Difference between revisions of "Part:BBa K2201241"

Latest revision as of 19:13, 30 October 2017

CrtEBI under constitutive promoter with an amber codon at amino acid position 318 of crtI

This parts contains the part BBa_K274110 with an amber codon at position 318 of the crtI enzyme. With the help of an photoinducable amino acid crtI can be regulated by irradiation with light.

Usage and Biology

Lycopene and β-carotene, both members of the carotenoid family, originate from the terpenoid biosynthetic pathway and are ubiquitous pigments found in bacteria, archaea, fungi and plants (Lee and Schmidt-Dannert, 2002). Their biological functions range from light absorption, thus playing a part in energy uptake and transduction, preventing photo-damage, antioxidant properties, and being precursors for multiple hormones, such as Vitamin A (Palozza and Krinsky, 1992; Vershinin, 1999; Lee and Schmidt-Dannert, 2002). Furthermore, carotenoids are used as colorants in food and cosmetics, but also as food additives, in pharmaceuticals and neutraceuticals (Lee and Schmidt-Dannert, 2002; Yuan et al., 2006). Due to their antioxidant properties, many studies investigate carotenoids with regards to cancer and degenerative diseases (Palozza and Krinsky, 1992; Mayne, 1996; Kirsh et al., 2006; Wang et al., 2009). Thus an upscale in production of carotenoids using microbial fermentation systems with recombinant carotenoid genes in non-carotenogenic microbes like E. coli have increased (Yuan et al., 2006; Yoon et al., 2009; Albermann et al., 2010).
As a proof-of-concept for our photoswitch application, we will incorporate the non-canonical amino acid AzoF into the binding site of crtI by an aaRS (BBa_K2201207), which catalyzes the production of lycopene. crtI is part of the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase (Schaub et al., 2012). By incorporation of AzoF the metabolic flux through this specific pathway can be controlled. In the OFF state, in which AzoF is in cis-form, the colorless substrate 15-cis-phytoene cannot bind and cannot be catalyzed to the red colored all-trans-lycopene. After irradiation AzoF isomerizes into its trans-form, which defines the ON state. The substrate can bind and can be catalyzed into lycopene which can easily be detected.

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 2037
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1573
Illegal NgoMIV site found at 1703
Illegal AgeI site found at 788
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

To investigate the influence of photoswitching on the lycopene production, we cultivated three biological replicates of the three variants and each with one of the AzoF conformations for 24 hours in a 6-wellplate at 37 °C and 400 rpm. The media was supplemented with 1 mM of AzoF and then split in to charges. Both were irradiated for 40 minutes and 100 % brightness, one with 367 nm and the other with 465 nm to photoswitch the amino acids. After the cultivation, we measured the OD₆₀₀ of each sample (Figure 1). The growth was not influenced in a noticeable way by the different AzoF variants, since the error bars overlap each other.

Figure 1: OD₆₀₀ of three biological and three technical replicated of the crtI variants after cultivation.<p> </div> We then extracted the lycopene from the cell pellet and measured the lycopene amount (Figure 2). It can be seen that the TAG353 variant with the trans-AzoF has the highest lycopene production, followed by the TAG353 with the cis-AzoF and TAG318 with the trans-AzoF nearly equal. The TAG318 variant with the cis-AzoF has the lowest lycopene amount.

Figure 2: Absorption spectrum of the four samples of the crtI variants, cultivated with AzoF supplemented to the media photoswitched to cis- or trans-conformation.

The absorption at 476 nm was measured and normed to the OD₆₀₀ of the samples. The relative lycopene production of each crtI and AzoF variant is shown in Figure 3 compared to the unmodified lycopene producer.

Figure 3: Absorption at 476 nm (indicator for lycopene) normalized to the OD₆₀₀ (indication for the cell density) to calculate the relative lycopene production of each crtI variant cultivated with AzoF in cis- and trans-conformation.

