Difference between revisions of "Part:BBa K3930016"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
<p>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 <i>S.cerevisiae</i>. | <p>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 <i>S.cerevisiae</i>. | ||
| − | 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>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 (ε-carotene) closer to the enzyme which will transform it into the molecule of interest, α-ionone.</p> |
| + | <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> | ||
<br> | <br> | ||
<h2>Characterization</h2> | <h2>Characterization</h2> | ||
Revision as of 15:19, 16 October 2021
Fusion between CrtY and phCCD1 with a LGS linker to produce β-ionone
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1641
Illegal BglII site found at 1824
Illegal BamHI site found at 1989 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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, lycopene is converted into a new product with a higher retention time upon induction (Figure 1). Considering the yellow color of pFRAMBOISE strains, as well as the in-line following α-ionone production results, this new peak most likely corresponds to ε-carotene, the expected precursor.
The CrtY side of our enzymatic fusion work those lab conditions
References
- 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.
- 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.