Team:TU Darmstadt/Results/Pathway

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Overview

We build up a pathway consisting of seven enzymes (TAL, 4CL, CHS, CHI, F3H, DFR, ANS), which we split into two operons. In addition we build up a biosensor for naringenin, which alowed us to prove this importnant intermediate.

Now, we are satiesfied to present 3 results.

1. Our naringenin operon (K1497016) functions well.

2. Our naringenin biosensor (K1497019 - K1497022) works in several constructions.

3. Our pelargonidin operon (K1497015) functions well and we could extract the desired anthocyanin.

Naringenin operon (K1497016)

This part is a composite of four genes each with the strong RBS (BBa_B0034).

 

- 4-Coumaryl ligase - 4CL (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.

Figure 1: Genetic map of the naringenin operon (K1479016)

In 4 steps naringenin is produced stereoselective out of tyrosine. Malonyl-CoA is consumed during the pathway.

Figure 2: Pathway of the naringenin operon. Starting with tyrosine and ending with naringenin.

 

We created the naringenin biosynthesis operon under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . We 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.

We were 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 we calculated 3 µM naringenin and for the operon with the constitutive promoter BBa_J23100 (BBa_K1497016) we calculated 1.9 µM naringenin.

 

 

Figure 3: 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 4: 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.

fdeR - Naringenin sensor (K1497019 - K1497022)

FdeR is a homo dimeric protein from Herbaspirillum seropedicae. In the presence of naringenin (or naringenin chalchone), FdeR activates the specific promoter region upstream of the fdeR region and induces a strong gene expression. In Herbaspirillum seropedicae the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enable growth with naringenin and the naringenin chalcone.

So in combination with GFP or another fluorescense protein this part can be used as an in vivo naringenin sensor.

Figure 5: Flow chart of the FdeR activated gfp expression. The constitutive expression of fdeR the FdeR proteins form homodimers. In the presence of naringenin, naringenin molecules bind to the FdeR homodimer and operate a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now enabled to bind to the uncharacterized promotor domain. Binding to the promotor domain induces expression of genes downstream of the fdeR promoter region.

Usage and Biology

You can use the reporters for measuring naringenin concentrations in your samples.

Depending on which fluorophor you want to detect, you can use one of three biosensors:


Figure 6: E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample BBa_K1497019 without fluorophor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.

Functional Parameter

The Biobrick BBa_K1497019 produces in E. coli B and K strains the FdeR Protein. We measured the fluorescense of GFP and mKate after the incubation with different concentrations of naringenin. The results are shown in Figure 7 & 8.

Figure 7: Characterization of BBa_K1497020. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well.
Figure 8: Characterization of BBa_K1497021. mKate (BBa_K1055000) fluorescence depends on the concentration of naringenin. We measured the mKate (BBa_K1055000) fluorescence after 16 h incubation with different concentrations of Naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.

Pelargonidin operon (K1497015)

Pelargonidin is an anthocyanin. Anthocyanins are water-soluble vacuolar pigments that appear yellow to dark-blue (pH-dependent), which are responsible for color of flowers and fruits and are health-promoting for humans.

We constructed a pelargonidin producing operon under the control of a T7 promoter (K1497015). The operon consists of 3 genes (flavonon-3beta-hydroxylase, dihydroflavonol 4-reductase, anthocyanindin synthase) each with strong RBS (Fig.1) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 2).

The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided from Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E. coli coding optimized and synthezised by Eurofins MWG opreon.

Futhermore, we designed two different ANS gene version by using XX to verify XXX



Figure 1: Genetic map of pelargonidin producing operon (K1497015). R: RBS; F3H: flavonon-3beta-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanindin synthase.
Figure 2: Pathway of the pelargonidin operon. Starting with naringenin and ending with pelargonidin.

Production

To analyze the pelagonidin production operon (K1497015), we transformed it into E.coli Bl21 (DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan et al., (2007). After the induction with 1mM Isopropyl-?-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9 media and fermented for 48h at 37°C in present of 0.1mM naringenin.

Figure 2: E.coli BL21 (DE3) pellet containing the pelargonidin producing operon after the fermentation. According to Yan et al. (2007) a pelargonidin producing E.coli should be red after a pelargenidin production. The operon with the engineered anthocyanindin synthase produces more pelargonidin.

Extraction

After the expression of pelagonidin producing operon with engineered ANS (K1497015) in present of 0.5mM narigenin we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet and supernatant and verified the pH-dependency of pelargonidin (Fig. 3).

Figure 3: Extracted pelargonidin from E.coli BL21 (DE3) under day light. The color of pelargonidin depends on pH value and solvent. This indicates the present of pelargonidin. Left: Methanol extraction; right: Dichlormethane extraction.

Application

Construction of Grätzel cells

We used the extracted Pelargonidin (produced from E.coli Bl21 (DE3) containing the pelagonidin producing operon, K1497015) to build a Grätzel cell. This was done according to Michael Grätzel & Brian O'Reagan. We were able to produce voltage of 380 mV and 0.5 mA under daylight conditions. 

Figure 4: Schematic representation of a Grätzel cell
Figure 5: Constructed Grätzel cell with extracted pelargonidin. We were able to produce a voltage of 380 mV and 0.5 mA under day light conditions.

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