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| | <h2>Improved by <b>Jiangnan_China</b> 2021</h2> | | <h2>Improved by <b>Jiangnan_China</b> 2021</h2> |
| | <p>We built a more efficient composite part <a href="https://parts.igem.org/Part:BBa_K3803016">BBa_K3803016</a> to achieve a functional improvement based on the parts <a href="https://parts.igem.org/Part:BBa_K2314913">BBa_K2314913</a> & <a href="https://parts.igem.org/Part:BBa_K2314324">BBa_K2314324</a> (Although they were uploaded as basic parts separately, they worked together as a composite part) from OUC_China in 2017 iGEM.</p> | | <p>We built a more efficient composite part <a href="https://parts.igem.org/Part:BBa_K3803016">BBa_K3803016</a> to achieve a functional improvement based on the parts <a href="https://parts.igem.org/Part:BBa_K2314913">BBa_K2314913</a> & <a href="https://parts.igem.org/Part:BBa_K2314324">BBa_K2314324</a> (Although they were uploaded as basic parts separately, they worked together as a composite part) from OUC_China in 2017 iGEM.</p> |
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| | <partinfo>BBa_K1583002 short</partinfo> | | <partinfo>BBa_K1583002 short</partinfo> |
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| − | <html>
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| − | <p>This basic part contains the <i>Mfp5</i> gene. It is used in the biobricks <a href="https://parts.igem.org/Part:BBa_K1583104">BBa_K1583104</a> and <a href="https://parts.igem.org/Part:BBa_K1583110">BBa_K1583110</a> as a fusion protein with the protein CsgA (<a href="https://parts.igem.org/Part:BBa_K1583000">BBa_K1583000</a>).The expression of this fusion protein can be controlled with the rhamnose inducible promoter (<a href="https://parts.igem.org/Part:BBa_K914003">BBa_K914003</a>).
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| − |
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| − | <p>CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from <i>E.coli K-12 MG1655</i>. Inspired by mussels, the Mfp5 (mussel foot protein) has high adhesive properties towards wet polar surfaces. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. By creating a fusion protein of Mfp5 and CsgA, the adhesive properties of the mussel foot protein is combined with the formation of nanowires.</p>
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| − |
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| − | <p>The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al, 2014).</p>
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| − | </html>
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| − | <!-- -->
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| − | <span class='h3bb'><h3>Sequence and Features</h3></span>
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| − | <partinfo>BBa_K1583002 SequenceAndFeatures</partinfo>
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| − | <html>
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| − |
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| − | <h3>Characterization</h3>
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| − | <p>
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| − | This basic part was used in the biobricks <a href="https://parts.igem.org/Part:BBa_K1583104">BBa_K1583104</a> and <a href="https://parts.igem.org/Part:BBa_K1583110">BBa_K1583110</a>. These biobricks were characterized in three different experiments:
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| − | <ul>
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| − | <li> Protein expression
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| − | <li> Fluorescence assay </li>
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| − | <li> Crystal Violet assay </li>
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| − | <li> Transmission electron microscopy </li></ul>
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| − | </p>
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| − |
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| − | <h4> Fluorescence assay </h4>
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| − | <p>
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| − | To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (<a href="https://parts.igem.org/Part:BBa_I13504"target="_blank">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112"target="_blank">BBa_K1583112</a>) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C.
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| − | </p>
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| − |
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| − | <p>
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| − | In figure 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID.
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| − | </p>
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| − |
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| − | <div class="photos-wrapper full-width">
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| − | <div class="photos photo-grid one-part no-shadow narrower">
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| − | <img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%">
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| − | <figcaption>
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| − | <b>Figure 1</b>: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments.
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <p>
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| − | As can be seen from Fig. 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with the csgA-GFPmut3 construct gave a clear increase in fluorescence signal in time. All other experiments, gave similar levels of fluorescence, slightly increasing in time. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.
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| − | </p>
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| − |
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| − | <p>
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| − | With this kinetic experiment, we have proven that the rhamnose promoter does indeed induce the expression of the <i>csgA</i> gene.
