Part:BBa_K2201241

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.

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