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Part:BBa_K2423007

Designed by: Oscar Brostrm   Group: iGEM17_Uppsala   (2017-10-01)
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CsADH2946 under control with BBa_J04500

This BioBrick contains the gene coding for the aldehyde dehydrogenase called CsADH2946, which is under regulation with BBa_J04500. This enzyme is a part of the second step in the zeaxanthin to crocin pathway. More specifically it catalyzes the reaction from crocetin dialdehyde to crocetin using NAD+ as a cofactor. The enzyme can be found naturally in Crocus Sativus (the plant that saffron is harvested from).

Since the gene of interest is under regulation BBa_J04500 it has to be induced with lactose or any closely related derivate such as IPTG. In our project we used IPTG to induce overexpression of protein.

Usage and Biology

Saffron, a well recognized, but expensive spice has not only uses in terms of cooking but compounds found in saffron have been shown to help with inflammation (1), neurodegenerative diseases (2) and more. Some of those compounds namely zeaxanthin, crocetin dialdehyde, crocetin and crocin are all a part of the same metabolic pathway in the plant specie Crocus Sativus. Not only are these compounds in saffron helpful in terms their medicinal properties, but also the fact that they are very colorful. These aspects was what drew us at iGEM Uppsala 2017 to work with the pathway from zeaxanthin to crocin in the BioBrick format, put also to integrate the metabolic steps that leads up to crocin (the pathway from farnesyl pyrophospate (FPP) to zeaxanthin) on the chromosome of Escherichia Coli. The enzyme presented on this page catalyzes the second reaction in the zeaxanthin to crocin pathway.

In more detail CsADH2946 is an aldehyde dehydrogenase (ALDH) that oxidizes the two aldehyde groups at each end of crocetin dialdehyde to carboxylic acids using NAD+ as a cofactor. The resulting molecule from this reaction is crocetin. CsADH2946 was discovered through transcriptomic analysis of the chromoplasts of Crocus Sativus (3). The active site of CsADH2946 can be found around a loop containing three cystenin residues in a row (C337, C338, C339; positions were determined from our homology model). The residues that are conserved were found by looking at the template (PDB: 4fqf) (4) used in our homology model.


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 360
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 767
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1284
    Illegal SapI.rc site found at 392

Characterization

We are the first to express and characterize CsADH2946 (Crocus Sativus aldehyde dehydrogenase 2946)! This aldehyde dehydrogenase gene from Crocus Sativus has previously only been identified as a candidate gene through proteome analysis, and has thus never been isolated or characterized before (3). We successfully made a sequence verified BioBrick of CsADH2946 with his-tag (<a href="http://2017.igem.org/Team:Uppsala/Parts">BBa_K2423007</a>). The BioBrick was also combined with the other steps in the pathway and inserted into the zeaxanthin producing E. coli strain for a complete pathway from FPP to crocin. See the result <a href="http://2017.igem.org/Team:Uppsala/Zea-Strain">here</a>! In summary, our experimental data and modeling results show that CsADH2946 is a very good enzyme for this reaction. 

       <img src="CraftingCrocinStep2Final.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 55%; height: auto; padding-top: 5%; padding-bottom: 3%;">

CsADH2946 was transformed and expressed in E. coli strain BL21(DE3*) and purified <a href="http://2017.igem.org/Team:Uppsala/Experiments">using IMAC</a> on an ÄKTA protein purification system. We used a gradient of imidazole concentration from 20–500 mM, in order to get our enzyme as separated as possible from other proteins that ends up in the fractions. The peak pointed at by the arrow in the chromatogram (figure 2) indicates protein that elutes at high imidazole concentration, i.e our desired his-tagged CsADH2946. The purification was followed by SDS-PAGE to analyse the fractions, control purity and verify the protein product. In figure 3a the band at around 60 kDa in the crude pellet indicate an overexpression of a protein in that size range. In the SDS gel of fractions 16–26 collected between 115–145 mL elution volume (figure 3b) there is a strong band at 60 kDa corresponding to the molecular weight of CsADH2946, indicating that our protein was successfully overexpressed and well-separated. 

       <img src="CraftingCrocinElutionStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width:80%; height: auto; padding-top: 3%;">
       <figcaption class="figure-caption figtext" style="text-align: center; padding-bottom: 2%;"> Figure 2. Chromatogram from IMAC-purification of CsADH2946. </figcaption>
       <img src="CraftingCrocinSDS-PAGE1.png" class="figure-img img-fluid picturerow">
       <img src="CraftingCrocinSDS-PAGE2.png" class="figure-img img-fluid picturerow">
       <figcaption class="figure-caption figtext" style="padding-bottom: 3%;"> Figure 3. a) SDS-PAGE gel of from IMAC purification. 1) Crude pellet. 2) Pellet after lysis. 3) Supernatant after lysis. 4) Flow through. 5) Wash with buffer A. 6) PageRuler protein prestained ladder. b) SDS-PAGE gel from IMAC purification. Fractions 16-26 were collected between 115 and 145 ml elution volume. A band at about 60 kDa is clearly visible. </figcaption>

