Difference between revisions of "Part:BBa K3930018"
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>This sequence codes for the CrtY enzyme, that transforms Lycopene into &beta;-carotene. The <i>CrtY</i> sequence comes from <i>Pantoea ananatis</i> and was codon optimized for its expression into <i>S.cerevisiae</i>. Its sequence is described into the publication of (López et al. 2020).
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<p>This sequence codes for the CrtY enzyme, that transforms Lycopene into &beta;-carotene. The <i>CrtY</i> sequence comes from <i>Pantoea ananatis</i> and was codon optimized for its expression into <i>S. cerevisiae</i>. Its sequence is described into the publication of López et al. (2020).
 
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<h2>Characterisation</h2>
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<h2>Characterization</h2>
 
<h3>Production of &beta;-carotene</h3>
 
<h3>Production of &beta;-carotene</h3>
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<p>The carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after this lysis was analyzed by RP-HPLC using a C18 column.In the LycoYeast-pFRAMBOISE-notfused strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE-notfused strains and resulting the &beta;-ionone production, this new peak most likely corresponds to &beta;-carotene, i.e., the expected precursor.
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<p>To demonstrate the CrtY activity, carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after this lysis was analyzed by RP-HPLC using a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE-notfused strains and resulting the &beta;-ionone production, this new peak most likely corresponds to &beta;-carotene, i.e., the expected precursor.
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Nonetheless, the negative control with no inducer also presents some </p>
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The negative control with no inducer also presents the same activity, meaning the induction system is not working here. Nonetheless, CrtY is functional to produce &beta;-carotene.</p>
 
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                     <b>Figure 5: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE-notfused</b>   
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                     <b>Figure 1: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE-notfused possessing the <i>CrtY gene</i></b>   
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                     <p>tr= retention time; 3 peaks are observed in a non-modified and a modified but not induced LycoYeast while 4 peaks are present in a LycoYeast-pFRAMBOISE-notfused strain.</p>
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                     <p>tr= retention time.</p>
 
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<p><b> The CrtY (BBa_K3930018) part works under those lab conditions </p></b>
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<p><b> We concluded that the CrtY (BBa_K3930018) part works under those lab conditions. </p></b>
 
<h2>References</h2>
 
<h2>References</h2>
 
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Latest revision as of 08:51, 17 October 2021


Gene coding for the lycopene cyclase CrtY 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]

Introduction

This sequence codes for the CrtY enzyme, that transforms Lycopene into β-carotene. The CrtY sequence comes from Pantoea ananatis and was codon optimized for its expression into S. cerevisiae. Its sequence is described into the publication of López et al. (2020).

Characterization

Production of β-carotene

To demonstrate the CrtY activity, carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after this lysis was analyzed by RP-HPLC using a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE-notfused strains and resulting the β-ionone production, this new peak most likely corresponds to β-carotene, i.e., the expected precursor. The negative control with no inducer also presents the same activity, meaning the induction system is not working here. Nonetheless, CrtY is functional to produce β-carotene.


Figure 1: Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE-notfused possessing the CrtY gene

tr= retention time.


We concluded that the CrtY (BBa_K3930018) part works under those lab conditions.

References

  1. López J, Bustos D, Camilo C, Arenas N, Saa PA, Agosin E. 2020. Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene. Front Bioeng Biotechnol. 8:578793. doi:10.3389/fbioe.2020.578793.
    2.