Difference between revisions of "Part:BBa K3930016"
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>This sequence codes for an enzymayivc fusion between CrtY that transforms Lycopene into &beta;-carotene, and phCCD1 that transforms &beta;-carotene into &beta;-ionone. Those two sequences are codon optimized for an expression into <i>S.cerevisiae</i>.
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<p>This sequence codes for an enzymatic fusion between CrtY that transforms Lycopene into &beta;-carotene, and phCCD1 that transforms &beta;-carotene into &beta;-ionone. Those two sequences are codon optimized for an expression into <i>S. cerevisiae</i>.
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The <i>CrtY</i> sequence comes from <i>Pantoea ananatis</i> and <i>phCCD1</i> comes from <i>Petunia hybrida</i> genome. We take advantage of the publication of (Chen et al. 2019) to design our enzymatic fusion, and the sequences are described into the publication of (López et al. 2020).
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<p>These two sequences are codon optimized for expression into <i>S. cerevisiae</i>. These two enzymes are fused by a long linker composed of 4 times 4 glycines followed by a serine (LGS). This linker brings the substrate (&epsilon;-carotene) closer to the enzyme which will transform it into the molecule of interest, &alpha;-ionone.</p>
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<p>The <i>CrtY</i> sequence comes from <i>Pantoea ananatis</i> and <i>phCCD1</i> comes from <i>Petunia hybrida</i> genome. We took advantage of the publication of Chen et al. (2019) to design our enzymatic fusion, and the sequences are described into the publication of López et al. (2020).</p>
 
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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 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 strains, as well as the in-line following &alpha;-ionone production results, this new peak most likely corresponds to &epsilon;-carotene, the expected precursor.</p>
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<p>All the experiments that characterized this part are related to the final construct pFRAMBOISE-fused <a href="https://parts.igem.org/Part:BBa_K3930001" class="pr-0" target="_blank">(BBa_K3930001)</a> which was cloned into the <i>S. cerevisiae</i> LycoYeast strain. For more information on the experimental background, please refer to this part.</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-fused strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE-fused strains, as well as the in-line following &beta;-ionone production results, this new peak most likely corresponds to &beta;-carotene, the expected precursor, which means that the CrtY part is functional.</p>
 
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                     <b>Figure 4: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE</b>  
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                     <b>Figure 1: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE</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-pFRABOISE strain</p>
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                     <p>tr= retention time.</p>
 
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<p><b> The CrtY side of our enzymatic fusion work those lab conditions</b></p>
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<p><b> We concluded the CrtY side of our enzymatic fusion works in those lab conditions. The phCCD1 part still needs to be characterized.</b></p>
 
<h2>References</h2>
 
<h2>References</h2>
 
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Latest revision as of 06:07, 17 October 2021


Fusion between CrtY and phCCD1 with a LGS linker to produce β-ionone Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1641
    Illegal BglII site found at 1824
    Illegal BamHI site found at 1989
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

This sequence codes for an enzymatic fusion between CrtY that transforms Lycopene into β-carotene, and phCCD1 that transforms β-carotene into β-ionone. Those two sequences are codon optimized for an expression into S. cerevisiae.

These two sequences are codon optimized for expression into S. cerevisiae. These two enzymes are fused by a long linker composed of 4 times 4 glycines followed by a serine (LGS). This linker brings the substrate (ε-carotene) closer to the enzyme which will transform it into the molecule of interest, α-ionone.

The CrtY sequence comes from Pantoea ananatis and phCCD1 comes from Petunia hybrida genome. We took advantage of the publication of Chen et al. (2019) to design our enzymatic fusion, and the sequences are described into the publication of López et al. (2020).


Characterization

Production of β-carotene

All the experiments that characterized this part are related to the final construct pFRAMBOISE-fused (BBa_K3930001) which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

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-fused strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE-fused strains, as well as the in-line following β-ionone production results, this new peak most likely corresponds to β-carotene, the expected precursor, which means that the CrtY part is functional.


Figure 1: Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE

tr= retention time.


We concluded the CrtY side of our enzymatic fusion works in those lab conditions. The phCCD1 part still needs to be characterized.

References

  1. Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459. doi:10.1021/acs.jafc.9b00860.
  2. 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.
    3.