Part:BBa_K3930017

Fusion between phCCD1 and the fyn anchor with linker to produce β-ionone Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 864
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This sequence codes for a phCCD1 enzyme, that transforms beta-carotene into beta-ionone, fused with a fyn anchor, which allows relocation of phCCD1 to the membrane. This sequence is codon optimized for its expression into S.cerevisiae. The phCCD1 sequence comes from Petunia hybrida and is described in the publication of (López et al. 2020).

Characterization

Production of β-ionone

β-ionone is a very volatile molecule. 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 1 shows the GC-MS spectrum for the LycoYeast-FRAMBOISE-notfused strain. A peak can be observed at the same retention time as the β-ionone standard for the induced LycoYeast-FRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The β-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). Thus, production of β-ionone, the main molecule of the violet odor, was successfully achieved with this construction.

Figure 1: GC-MS analysis of the dodecane layer of the LycoYeast-pFRAMBOISE-notfused

β-ionone is produced in vivo by our strain LycoYeast-pFRAMBOISE-notfused. On the right are presented the matching mass spectra between the standard and the observed peak.

We concluded the fyn-phCCD1 part works under those lab conditions

References

1. 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.