Figure 3 shows the effect on the lycopene production based on the incorporation of photoswitched AzoF. The trans-conformation seems to favor the binding activity of the active site, while the cis-conformation seems to reduce the binding activity. The highest difference in the lycopene production is present at the TAG353 variant. Here the cotransformant shows a lycopene production similar to the unmodified lycopene producer when cultivated with trans-AzoF while the productivity is reduced to nearly a third when cultivated with cis-AzoF. The AzoF-variants do not seem to influence the lycopene production when no amber-codon is present in crtI. Concluding, we provided strong evidence that that the observed difference in lycopene production in the three variants is caused by the incorporation and photoswitching of AzoF.

References
Albermann, C., Trachtmann, N., and Sprenger, G.A. (2010). A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome. Biotechnol. J. 5: 32–38.
Bose, M., Groff, D., Xie, J., Brustad, E., and Schultz, P.G. (2006). The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. coli. J. Am. Chem. Soc. 128: 388–389.
Brieke, C., Rohrbach, F., Gottschalk, A., Mayer, G., and Heckel, A. (2012). Light-Controlled Tools. Angew. Chem. Int. Ed. 51: 8446–8476.
Choi, S.-K., Osawa, A., Maoka, T., Hattan, J.-I., Ito, K., Uchiyama, A., Suzuki, M., Shindo, K., and Misawa, N. (2013). 3-β-Glucosyl-3’-β-quinovosyl zeaxanthin, a novel carotenoid glycoside synthesized by Escherichia coli cells expressing the Pantoea ananatis carotenoid biosynthesis gene cluster. Appl. Microbiol. Biotechnol. 97: 8479–8486.
Fraser, P.D., Misawa, N., Linden, H., Yamano, S., Kobayashi, K., and Sandmann, G. (1992). Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. J. Biol. Chem. 267: 19891–19895.
Iwata-Reuyl, D., Math, S.K., Desai, S.B., and Poulter, C.D. (2003). Bacterial Phytoene Synthase: Molecular Cloning, Expression, and Characterization of Erwinia herbicola Phytoene Synthase. Biochemistry (Mosc.) 42: 3359–3365.
Kirby, J. and Keasling, J.D. (2009). Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering. Annu. Rev. Plant Biol. 60: 335–355.
Kirsh, V.A., Mayne, S.T., Peters, U., Chatterjee, N., Leitzmann, M.F., Dixon, L.B., Urban, D.A., Crawford, E.D., and Hayes, R.B. (2006). A Prospective Study of Lycopene and Tomato Product Intake and Risk of Prostate Cancer. Cancer Epidemiol. Prev. Biomark. 15: 92–98.
Klán, P., Šolomek, T., Bochet, C.G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. (2013). Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 113: 119–191.
Lange, B.M., Rujan, T., Martin, W., and Croteau, R. (2000). Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes. Proc. Natl. Acad. Sci. U. S. A. 97: 13172–13177.
Lee, P. and Schmidt-Dannert, C. (2002). Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl. Microbiol. Biotechnol. 60: 1–11.
Mayne, S.T. (1996). Beta-carotene, carotenoids, and disease prevention in humans. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 10: 690–701.
Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K. (1990). Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172: 6704–6712.
Palozza, P. and Krinsky, N.I. (1992). [38] Antioxidant effects of carotenoids in Vivo and in Vitro: An overview. Methods Enzymol. 213: 403–420.
Rodríguez-Villalón, A., Pérez-Gil, J., and Rodríguez-Concepción, M. (2008). Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways. J. Biotechnol. 135: 78–84.
Rohdich, F., Zepeck, F., Adam, P., Hecht, S., Kaiser, J., Laupitz, R., Gräwert, T., Amslinger, S., Eisenreich, W., Bacher, A., and Arigoni, D. (2003). The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. U. S. A. 100: 1586–1591.
Rohmer, M. (1999). The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep. 16: 565–574.
Schaub, P., Yu, Q., Gemmecker, S., Poussin-Courmontagne, P., Mailliot, J., McEwen, A.G., Ghisla, S., Al-Babili, S., Cavarelli, J., and Beyer, P. (2012). On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase. PLoS ONE 7: e39550.
Vershinin, A. (1999). Biological functions of carotenoids - diversity and evolution. BioFactors 10: 99–104.
Wang, Q., Parrish, A.R., and Wang, L. (2009). Expanding the Genetic Code for Biological Studies. Chem. Biol. 16: 323–336.
Yoon, S.-H., Lee, S.-H., Das, A., Ryu, H.-K., Jang, H.-J., Kim, J.-Y., Oh, D.-K., Keasling, J.D., and Kim, S.-W. (2009). Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. J. Biotechnol. 140: 218–226.
Yuan, L.Z., Rouvière, P.E., LaRossa, R.A., and Suh, W. (2006). Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab. Eng. 8: 79–90.
Zimmerman, G., Chow, L.-Y., and Paik, U.-J. (1958). The Photochemical Isomerization of Azobenzene1. J. Am. Chem. Soc. 80: 3528–3531.

