Difference between revisions of "Part:BBa K3930023"

Latest revision as of 09:51, 17 October 2021

Galactose inducible promoter Sequence and Features

Introduction

The promoter Gal1 is a galactose inducible promoter. This sequence comes from Sacharomyces cerevisiae genome and was assembled by IDT. This part is related to the extensively used and characterized part (BBa_J63006). Nonetheless, our promoter lacks a Kozak consensus sequence. This part is the twin of (BBa_K1969004) part, but no experience results were obtained for it. We demonstrated with our part that this Gal 1 promoter is functional in S. cerevisiae.

Characterization

Production of ε-carotene

All the experiments that characterized this part are related to the final construct pVIOLETTE (BBa K3930003), which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

The Gal1 promoter was placed upstream of the (BBa_K3930024) part, allowing the synthesis of ε-carotene and α-ionone. The characterization of the Gal1 promoter is therefore performed by observing the production of these two molecules when galactose is added to the culture medium.

The carotenoids are contained in the cells. They 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 lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pVIOLETTE strains (which express the LcyE-ofCCD1 fusion upon galactose induction). Figure 1 shows that lycopene is converted into a new product with a higher retention time. Considering the α-ionone production results, we concluded this new peak most likely corresponds to ε-carotene, produced only in the presence of galactose, thus indicating the functionality of the Gal1 promoter.

Figure 1: Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE upon galactose induction

tr= retention time; 3 peaks are observed in a non-modified and a modified but not induced LycoYeast, while a 4th peak is present in a LycoYeast-pVIOLETTE induced strain.

Production of α-ionone

The α-ionone prodution is also a control molecule of Gal1 promoter functionality, and this molecule 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 2 shows the GC-MS spectrum for the LycoYeast-pVIOLETTE strains expressing the part LcyE-ofCCD1 upon galactose induction. A peak is observed at the same retention time as the α-ionone standard for the induced LycoYeast-pVIOLETTE strain. The mass spectra associated with this peak matches 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 α-ionone, the main molecule of the violet odor, was successfully achieved thanks to the Gal1 promoter.

Figure 2: GC-MS analysis of the dodecane layer from the LycoYeast-pVIOLETTE expressing LcyE-ofCCD1

α-ionone is produced in vivo by our strain upon galactose induction. Panel 1 = α-ionone standard ; Panel 2 = LycoYeast WT ; Panel 3 = Lycoyeast-pVIOLETTE not induced ; Panel 4 = Lycoyeast-pVIOLETTE induced. On the right are presented the standard α-ionone mass spectra which matches to the observed new peak molecule.

This pGAL1 promoter without Kozak sequence is functional under these lab conditions.