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LCYe-ofCCD1m fusion with a LGS linker to produce α-ionone in Saccharomyces cerevisiae Sequence and Features

Introduction

This sequence codes for an enzymatic fusion between LcyE, converting lycopene into ε-carotene, and ofCCD1, transforming ε-carotene into α-ionone.

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 LcyE sequence comes from Latuca sativa and ofCCD1 comes from Osmanthus fragrans genome. We took advantage of the publication from Chen et al. (2019) to design our enzymatic fusion and to retrieve the gene sequences.

Characterization

Production of ε-carotene

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

The promoter controls in the pVIOLETTE construct the expression of the CrtY gene, which converts lycopene to β-carotene. The production of β-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids produced by the LcyE part of our enzymatic fusion, 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), 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. The LcyE part of our enzymatic fusion is functional.

Figure 1: Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE expressing LcyE-ofCCD1

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 should be produced by the ofCCD1 part of our enzymatic fusion, and 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 . 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 with the construction LcyE-ofCCD1.

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.

We concluded that the enzymatic fusion LcyE-ofCCD1 is fully functionnal under these lab conditions.

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.