@@ Line 5: / Line 5: @@
 This parts contains the part BBa_K274110 with an amber codon at position 318 of the crtI enzyme. With the help of an photoinducable amino acid crtI can be regulated by irradiation with light.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+Lycopene and <i>β</i>-carotene, both members of the carotenoid family, originate from the terpenoid biosynthetic pathway and are ubiquitous pigments found in bacteria, archaea, fungi and plants (Lee and Schmidt-Dannert, 2002). Their biological functions range from light absorption, thus playing a part in energy uptake and transduction, preventing photo-damage, antioxidant properties, and being precursors for multiple hormones, such as Vitamin A (Palozza and Krinsky, 1992; Vershinin, 1999; Lee and Schmidt-Dannert, 2002). Furthermore, carotenoids are used as colorants in food and cosmetics, but also as food additives, in pharmaceuticals and neutraceuticals (Lee and Schmidt-Dannert, 2002; Yuan <i>et al.</i>, 2006). Due to their antioxidant properties, many studies investigate carotenoids with regards to cancer and degenerative diseases (Palozza and Krinsky, 1992; Mayne, 1996; Kirsh <i>et al.</i>, 2006; Wang <i>et al.</i>, 2009).
+Thus an upscale in production of carotenoids using microbial fermentation systems with recombinant carotenoid genes in non-carotenogenic microbes like <i>E. coli </i> have increased (Yuan <i>et al.</i>, 2006; Yoon <i>et al.</i>, 2009; Albermann <i>et al.</i>, 2010). <br>
+As a proof-of-concept for our photoswitch application, we will incorporate the non-canonical amino acid AzoF into the binding site of <i>crtI</i> by an aaRS <html> (<a target="_blank" href="https://parts.igem.org/Part:BBa_K2201207">BBa_K2201207</a>)</html>, which catalyzes the production of lycopene. <i>crtI</i> is part of the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase (Schaub <i>et al.</i>, 2012). By incorporation of AzoF the metabolic flux through this specific pathway can be controlled. In the OFF state, in which AzoF is in <i>cis</i>-form, the colorless substrate 15-<i>cis</i>-phytoene cannot bind and cannot be catalyzed to the red colored all-<i>trans</i>-lycopene. After irradiation AzoF isomerizes into its <i>trans</i>-form, which defines the ON state. The substrate can bind and can be catalyzed into lycopene which can easily be detected.
-<!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2201241 SequenceAndFeatures</partinfo>
@@ Line 18: / Line 19: @@
 <div class="figure small">
-<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_OD_after_irradiation.png">
+https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_OD_after_irradiation.png
 <p class="figure subtitle"><b>Figure 1:</b> OD<sub>600</sub> of three biological and three technical replicated of the <i>crtI</i> variants after cultivation.<p>
 </div>
@@ Line 25: / Line 26: @@
 <div class="figure small">
-<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/95/T--Bielefeld-CeBiTec--YKE_lycopene8.png">
+https://static.igem.org/mediawiki/2017/9/95/T--Bielefeld-CeBiTec--YKE_lycopene8.png <br>
-<p class="figure subtitle"><b>Figure 2:</b> Absorption spectrum of the four samples of the <i>crtI</i> variants, cultivated with AzoF supplemented to the media photoswitched to <i>cis</i>- or <i>trans</i>-conformation.<p>
