Coding

Part:BBa_K3930009

Designed by: Thomas Gaudin   Group: iGEM21_Toulouse_INSA-UPS   (2021-10-07)


Gene coding for the DBR1 enzyme Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 856
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 856
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 856
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This sequence codes for the DBR1 enzyme, an enzyme that transform β-ionone into dihydro-β-ionone a molecule with a violet odor. The sequence derived from the Artemisia annua genome, but its sequence is codon optimized for an expression into S. cerevisiae. We took advantage of the publication of Zhang et al. (2018).

Characterization

Production of dihydro-β-ionone

All the experiments that characterized this part are related to the final construct pFLEUR (BBa_K3930000) which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

The strain LycoYeast-pFLEUR (BBa_K3930000), carrying the DBR1 gene, is cultivated and induced for the expression of DBR1 by adding copper to the culture medium in late exponential phase. After waiting for the enzymes to be produced, the yeast cells were lysed in TRIS-HCl pH 6.5 buffer. The activity was studied by GC-MS using β-ionone as substrate, adapting the method described by Zhang et al. (2018). Assays contained 50 mM TRIS-HCl (pH 6.5), 1 mM NADPH, 2 mM dithiothreitol (DTT), 1 mM β-ionone and 500 μL of the yeast lysate in a total volume of 1 mL at 45 °C for 1.5 h. At the beginning and end of the incubation, 200 µL of the mix were collected and then a liquid-liquid extraction was carried out by vortexing the sample with 200 µL of dodecane. The dodecane phase was then analyzed by GC-MS.

Figure 1 shows the GC-MS spectrum for the LycoYeast-pFLEUR strain expressing DBR1. A peak can be observed at the same retention time as the dihydro-β-ionone standard for the induced LycoYeast-pFLEUR strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The dihydro-β-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). The production of dihydro-β-ionone in vitro was successfully achieved with a lysate of a LycoYeast expressing the DBR1 part.


Figure 1: GC-MS analysis of the dodecane layer of the LycoYeast-pFLEUR induce for DBR1

dihydro-β-ionone is produced in vitro by our strain when it is induced by copper. On the right are presented the mass spectra that correspond between the standard and the observed peak. First panel is the β-ionone standard. Second panel is the dihydro-β-ionone standard. Third panel is the 1.5 hours induced LycoYeast-pFLEUR. Forth panel is the induced LycoYeast-pFLEUR at starting point.

We concluded this DBR1 parts (BBa_K3930009) works under our lab conditions.


References

  1. Zhang X, Liao S, Cao F, Zhao L, Pei J, Tang F. 2018. Cloning and characterization of enoate reductase with high β-ionone to dihydro-β-ionone bioconversion productivity. BMC Biotechnol. 18:26. doi:10.1186/s12896-018-0438-x.


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