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Team:TU Darmstadt/Results/Pathway
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Revision as of 01:00, 18 October 2014
Overview
We constructed a biosynthetic pathway consisting of seven enzymes (TAL, 4CL, CHS, CHI, F3H, DFR, ANS), which were split into two separate operons. In addition we designed a biosensor, which allowed us to measure the concentration of the key intermediate naringenin.
Now, we are proud to present the following results:
1. Our naringenin operon (K1497016) works well.
2. Our naringenin biosensor (K1497019 - K1497022) works in several constructions.
3. We engineered an improved anthocyanidin synthase eANS (K1497002) by using rational design methods.
4. Our pelargonidin operon (K1497015) works well and we could extract the desired pelargonidin.
5. We constructed a functional Grätzel cell (DSC) with our produced and extracted pelargonidin.
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)
- Chalcone isomerase - CHI (BBa_K1497000)
- Chalcone synthase - CHS (BBa_K1497001)
Together, these genes define the naringenin biosynthesis operon without a promotor. In addition of a promotor part the device is able to produce S-naringenin. This device is working in E. coli K and B strains.
Naringenin is produced stereoselectively from tyrosine. Malonyl-CoA is consumed during the biosynthesis process.
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 16 h of incubation 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 the cell transformed with BBa_K1497017 exhibited GFP fluorescence.
We were also able to measure the GFP fluorescence quantitatively and to calculate the production yield of both operons with the help of a calibration curve for the naringenin sensor (Figure 4). We calculated a concentration of 1.9 µM (BBa_K1497016) and 3 µM (BBa_K1497017) naringenin repectively.
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.
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:
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.
In addition, we proved the expression of GFP by running a SDS-PAGE (12,5% polyacrylamide gel).
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 8 & 9.
eANS (K1497002)
The anthocyanidin synthase from Fragaria x ananassa (ANS, EC 1.14.11.19) catalyzes many reactions in the anthocyanidin pathway. We used its functionality by catalyzing the conversion of the leucoanthocyanidin (2R,3S,4S)-cis-lucopelargonidin to the anthocyanidin pelargonidin. It also catalyzes the conversion of the leucoanthocyanidin to flavonol (kampferol). In order to avoiding this side reaction and enhance metabolic flux, we designed a protein scaffold (BBa_K1497031).
Earlier studies hypothesized that ANS may be involved in metabolic channeling in their native organisms. By modelling the mechanical movements of ANS, we discovered a strong flexibility at the C-terminus. Subsequently we modelled ANS' structure and its movements. Thereby we detected a tail at the C-terminus of the enzyme fluctuating and covering the active site (Fig. 15). In oder to improve the enzyme we decided to remove the tail and to construct our pelargonidin operon with this engineered ANS (eANS). The laboratory results confirmed the previous modeling results. The engineered ANS exhibited better yields than the original one when used in an operon producing pelargonidin (BBa_K1497015).
In order to optimize the metabolic channeling of ANS, we chose a rational protein engineering approach.
The first step of our multi scale and rational engineering project was the creation of
a sophisticated 3D model with YASARA structure. This model was then used for a structural refinement with the SCWRL alghorithm and was energy minimized with YASARA nova force field.
Afterwards, we started a true mechanical engineering approach to determine the movements within the protein. Therefore, a Gaussian Network Model (GNM) (Fig. 11) and an Anisotropic Network Model (Fig. 12) were implemented.
Those are simple models which simulate the mechanical behavior of the protein. Moreover, Linear Response Theory (LRT) (Fig. 14) was used to simulate the substrate binding inside the pocket and thus trigger an induced fit mechanism.
Afterwards we collect our data, defined rational mutations and finally constructed eANS. With this eANS version another MD simulation was started and the sequence of the protein was given to the wetlab for in vitro construction and in vivo characterization.
Pelargonidin operon (K1497015)
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.16) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 17).
The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided by Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E.coli codon optimized and synthezised by Eurofins MWG Operon.
Futhermore, We engineered an improved anthocyanidin synthase (eANS) by using rational design methods.
Production
To analyze the pelargonidin 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-beta-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9 media and fermented for 48h at 30°C in the presence of 0.1mM naringenin.
Extraction
After the production of pelargonidin producing operon with eANS (K1497015) in the presence of 0.5mM narigenin, we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet. We verified the pH-dependency of pelargonidin color (Fig. 19).
Application
Construction of Grätzel cells
We used the extracted Pelargonidin (produced from E.coli BL21 (DE3) containing the pelargonidin producing operon, K1497015) to build a Grätzel cell. This was done according to Michael Grätzel & Brian O'Reagan. We were able to produce a voltage of 380 mV and a current 0.5 mA under daylight conditions.