+<b>Figure 2:</b> Absorption spectrum of the four samples of the <i>crtI</i> variants, cultivated with AzoF supplemented to the media photoswitched to <i>cis</i>- or <i>trans</i>-conformation.
 </div>
@@ Line 32: / Line 33: @@
 <div class="figure small">
-<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_switch_proof.png">
+https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--YKE_switch_proof.png <br>
-<p class="figure subtitle"><b>Figure 3:</b> Absorption at 476&nbsp;nm (indicator for lycopene) normalized to the OD<sub>600</sub> (indication for the cell density) to calculate the relative lycopene production of each <i>crtI</i> variant cultivated with AzoF in <i>cis</i>- and <i>trans</i>-conformation.
+<b>Figure 3:</b> Absorption at 476&nbsp;nm (indicator for lycopene) normalized to the OD<sub>600</sub> (indication for the cell density) to calculate the relative lycopene production of each <i>crtI</i> variant cultivated with AzoF in <i>cis</i>- and <i>trans</i>-conformation.
-</div>
+</div><br>
 Figure 3 shows the effect on the lycopene production based on the incorporation of photoswitched AzoF. The <i>trans</i>-conformation seems to favor the binding activity of the active site, while the <i>cis</i>-conformation seems to reduce the binding activity. The highest difference in the lycopene production is present at the TAG353 variant. Here the cotransformant shows a lycopene production similar to the unmodified lycopene producer when cultivated with <i>trans</i>-AzoF while the productivity is reduced to nearly a third when cultivated with <i>cis</i>-AzoF. The AzoF-variants do not seem to influence the lycopene production when no amber-codon is present in <i>crtI</i>. Concluding, we provided strong evidence that that the observed difference in lycopene production in the three variants is caused by the incorporation and <html><a href="http://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">photoswitching</a> </html>of AzoF.
+<br>
+<br>
+<b>References</b><br>
+<b>Albermann, C., Trachtmann, N., and Sprenger, G.A.</b> (2010). A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome. Biotechnol. J. 5: <b>32–38.</b><br>
+<b>Bose, M., Groff, D., Xie, J., Brustad, E., and Schultz, P.G.</b> (2006). The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. coli. J. Am. Chem. Soc. 128: <b>388–389.</b><br>
+<b>Brieke, C., Rohrbach, F., Gottschalk, A., Mayer, G., and Heckel, A.</b> (2012). Light-Controlled Tools. Angew. Chem. Int. Ed. 51: <b>8446–8476.</b><br>
+<b>Choi, S.-K., Osawa, A., Maoka, T., Hattan, J.-I., Ito, K., Uchiyama, A., Suzuki, M., Shindo, K., and Misawa, N. </b>(2013). 3-β-Glucosyl-3’-β-quinovosyl zeaxanthin, a novel carotenoid glycoside synthesized by Escherichia coli cells expressing the Pantoea ananatis carotenoid biosynthesis gene cluster. Appl. Microbiol. Biotechnol. 97: <b>8479–8486.</b><br>
+<b>Fraser, P.D., Misawa, N., Linden, H., Yamano, S., Kobayashi, K., and Sandmann, G.</b> (1992). Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. J. Biol. Chem. 267: <b>19891–19895.</b><br>
+<b>Iwata-Reuyl, D., Math, S.K., Desai, S.B., and Poulter, C.D. </b>(2003). Bacterial Phytoene Synthase:  Molecular Cloning, Expression, and Characterization of Erwinia herbicola Phytoene Synthase. Biochemistry (Mosc.) 42: <b>3359–3365.</b><br>