To verify the activity of our purified enzyme CsADH2946 to convert crocetin dialdehyde to crocetin, an <a href="http://2017.igem.org/Team:Uppsala/Experiments">activity measurement assay</a> was performed on a plate reader measuring absorbance of the substrate and product of the reaction. For the experiment we used a 96-well plate in which we included wells with enzyme from pooled fractions + substrate, as well as positive and negative controls, see table 1 for the specifics. </p> 

       <figcaption class="figure-caption figtext" style="text-align:center; padding-top: 3%;"> Table 1. Content of wells used for activity measurement of CsADH2946.</figcaption>
       <img src="CraftingCrocinTableStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-bottom: 3%;">

As can be seen in figure 4, the absorbance of the product crocetin increases over time in well 2 containing enzyme and the substrate crocetin dialdehyde. After 9 hours of reaction, the blue curve corresponding to the enzyme + substrate mixture has increased its absorbance in the exact range of the product. The negative and positive control curves look similar to time point zero, apart from some precipitation of product and substrate indicated by the decreased curves. A definite evidence that we succeeded to produce a functional CsADH2946 enzyme. Using this data, we could <a href="http://2017.igem.org/Team:Uppsala/Model#KM">estimate KM</a> = 20.7842 µM ± 3.5264.

In addition, in figure 5 we can see that well 2 containing enzyme and crocetin dialdehyde has changed color compared to the negative control, to become more yellow like the product crocetin in well 8. This also shows that CsADH2946 was produced and that it converts crocetin dialdehyde into crocetin. 

       <img src="CraftingCrocinActivityStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 50%; height: auto; padding-top: 3%; padding-bottom: 2%;">
       <figcaption class="figure-caption figtext" style="padding-left: 20%; padding-right: 20%"> Figure 4. Activity measurement curve. The dotted lines describe wells at the starting time and the fully drawn lines describe the absorbance after 9 hours. The blue lines indicate wells containing protein and crocetin dialdehyde, red lines describe positive control with only crocetin dialdehyde and black lines correspond to the negative control only containing the desired product crocetin.</figcaption>


       <img src="CraftingCrocinWellStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 60%; height: auto; padding-top: 5%; padding-bottom: 2%;">
       <figcaption class="figure-caption figtext" style="padding-left: 20%; padding-right: 20%; padding-bottom: 3%"> Figure 5. 96-well plate for activity measurements post 24 hours reaction and plate reading. The plate include pooled enzyme fractions 10–15 + substrate crocetin dialdehyde (well 1), pooled enzyme fractions 16–23 + crocetin dialdehyde (well 2), flow through + crocetin dialdehyde (well 3), negative control with only crocetin dialdehyde (well 4), pooled enzyme fractions 10-15 + product crocetin (well 5) pooled enzyme fractions 16–23 + crocetin (well 6), flow through + crocetin (well 7) and positive control with only crocetin (well 8).</figcaption>


Since the enzyme is poorly characterized, we created a homology model and performed stability simulations to verify that our model was reasonable. The homology modeling revealed that CsADH2946 is in fact tetrameric, which helped us in the purification and characterization process. We performed a <a href="http://2017.igem.org/Team:Uppsala/Model#pulling">pulling simulation</a> between the enzyme and its substrate in order to estimate binding energy and calculate a theoretical Kd (=4.9321 µM). The resulting structure of the homology modeling and a plot of the pulling simulation can be seen in figure 6. Using the results from the activity measurement, the earlier unknown Michaelis-Menten kinetic parameters of the reaction could also be estimated using a Bayesian inference algorithm. With this method we got KM (=20.7842 µM). Read more about the homology modeling, dynamics modeling and the kinetic parameter estimation in the <a href="http://2017.igem.org/Team:Uppsala/Model">Modeling section</a>. 

       <img src="CraftingCrocinModelingStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 65%; height: auto; padding-top: 5%;">
       <figcaption class="figure-caption figtext" style="padding-left: 20%; padding-right: 20%; padding-bottom: 3%;"> Figure 6. Homology model of CsADH2946 and a plot demonstrating the pulling of the substrate crocetin dialdehyde from the active site of CsADH2946.

</figcaption>


References

1. Papandreou MA, Kanakis CD, Polissiou MG, Efthimiopoulos S, Cordopatis P, Margarity M, et al. Inhibitory Activity on Amyloid-β Aggregation and Antioxidant Properties of Crocus sativus Stigmas Extract and Its Crocin Constituents. J Agric Food Chem. 2006 Nov 1;54(23):8762–8.

2. Chen L, Qi Y, Yang X. Neuroprotective effects of crocin against oxidative stress induced by ischemia/reperfusion injury in rat retina. Ophthalmic Res. 2015;54(3):157–68.

3. Gómez-Gómez L, Parra-Vega V, Rivas-Sendra A, Seguí-Simarro JM, Molina RV, Pallotti C, et al. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma. Int J Mol Sci [Internet]. 2017 Jan 1;18(1). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297711/

4. Lang BS, Gorren ACF, Oberdorfer G, Wenzl MV, Furdui CM, Poole LB, et al. Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2. J Biol Chem. 2012 Nov 2;287(45):38124–34.

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