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| − | </p>
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| − |
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| − | <h4> Crystal violet assay </h4>
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| − | <p>
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| − | The assay above showed that the bacteria that we engineered for the project is capable of producing the CsgA proteins after induction with L-rhamnose. However, this did not yet prove that curli are formed. In order to assess whether our bacteria producting the fusion protein of Mfp5 and CsgA can still produce these nanowires, our team adapted the protocol from Zhou et al. (2013) that employs crystal violet (methyl violet 10B) for dying the biofilm-making bacteria that attaches to the surface. In the experiment, our Mfp5_CsgA-producing strain of <i>E. coli</i> was induced at a high (0.5% w/v), low (0.2% w/v) and no (0% w/v) concentration of L-rhamnose. Furthermore, <i>csgA</i> deficient bacteria transformed with an empty plasmid (pSB1C3) were used as control. In the end, the wells were diluted with ethanol so all the content can dissolve in the liquid phase. We measured the absorbance at 590 nm of wavelength for all the samples, obtaining the following results (figure 2.).
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| − | </p>
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| − |
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| − | <figure><img class="featurette-image img-responsive center-block" src="https://static.igem.org/mediawiki/2015/f/f1/TU_Delft_mfp5.png" style="width:70%; background-size: cover;" alt="Generic placeholder image"><figcaption><b>Figure 2</b>: Microtiter Plate Assay results for testing biofilm formation. All the measurements were conducted in triplicates. CTRL0, CTRL2 and CTRL5 are <i>E. coli K-12 MG1655 PRO ΔcsgA ompR234</i> cells with pSB1C3, used as a control. MFP50, MFP52 and MFP55 are <i>E. coli K-12 MG1655 PRO ΔcsgA ompR234</i> cells expressing a plasmid that contains the Mfp5_CsgA fusion protein under an rhamnose inducible promoter. The termination 0, 2 and 5 denote the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).</figcaption></figure>
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| − |
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| − | <p> To confirm that there is a real change between the analysed samples and the empty plasmid control, a significance analysis was performed for α=0.05 (Table 1.).</p>
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| − |
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| − | <br><ul><caption>
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| − | <b>Table 1</b>: Significance analysis of the samples analysed, with a significance α value of 5%. All the samples display a significant difference when compared to the empty plasmid homologe sample (CTRL).
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| − | </caption>
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| − | </html>
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| − |
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| − | {|style="color:black" cellpadding="6" cellspacing="2" border="1" align="middle"
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| − | ! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Sample'''</FONT>
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| − | ! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''p-value'''</FONT>
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| − | ! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Significant difference (5%)'''</FONT>
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| − | |-
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| − | |'''CTRL2 & MFP52'''
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| − | |0.0043
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| − | |Yes
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| − | |-
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| − | |-
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| − | |'''CTRL5 & MFP55'''
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| − | |0.0016
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| − | |Yes
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| − | |}
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| − | <html><br></ul> <!--Table formatting originally from https://parts.igem.org/Part:BBa_K1150020 -->
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| − |
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| − | <p>
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| − | The significance analysis shows that cells containing the Mfp5_CsgA_His (BBa_K1583104) biobrick can efficiently create a curli, when compared with an empty plasmid control (i.e. without csgA expression).
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| − | </p> <br>
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| − |
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| − | <h4>Transmission electron microscopy</h4>
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| − |
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| − | <p>
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| − | Using TEM the formation of curli of the biobrick <a href="https://parts.igem.org/Part:BBa_K1583100">BBa_K1583100</a> was visualized (Figure 3). Although this is a different biobrick containing only CsgA (compared to the fusion protein of Mfp5_CsgA_His), curli formation can be presumed to be similar for this biobrick suggested by the Crystal Violet assay.</p>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/parts/d/d3/TEM_7300x_no_induction.png" width="40%" height="100%">
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| − | <img src="https://static.igem.org/mediawiki/parts/a/ac/TEM_7300x%2Binduction.png" width="40%" height="100%">
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| − | <figcaption>
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| − | <b>Figure 3</b>: TEM images (magnification 7300 x) of cells containing <a href="https://parts.igem.org/Part:BBa_K1583100"target="_blank">BBa_K1583100</a>. The left picture shows uninduced cells (0% Rhamnose).The picture on the right shows cells incubated with 1% (w/v) rhamnose.
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <p>
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| − | We did not observe formation of curli nanowires in the uninduced cultures of our strain. However, cells from induced cells clearly produced them, as supported by the TEM images.
