Difference between revisions of "Part:BBa K3930018"
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<h2>Characterisation</h2>
 
<h2>Characterisation</h2>
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<h3>Production of &beta;-ionone</h3>
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<h3>Production of &beta;-carotene</h3>
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<p>The &beta;-ionone is very volatile. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest.The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020).Figure 5 shows the GC-MS spectrum for the LycoYeast-FRAMBOISE-notfused strain. A peak can be observed at the same retention time as the &beta;-ionone standard for the induced LycoYeast-FRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The &beta;-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology).The production of &beta;-ionone, the main molecule of the violet odour, was successfully achieved with this construction.</p>
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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, Figure 4 shows that lycopene is converted into a new product with a higher retention time upon induction. Considering the yellow color of pFRAMBOISE-notfused strains, as well as the in-line following &beta;-ionone production results, this new peak most likely corresponds to &beta;-carotene, the expected precursor.
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Nonetheless, it seems that the negative control from the Teto7 promoter doesn't work when there is no inducer added in the media of culture</p>
 
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                         <a href="https://2021.igem.org/wiki/images/a/ac/T--Toulouse_INSA-UPS--2021_results_Carotenoid_analysis.png" class="internal" title="Enlarge"></a>
 
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                     <b>Figure 5: </b> <b>GC-MS analysis of the dodecane layer of the LycoYeast-pFRAMBOISE-notfused</b>  
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                     <b>Figure 5: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE-notfused</b>
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                     <p>&beta;-ionone is produced in vivo by our strain LycoYeast-pFRAMBOISE-notfused. On the right are presented the mass spectra that correspond between the standard and the observed peak.</p>
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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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Revision as of 08:35, 15 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 is codon optimized for an expression into S.cerevisiae. Its sequence is described into the publication of (López et al. 2020).

Characterisation

Production of β-carotene

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, Figure 4 shows that lycopene is converted into a new product with a higher retention time upon induction. Considering the yellow color of pFRAMBOISE-notfused strains, as well as the in-line following β-ionone production results, this new peak most likely corresponds to β-carotene, the expected precursor. Nonetheless, it seems that the negative control from the Teto7 promoter doesn't work when there is no inducer added in the media of culture


Figure 5: Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE-notfused

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.


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.