Part:BBa_K1497016

Constitutive naringenin producing operon

Naringenin is the main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolites in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are involved in the degradation of caffeine and increase the effect of caffeine after the inhibition with naringenin. The biosynthesis of naringenin is encoded by four genes and these proteins convert L-tyrosine to the bioactive enantiomer S-naringenin.

Figure 1 Reaction Scheme of the naringenin producing operon. The substrate for the reaction is L-tyrosine. The substrate is metabolized to S-naringenin in serval steps.

Usage and Biology

This part is a composite of four genes each with the strong RBS (BBa_B0034). 4-coumaryl ligase - 4-CL BBa_K1033001 Tyrosine ammonia lyase - TAL BBa_K1033000 Chalchone isomerase - CHI BBa_K1497100 Chalchone synthase - CHS BBa_K1497101 Together, these genes build the naringenin biosynthesis operon without a promotor. In addition of a promotor part the device is able to build S-naringenin. These device is working in E. coli K and B strains.

iGEM TU Darmstadt 2014 :)

Figure 2 Genetic map of the naringenin operon with T7 promoter (BBa_K1497017). This device build naringenin in E. coli BL21(DE3) in present of the inductor IPTG .

Functional Parameters

The iGEM Team TU Darmstadt 2014 created the naringenin biosynthesis operons under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . They measured the naringenin production after a 16 h incubation time with the naringenin biosensor BBa_K1497020. The cell pellets from E. coli BL21(DE3) – pSB1C3-fdeR-gfp with and without T7-naringenin operon (BBa_K1497017) are shown in figure 3. Only in the cell pellet with BBa_K1497017 exhibited GFP fluorescence. The Darmstadt team was also able to measure the GFP fluorescence quantitatively and to calculate with the help of a calibration curve for the naringenin sensor the production yield of both operons (Figure 4). For BBa_K1497017 was 3 µM naringenin calculated and for the operon with the constitutive promoter BBa_J23100 (BBa_K1497016) was 1.9 µM naringenin calculated.

iGEM TU Darmstadt 2014 :)

Figure 2 Cell pellets with and without T7-Naringenin operon from E. coli BL21(DE3)-pSB1C3-fdeR-gfp. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence.

Figure 1 Fluorescence of cells with and without the T7-naringenin operon BBa_K1497017 from E. coli BL21(DE3)-pSB1C3-fdeR-gfp and J23100-naringenin operon (BBa_K1497016) from E. coli Top10-pSB1C3-fdeR-gfp, respectively. E. coli BL21(DE3)-pSB1C3-fdeR-gfp without T7-naringenin operon showed no detectable fluorescence. Only in the cells with the functional operon is the GFP fluorescence measurable. The estimated yields are 3 µM for BBa_K1497017 and 1,9 µM for BBa_K1497016.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1147
  Illegal NheI site found at 4674
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1714
  Illegal BglII site found at 4683
  Illegal XhoI site found at 3681
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1820
  Illegal NgoMIV site found at 2652
  Illegal NgoMIV site found at 4654
  Illegal NgoMIV site found at 5230
  Illegal AgeI site found at 1915
  Illegal AgeI site found at 2081
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3110
  Illegal BsaI site found at 4603
  Illegal BsaI.rc site found at 1346
  Illegal BsaI.rc site found at 4085