Difference between revisions of "Part:BBa K4440002"

 
 
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Yeast optimized tyrosinase producing gene
 
Yeast optimized tyrosinase producing gene
  
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===Usage and Biology===
 
===Usage and Biology===
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The Tyr1 gene encodes tyrosinase, an enzyme that mediates the synthesis of melanin precursors (L-Dopa, dopaquinone) from the amino acid tyrosine. Aromatic acids are synthesized in <i>S. cerevisiae</i> natively in the shikimate pathway (Fig. 1), but this yeast does not have the capacity to synthesize melanin. Hence, additional enzymes must be introduced (Fig. 2).
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[[Image:Engenering Tyrosine path.svg|600px|thumb|center|<b>Fig 1.</b> Pathway of tyrosine synthesis in yeast.]]
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[[Image:Melanin pathway.svg|600px|thumb|center|<b>Fig 2.</b>  Reaction of melanin biosynthesis catalyzed by tyrosinase.]]
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Most tyrosinase enzymes are heterodimeric complexes, but the one from <i>Bacillus megaterium</i>, which is presented here, functions as a homodimer, simplifying its use. We found that tyrosinase catalyzes all reactions in the pathway (tyrosine - L-Dopa, L-Dopa - Dopaquinone), and research and modeling demonstrated it to be highly efficient and active in yeast cells (Tisma <i>et al.</i>, 2009)
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The tyr1 coding sequence from <i>Bacillus megaterium</i> (Xiaolin Shen <i>et. al</i>, 2019) was taken, followed by removal of selected restriction sites and codon optimization for use in yeast.
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In order to test the activity of tyrosinase in yeast, the tyrosinase was overexpressed using the inducible <i>GAL1</i> promoter. This led to a change in the color of the culture indicating that some pigments are produced (Fig. 3). These experiments showed that tyrosinase Tyr1 from <i>B. megaterium</i> is active in yeast.
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[[Image:Tyr1 part.svg|400px|thumb|center|<b>Fig 3.</b> Observable color change in yeast expressing Tyr1 from <i>B. megaterium</i>.]]
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<b>References:</b>
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<ul>
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<li> Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J., & Larsson, G. (2016). Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior. <i>Scientific Reports</i> 2016 6:1, 6(1), 1–9. https://doi.org/10.1038/srep36117 </li>
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<li> Tišma, M., Zelić, B., Vasić-Rački, D., Žnidaršič-Plazl, P., & Plazl, I. (2009). Modeling of laccase-catalyzed l-DOPA oxidation in a microreactor. <i>Chemical Engineering Journal</i>, 149(1–3), 383–388. https://doi.org/10.1016/J.CEJ.2009.01.025 </li>
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</ul>
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Latest revision as of 02:09, 12 October 2022


Tyr1

Yeast optimized tyrosinase producing gene

Usage and Biology

The Tyr1 gene encodes tyrosinase, an enzyme that mediates the synthesis of melanin precursors (L-Dopa, dopaquinone) from the amino acid tyrosine. Aromatic acids are synthesized in S. cerevisiae natively in the shikimate pathway (Fig. 1), but this yeast does not have the capacity to synthesize melanin. Hence, additional enzymes must be introduced (Fig. 2).

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Fig 1. Pathway of tyrosine synthesis in yeast.
Error creating thumbnail: File missing
Fig 2. Reaction of melanin biosynthesis catalyzed by tyrosinase.

Most tyrosinase enzymes are heterodimeric complexes, but the one from Bacillus megaterium, which is presented here, functions as a homodimer, simplifying its use. We found that tyrosinase catalyzes all reactions in the pathway (tyrosine - L-Dopa, L-Dopa - Dopaquinone), and research and modeling demonstrated it to be highly efficient and active in yeast cells (Tisma et al., 2009)

The tyr1 coding sequence from Bacillus megaterium (Xiaolin Shen et. al, 2019) was taken, followed by removal of selected restriction sites and codon optimization for use in yeast.

In order to test the activity of tyrosinase in yeast, the tyrosinase was overexpressed using the inducible GAL1 promoter. This led to a change in the color of the culture indicating that some pigments are produced (Fig. 3). These experiments showed that tyrosinase Tyr1 from B. megaterium is active in yeast.

Error creating thumbnail: File missing
Fig 3. Observable color change in yeast expressing Tyr1 from B. megaterium.

References:

  • Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J., & Larsson, G. (2016). Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior. Scientific Reports 2016 6:1, 6(1), 1–9. https://doi.org/10.1038/srep36117
  • Tišma, M., Zelić, B., Vasić-Rački, D., Žnidaršič-Plazl, P., & Plazl, I. (2009). Modeling of laccase-catalyzed l-DOPA oxidation in a microreactor. Chemical Engineering Journal, 149(1–3), 383–388. https://doi.org/10.1016/J.CEJ.2009.01.025


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]