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| − | </p>
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| − |
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| − | <h3>Reference</h3>
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| − | <p>Wegerer, A., Sun, T., and Altenbuchner, J. (2008). Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, BMC Biotechnology, 8:2 </p>
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| − | <p>Zhou, Kang, Kangjian Qiao, Steven Edgar, and Gregory Stephanopoulos. 2015. “Distributing a Metabolic Pathway among a Microbial Consortium Enhances Production of Natural Products.” Nature Biotechnology 33(4): 377–83. </p>
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| − | </html>
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| − |
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| − | <html>
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| − | <h2>Improved by team <b>Greatbay_SCIE</b></h2>
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| − |
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| − | <h3>Characterisation</h3>
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| − | <p>
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| − | BBa_K1583002 was characterized in following experiments:
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| − | </p>
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| − |
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| − | <ul>
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| − | <li>Protein expression</li>
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| − | <li>Protein purification</li>
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| − | </ul>
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| − |
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| − | <h3>Protein expression</h3>
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| − | <figure>
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| − | <img width="450px" src="https://2019.igem.org/wiki/images/4/4b/T--Greatbay_SCIE--006-fig1.png">
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| − | </figure>
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| − |
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| − | <figcaption>
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| − | Figure 4. The circuit of the protein, with a His-tag.
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| − | </figcaption>
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| − |
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| − | <p>
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| − | In our project, Mfp5 was used as an adhesive part to create recombinant proteins. We clone Mfp5 alone into the pET28b expression vector as control. We expressed pET28b-mfp5 in
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| − | <i>E.coli</i>
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| − | BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE.
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| − |
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| − | Results showed that no obvious protein bands of Mfp5 (~10 kDa) could be observed on lane mfp5 compared with lane pET28b (pET28b empty vector)(Figure 5A), which means the expression of this protein couldn’t be detected in BL21(DE3) Rosetta. Quantitative densitometry analysis of SDS-PAGE indicated that Mfp5 almost not expressed under this expression conditions (Figure 5B).
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| − | </p>
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| − |
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| − | <figure>
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| − | <img width="600px" src="https://static.igem.org/mediawiki/parts/5/53/T--Greatbay_SCIE--Detection_of_expression_level.jpeg">
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| − | </figure>
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| − |
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| − | <figcaption>
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| − | Figure 5. Detection of the expression level of all recombinant proteins by SDS-PAGE.
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| − | (A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins.
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| − | (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots.
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| − | </figcaption>
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| − |
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| − | <h3>Protein purification</h3>
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| − | <p>
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| − | The predicted size of mfp5 is 9.97 kDa and the isoelectric point is 10.19. Though no bands of interest could be observed on gels detecting the expression of Mfp5 (Figure 5A), we straightly go protein purification under denaturing conditions. As shown in Figure 6, no bands around 10kDa could be observed on Lane E1 and E3, which were eluted protein samples.
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| − | </p>
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| − |
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| − | <figure>
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| − | <img width="300px" src="https://2019.igem.org/wiki/images/7/7d/T--Greatbay_SCIE--006-fig3new.png">
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| − | </figure>
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| − |
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| − | <figcaption>
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| − | Figure 6. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein Mfp5 by cobalt-resin columns. Lanes: M, protein molecular weight marker; WC, whole-cell sample of recombinant proteins; IS, insoluble fragments. E, eluted proteins. 12% SDS-PAGE gels were used for the analyses.
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| − | </figcaption>
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| − |
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| − | <html>
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| − | <h2>Improved by team <b>ShanghaiTech_China</b></h2>
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| − | <p>We got the plasmid pET28a-Mfp5-1 by placing an order with GENEWIZ. (Mfp5-1 is the same as Mfp5. We call it Mfp5-1 for we expressed another protein(Mfp5-2) which contains two copies of Mfp5.) Then we transformed this plasmid to express Mfp5-1 in <i>E.coli</i> BL21(DE3). <b>We purified Mfp5-1 and modified the tyrosine residues of Mfp5-1 successfully.</b> Our protocols and related results are shown below.</p>
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| − |
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| − | <h3>Exploration of Cinditions of Induction</h3>
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| − | <p>Before protein purification, we decided to explore the proper induction conditions. So we made two control groups and four experimental groups. In Figure 1, we could find that mfp5-1 was successfully expressed in four groups since there was an obvious band between 15-20 kDa compared with blank control(Blank BL21(DE3)) and negative control(Before induction). </p>
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| − |
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| − | <p>Notably, there was a slight band in negative control(Before induction). We inferred that leaked expression of mfp5-1 might happen in negative control.
