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Revision as of 22:14, 14 October 2021
LCYe-ofCCD1m fusion with a LGS linker to produce α-ionone in Saccharomyces cerevisiae
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 282
Illegal BglII site found at 812
Illegal BamHI site found at 396
Illegal BamHI site found at 2535
Illegal BamHI site found at 3387 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
This sequence codes for an enzymayivc fusion between LcyE, that transforms Lycopene into ε-carotene, and ofCCD1 that transforms ε-carotene into α-ionone. Those two sequences are codon optimized for an expression into S.cerevisiae.
The LcyE sequence comes from Latuca sativa and ofCCD1 comes from Osmanthus fragrans genome. We take advantage of the publication of (Chen et al. 2019) to design our enzymatic fusion and to get the gene sequences.
Characterisation
Production of ε-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-pVIOLETTE strains, Figure 4 shows that lycopene is converted into a new product with a higher retention time upon induction. Considering the yellow color of pVIOLETTE strains, as well as the in-line following α-ionone production results, this new peak most likely corresponds to ε-carotene, the expected precursor.
Production of α-ionone
The α-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-VIOLETTE strain. A peak can be observed at the same retention time as the α-ionone standard for the induced LycoYeast-VIOLETTE strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The α-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology).The production of alpha-ionone, the main molecule of the violet odour, was successfully achieved with this construction.
The enzymatic fusion LcyE-ofCCD1 works under those lab conditions
References