Difference between revisions of "Part:BBa K3930016"
| Line 13: | Line 13: | ||
<h2>Characterization</h2> | <h2>Characterization</h2> | ||
<h3>Production of β-carotene</h3> | <h3>Production of β-carotene</h3> | ||
| − | <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, | + | <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, 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.</p> |
<br> | <br> | ||
<div class="center"> | <div class="center"> | ||
| Line 23: | Line 23: | ||
<a href="https://2021.igem.org/wiki/images/a/ac/T--Toulouse_INSA-UPS--2021_results_Carotenoid_analysis.png" class="internal" title="Enlarge"></a> | <a href="https://2021.igem.org/wiki/images/a/ac/T--Toulouse_INSA-UPS--2021_results_Carotenoid_analysis.png" class="internal" title="Enlarge"></a> | ||
</div> | </div> | ||
| − | <b>Figure | + | <b>Figure 1: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pFRAMBOISE</b> |
| − | <p>tr= retention time | + | <p>tr= retention time.</p> |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 08:21, 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.
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.