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| − | </p>
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| − | <figure>
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| − | <img width="500px" src="https://static.igem.org/mediawiki/parts/a/ae/Induction_condition_exploration.png">
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| − | <figcaption>
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| − | Fig.1: SDS-PAGE of mfp5-1 expression in different induction conditions
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <h3>IPTG Inducion</h3>
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| − | <p>After obtaining the single colony of transformed bacteria on the solid medium, we need to inoculate them in liquid culture to expand culture and then induce them with IPTG to get enough protein-producing bacteria:
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| − | </p>
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| − | <p>
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| − | 1. the colony was inoculated into 5ml of 30µ l/ml kanamycin LB liquid medium and cultured on a 37 ° C,220 rpm shaker for 8 hours.<br>
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| − | 2. Then inoculated 5ml of bacterial solution in each 1L of 30µ L/mL kanamycin LB liquid medium.<br>
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| − | 3. When the OD value of the bacterial solution at 600nm reached 0.8-1.0, 500µ L IPTG solution of 1M was added to each L of bacterial solution, and the culture was performed on a 37 ° C,220 rpm shaker for 8-10 hours.<br>
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| − | 4. Finally, the obtained bacterial solution was centrifuged at 3500RCM for 20 minutes to obtain the precipitated bacteria.<br></p>
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| − |
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| − | <h3>Protein Purification</h3>
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| − | <p>Since the recombinant Mfp5 expressed in E.coli is mostly in inclusion bodies, we need to add denaturants(here we used guanidine hydrochloride refer to the literature<sup>[9]</sup>) during purification to maintain the soluble state of the protein. In order to obtain high purity protein, we added 6*His tag to Mfp5CDS and purified it by nickel ion affinity chromatography. The buffer formula and purification steps are as follows:
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| − | </p>
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| − | <b><u>Buffer preparation</u></b>
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| − | *<b>Lysis buffer</b>: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, pH 7.4<br>
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| − | *<b>Wash buffer</b>: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 50 mM imidazole, pH 7.4<br>
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| − | *<b>Elution buffer</b>: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4<br>
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| − |
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| − | <b><u>Mfp5 purification</u></b>
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| − |
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| − | <p>Suspended cell pellets with 10 ml of lysis buffer per gram of wet cells and lysed it at 37°C, 220 rpm for 20 min. The cell suspension was then sonicated on ice for 30 min with a sonicator using 5 s on/ 5 s off cycles. Sonicated cell suspension was incubated at 37°C, 220 rpm, 40 min for ful dissolution of Mfp5.
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| − | </p>
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| − | <p>The suspension was centrifuged at 20000 g for 20 min at 18°C, and the supernatant was collected to be incubated with nickel media at 37°, 30 rpm for 1 hour. Then put the mixture into the column to let it flow through. After washing with 5-10 column volumes of wash buffer, Mfp5 was eluted by 1-2 column volumes of elution buffer.
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| − | </p>
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| − | <h3>SDS-PAGE Analysis</h3>
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| − | <p>We ran SDS-PAGE after each mfp5-1 purification. Fig.2a is the results of our first purification. It was obvious that a great amount of mfp5-1 was expressed in BL21 (compared samples before induction and after induction), and it totally dissolved in the lysis buffer. After elution, we got purified mfp5-1, but some mfp5-1 still remained on nickel medium. Since Fig.2a lacked flow through and washing samples, we did another purification, which was Fig.2b.
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| − | </p>
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| − |
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| − |
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| − | <p>
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| − | Fig.2b almost shows the same results as Figure 2 A. We could find the band of mfp5-1 in flow-through, indicating that the amount of mfp5-1 had saturated nickel medium. Moreover, low concentration imidazole could elute some mfp5-1 as well.
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| − | </p>
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| − | <p>
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| − | We found there was an unexpected band between 20-25 kDa in washing and elution samples(Fig.2b), which is not shown in Figure 2 A. It may be caused by the nickel medium since we used it to purify mfp5-2 before purification of mfp5-1 and did not clear it totally. The band of mfp5-2 is actually between 20-25 kDa.
