1.1.1.307 EC 1.1.1.307]) is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:
 +
 +
<center>'''D-xylose + NAD(P)H + H<sup>+</sup> ⇌ xylitol + NAD(P)<sup>+</sup>'''</center>
 +
 +
In S. cerevisiae and S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 1, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway. This XR reaction is reversible.
 +
 +
[[Image:Xylose_reductase_kinetic_parameters.png|350px|thumb|left|'''Figure 2:''' 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. 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. This presents opportunities for expressing this enzyme in microorganisms that struggle to metabolise xylose in the presence of glucose (e.g. E. coli) for improved xylose uptake.
 +
 +
In such case, as the reaction is reversible from xylitol to D-xylose, expressing 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.
 +
 +
===<h2>Improvements: TheKingsSchool_AU_HS</h2>===
 +
 +
The improved part for the coding sequence is [https://parts.igem.org/Part:BBa_K4324100 BBa_K4324100], and a functional composite part with a promoter, RBS and terminator is at [https://parts.igem.org/Part:BBa_K4324000 BBa_K4324000].
 +
 +
[[Image:Aldose reductase michaelis constants.png|400px|thumb|center|'''Figure 3:''' Aldose reductase (GRE3) Michaelis constants, from [https://journals.asm.org/doi/epdf/10.1128/aem.61.4.1580-1585.1995 Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae] by Kuhn A., van Zyl C., van Tonder A., Prior B.A.]]
 +
 +
Inspecting the enzyme kinetics of aldose reductase (GRE3), the Km value of D-xylose is 27.90mM whilst for D-glucose, it is only 9.34mM. This reveals that aldose reductase has a higher affinity for glucose than it does for xylose. However, as our project sought to increase the uptake of xylose for E. coli, it was necessary that the enzyme have a stronger affinity towards xylose.
 +
 +
By instead utilising xylose reductase (XYL1) from S. stipitis, which has a higher affinity for xylose as shown in the Usage and Biology section, its ability to increase the efficiency of the xylose metabolism pathway (XR-XDH pathway)has been improved.
 +
 +
Furthermore, this part is from the GRE3 gene and can only be expressed in S. cerevisiae. Our improved part took the XYL1 gene from S. stipitis and codon-optimised its sequence for expression in E. coli. Furthermore, we added an appropriate lac promoter, RBS and T1 terminator to enable its expression in E. coli through IPTG induction.

Latest revision as of 14:13, 8 October 2022

Aldose reductase - GRE3

GRE3 is a gene, coding for an aldose reductase. The reductase catalyzes the conversion from xylose to xylitol in dependance of NADPH.
Figure 1 aldose reductase (coded by GRE3)

Characteristics


Molecular Weight 37118.78
Residues 327
Charge 3.5
Isoelectric Point 7.0925
A280 Molar Extinction Coefficients 45380 (reduced) 45755 (cystine bridges)
Improbability of expression in inclusion bodies 0.605
[Data taken from PEPSTATS]

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
    COMPATIBLE WITH RFC[1000]

Usage and Biology: TheKingsSchool_AU_HS

Aldose reductase is also notated as NAD(P)H-dependent D-xylose reductase, or xylose reductase (XR).

Figure 1: 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) 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. cerevisiae and S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 1, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway. This XR reaction is reversible.

Figure 2: 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. 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. This presents opportunities for expressing this enzyme in microorganisms that struggle to metabolise xylose in the presence of glucose (e.g. E. coli) for improved xylose uptake.

In such case, as the reaction is reversible from xylitol to D-xylose, expressing 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.

Improvements: TheKingsSchool_AU_HS

The improved part for the coding sequence is BBa_K4324100, and a functional composite part with a promoter, RBS and terminator is at BBa_K4324000.

Figure 3: Aldose reductase (GRE3) Michaelis constants, from Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae by Kuhn A., van Zyl C., van Tonder A., Prior B.A.

Inspecting the enzyme kinetics of aldose reductase (GRE3), the Km value of D-xylose is 27.90mM whilst for D-glucose, it is only 9.34mM. This reveals that aldose reductase has a higher affinity for glucose than it does for xylose. However, as our project sought to increase the uptake of xylose for E. coli, it was necessary that the enzyme have a stronger affinity towards xylose.

By instead utilising xylose reductase (XYL1) from S. stipitis, which has a higher affinity for xylose as shown in the Usage and Biology section, its ability to increase the efficiency of the xylose metabolism pathway (XR-XDH pathway)has been improved.

Furthermore, this part is from the GRE3 gene and can only be expressed in S. cerevisiae. Our improved part took the XYL1 gene from S. stipitis and codon-optimised its sequence for expression in E. coli. Furthermore, we added an appropriate lac promoter, RBS and T1 terminator to enable its expression in E. coli through IPTG induction.