+<b>Kirby, J. and Keasling, J.D. (2009).</b> Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering. Annu. Rev. Plant Biol. 60:<b> 335–355.</b><br>
+<b>Kirsh, V.A., Mayne, S.T., Peters, U., Chatterjee, N., Leitzmann, M.F., Dixon, L.B., Urban, D.A., Crawford, E.D., and Hayes, R.B.</b> (2006). A Prospective Study of Lycopene and Tomato Product Intake and Risk of Prostate Cancer. Cancer Epidemiol. Prev. Biomark. 15: <b>92–98.</b><br>
+<b>Klán, P., Šolomek, T., Bochet, C.G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J.</b> (2013). Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 113: <b>119–191.</b><br>
+<b>Lange, B.M., Rujan, T., Martin, W., and Croteau, R. </b>(2000). Isoprenoid biosynthesis: The evolution of two ancient and  distinct pathways across genomes. Proc. Natl. Acad. Sci. U. S. A. 97: <b>13172–13177.</b><br>
+<b>Lee, P. and Schmidt-Dannert, C.</b> (2002). Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl. Microbiol. Biotechnol. 60:<b> 1–11.</b><br>
+<b>Mayne, S.T. </b>(1996). Beta-carotene, carotenoids, and disease prevention in humans. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 10: <b>690–701.</b><br>
+<b>Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K. </b>(1990). Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172: <b>6704–6712.</b><br>
+<b>Palozza, P. and Krinsky, N.I. </b>(1992). [38] Antioxidant effects of carotenoids in Vivo and in Vitro: An overview. Methods Enzymol. 213: <b>403–420.</b><br>
+<b>Rodríguez-Villalón, A., Pérez-Gil, J., and Rodríguez-Concepción, M. </b>(2008). Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways. J. Biotechnol. 135: <b>78–84.</b><br>
+<b>Rohdich, F., Zepeck, F., Adam, P., Hecht, S., Kaiser, J., Laupitz, R., Gräwert, T., Amslinger, S., Eisenreich, W., Bacher, A., and Arigoni, D. </b>(2003). The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. U. S. A. 100: <b>1586–1591.</b><br>
+<b>Rohmer, M. </b>(1999). The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep. 16: <b>565–574.</b><br>
+<b>Schaub, P., Yu, Q., Gemmecker, S., Poussin-Courmontagne, P., Mailliot, J., McEwen, A.G., Ghisla, S., Al-Babili, S., Cavarelli, J., and Beyer, P.</b> (2012). On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase. PLoS ONE 7: <b>e39550.</b><br>
+<b>Vershinin, A.</b> (1999). Biological functions of carotenoids - diversity and evolution. BioFactors 10: <b>99–104.</b><br>
+<b>Wang, Q., Parrish, A.R., and Wang, L. </b>(2009). Expanding the Genetic Code for Biological Studies. Chem. Biol. 16: <b>323–336.</b><br>
+<b>Yoon, S.-H., Lee, S.-H., Das, A., Ryu, H.-K., Jang, H.-J., Kim, J.-Y., Oh, D.-K., Keasling, J.D., and Kim, S.-W. </b>(2009). Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. J. Biotechnol. 140: <b>218–226.</b><br>
+<b>Yuan, L.Z., Rouvière, P.E., LaRossa, R.A., and Suh, W. </b>(2006). Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab. Eng. 8: <b>79–90.</b><br>
+<b>Zimmerman, G., Chow, L.-Y., and Paik, U.-J.</b> (1958). The Photochemical Isomerization of Azobenzene1. J. Am. Chem. Soc. 80: <b>3528–3531.</b><br>
-</div>
-<div class="bevel bl"></div>
-</div>
 <partinfo>BBa_K2201241 parameters</partinfo>