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| − | </p>
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| − | <figure>
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| − | <img width="447px" src="https://static.igem.org/mediawiki/parts/a/a2/Mfp5_first_purification.png">
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| − | <img width="413px" src="https://static.igem.org/mediawiki/parts/0/06/Mfp5_second_purification.png">
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| − | <figcaption>
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| − | Fig.2a(left): SDS-PAGE of first purification of mfp5-1<br>
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| − | Fig.2b(right): SDS-PAGE of second purification of mfp5-1
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| − | </figcaption>
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| − | </figure>
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| − | </html>
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| − |
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| − | <html>
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| − | <h2>Modification of Tyrosine Residues</h2>
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| − | <p>Only when the tyrosine residue of Mfp5 is converted into dopamine can the protein become adherent. This step requires tyrosinase catalysis, so we first replace the purified protein buffer until the environment is suitable for tyrosinase to work, and then add enzymes to complete the modification. The buffer formula and modification steps are as follows:
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| − | <br>
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| − | <b><u>Buffer preparation</u></b><br>
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| − | *<b>Gradient buffer</b> 1: 4 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4<br>
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| − | *<b>Gradient buffer</b> 2: 2 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4<br>
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| − | *<b>Gradient buffer</b> 3: 0.5 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4<br>
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| − | *<b>Dialysis buffer</b>: 100 mM sodium acetate, pH 5.5<br>
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| − | *<b>Storage buffer</b>: 5% acetic acid<br>
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| − | </p>
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| − | <p>
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| − | <b><u>Mfp5 modification</u></b><br>
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| − | Purified protein solutions were first dialyzed in gradient buffer 1, gradient buffer 2, gradient buffer 3 at 4°C for 8 hours each successively to decrease the concentration of guanidine hydrochloride.
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| − | </p>
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| − | <p>
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| − | Then protein solutions were dialyzed in dialysis buffer at 4°C for 8 hours to entirely replace its buffer. Dialyzed proteins were then diluted to a concentration of 4 mg/ml in dialysis buffer with 100 mM ascorbic acid and filtered. Tyrosinase was added to a final concentration of 250 U/ml, and the mixture was incubated at 37°C, 220 rpm for 30 min. After the reaction, the solution was filtered, and enzyme activity in the solution was quenched by adding 0.2 ml of 6 N HCl per ml of reaction. The sample was then dialyzed in 5% acetic acid and stored in 4°C.
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| − | </p>
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| − | </html>
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| − |
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| − | <html>
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| − | <h2>Yield Calculation</h2>
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| − | <p>
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| − | After nickel ion affinity chromatography elution, we tested the OD value of the washing sample at 280nm and calculated the average yield. The result turned out to be 24mg per liter of bacteria solution. After modification, we tested the OD value again and found out the yield became 8mg per liter of bacteria solution or so, and the loss during modification is up to 2/3.
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| − | </p>
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| − | <h2>Mfp5-1 Adhesion Analysis</h2>
| |
| − | <p>
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| − | The adhesion test experiments of Mfp5-1&Mfp5-2 were carried out simultaneously as a control experiment, so the data of both Mfp5-1 and Mfp5-2 would be presented here.
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| − | </p>
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| − | <p>
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| − | To characterize as much as possible the viscosity of tyrosine enzyme-modified mfp5-1 and mfp5-2, take 20ul of the concentrated protein solution(concentration: mfp5-1=50mg/ml,mfp5-2=40mg/ml) to attach the PCR tube and the tube cover(Fig.3 top left and top right) (The PCR tube cover is trimmed and polished to a round face with a diameter of 7mm)to the center of the petri dish and perform a viscidity test after 48 hours of drying in a 37-degree oven.
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| − | </p>
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| − | <p>
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| − | Lift the petri dish at a certain height through the PCR tube and add the glass beads one by one to the petri dish until the last glass bead is added to cause the petri dish to fall off with the PCR tube(Fig.3 bottom left and bottom right). The viscosity of the added protein can be calculated by weighing the total weight of the Petri dish and glass beads.
