Part:BBa_K3930024

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This sequence codes for an enzymayivc 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.

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

Figure 4: Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE

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

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.

Figure 5: GC-MS analysis of the dodecane layer of the LycoYeast-pVIOLETTE

α-ionone is produced in vivo by our strain when it is induced by galactose. On the right are presented the mass spectra that correspond between the standard and the observed peak.

The enzymatic fusion LcyE-ofCCD1 works under those 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.