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| − | </p>
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| − | <figure>
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| − | <img width="500px" src="https://static.igem.org/mediawiki/parts/4/46/Adhesion_test_experiment.jpeg">
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| − | <figcaption>
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| − | Fig.3: Adhesion test experiment of Mfp5-1&Mfp5-2.
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| − | </figcaption>
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| − | </figure>
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| − | <p>
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| − | Each experiment was recorded with a video camera, and every moment of falling off was recorded.
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| − | </p>
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| − | <a href="https://static.igem.org/mediawiki/parts/3/34/Adhesion_test_vdieo.mp4">Video.1</a>
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| − | <p>
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| − | We calculated the results by the following steps:</p>
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| − | <figure>
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| − | <img width="900px" src="https://static.igem.org/mediawiki/parts/5/5e/Adhesion_calculation_2.png">
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| − | <figcaption>
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| − | Fig.4:Calculation process of Mfp5 viscosity
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| − | </figcaption>
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| − | <figure>
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| − | <br style="clear: both"/>
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| | </html> | | </html> |
This part is gene XYL1, which encoding NAD(P)H-dependent D-xylose reductase, it's short name is xylose reductase (XR) .It from Pichia stipitis (taxonomic classification has been changed to Scheffersomyces stipitis). The function of the protein is reduces D-xylose into xylitol, has a preference for NADPH, but can also utilize NADH as cosubstrate, facilitated xylose assimilation in yeast.
This protein is involved in the pathway D-xylose degradation, which is part of Carbohydrate metabolism. Xylose reductase (XR) from Pichia stipitis is one of the enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization.
Catalytic activity i
Xylitol + NAD(P)+ = D-xylose + NAD(P)H.
Kinetics i
KM=142 mM for xylose KM=38 µM for NADH KM=3 µM for NADPH
In our project, we expressed the XYL1 and XYL2 genes, which are associated with xylose metabolism in Saccharomyces cerevisiae EBY100. The whole process of xylose metabolism is as follows:
Xylose is reduced to xylulose under the action of NADPH-dependent xylose reductase (xylosereductase XR). Xylitol is then oxidized to xylulose under the action of xylitol dehydrogenase, which relies on NAD. Xylulose is then phosphorylated by xylulokinase to form 5-phosphate xylose and then into the pentose phosphate pathway, PPP. The intermediate product of PPP pathway 6-phosphate glucose and 3-phosphoric acid glycerol aldehyde through glycolysis pathway to form pyruvate. L-lactate dehydrogenase and coenzyme NADH reduced pyruvate to L-lactic acid. Saccharomyces cerevisiae is not able to use xylose as a result of the lack of enzymes that convert xylose into xylose, but can use the isomers of xylose-xylulose. In Saccharomyces cerevisiae, Xylulose is also first phosphorylated to form 5-phosphate sugar, which enters the PPP pathway and forms lactic acid by glycolysis.
We have constructed engineered bacteria that can use xylose as a carbon source, which can be used as xylose and fermented xylose to produce ethanol, which is the result of our experiment.
[1]Kötter, Peter, and M. Ciriacy. "Xylose fermentation by Saccharomyces cerevisiae." Applied Microbiology & Biotechnology 38.6(1993):776-783.
[2]Yu, S., H. Jeppsson, and Barbel Hahnhagerdal. "Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains.." Applied Microbiology and Biotechnology (1995): 314-320.
In short, XYL1 encoding xylose reductase (XR) from Pichia stipitis (taxonomic classification has been changed to Scheffersomyces stipitis).
Reduces D-xylose into xylitol, facilitated xylose assimilation in yeast
The content of xylose in the medium:
https://static.igem.org/mediawiki/parts/2/2a/T--OUC-China--xylose content.png
the growth curve of xylose strain:
https://static.igem.org/mediawiki/parts/2/2a/T--OUC-China--xylose OD.png
Xylose reductase (XR) will first transform xylose to xylitol, and xylitol dehydrogenase (XDH) can further convert xylitol to xylulose. Then, xylulose will be converted to xylulose 5-phosphate (X5P) by the native xylulose kinase (XK). Apart from the original XR and XDH genes, considering the low copy number of native XK gene in S. cerevisiae, we introduced an extra XK gene to improve the xylose utilization ability.
For our own product yield, our production also improved after substituting our three-gene cassette for OUC_China's two-gene one. The improved production was very obvious after the parts improvement based on our HPLC results.
