Difference between revisions of "Part:BBa K3570000"
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The <b>HMG1</b> (3-hydroxy-3-methylglutaryl coenzyme A) enzyme is considered as a rate-limiting step in the mevalonate pathway. To counteract this, authors [2] amplified it's catalytic domain and named it <b>tHMG1</b>. The overexpression of tHMG1 and <b>CrtE</b> (GGPP synthase) in <i>S. Cerevisiae</i> led to a significant improvement of carotenoid production because the direct precursor GGPP was increased[3].</p>
 
The <b>HMG1</b> (3-hydroxy-3-methylglutaryl coenzyme A) enzyme is considered as a rate-limiting step in the mevalonate pathway. To counteract this, authors [2] amplified it's catalytic domain and named it <b>tHMG1</b>. The overexpression of tHMG1 and <b>CrtE</b> (GGPP synthase) in <i>S. Cerevisiae</i> led to a significant improvement of carotenoid production because the direct precursor GGPP was increased[3].</p>
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The bidirectional TDH3-TEF1 promoter was used in this construction. This sequence was identified from personal communication with Dr. Gilles Truan. TDH3 promoter is a gene-specific promoter from the yeast TDH3 gene[4], as well as TEF1 promoter is a gene-specific promoter from the yeast TDH3 gene[5]. This choice
  
  
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*[2]- Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology, 49(1), 66–71. https://doi.org/10.1007/s002530051138
 
*[2]- Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology, 49(1), 66–71. https://doi.org/10.1007/s002530051138
 
*[3]- Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350. https://doi.org/10.1128/aem.02759-06
 
*[3]- Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350. https://doi.org/10.1128/aem.02759-06
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*[4]- [https://www.yeastgenome.org/locus/S000003424 SGD:S000003424]
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*[5]- [https://www.yeastgenome.org/locus/S000006284 SGD:S000006284]
  
 
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Revision as of 18:55, 13 October 2020


GGPP production enhancement in S. cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 683
    Illegal NheI site found at 3586
    Illegal NheI site found at 4491
    Illegal NheI site found at 5834
    Illegal PstI site found at 50
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1641
    Illegal BglII site found at 3716
    Illegal BglII site found at 5725
    Illegal BglII site found at 5785
    Illegal BamHI site found at 3376
    Illegal XhoI site found at 705
    Illegal XhoI site found at 3415
    Illegal XhoI site found at 3547
    Illegal XhoI site found at 4366
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
    Illegal NgoMIV site found at 870
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

The ultimate goal of this biobrick is to enhance the mevalonate pathway in S. Cerevisiae to increase the pool of geranylgeranyl pyrophosphate (GGPP). The surplus of GGPP can be used to make S. Cerevisiae produce provitamin A (𝛽-carotene), geraniol or limonene using BBa_K3570001, BBa_K3570002 or BBa_K3570003 biobricks respectively(figure 1).

Fig. 1: Metabolic pathway of 𝛽-carotene, limonene and geraniol from Acetyl-CoA in engineered yeast.

Design

According to Rabeharindranto et al. 2019, the enhancement of the mevalonate pathway can be achieved by overexpressing the HMG1 and CrtE genes. The construction as it is presented here differs from the publication in the choice of the promoter. The promoter TDH1 has been chosen instead of Gal1/10 because it was needed elsewhere in our project iGEMINI. We thus created the plasmids containing a truncated version of the HMG1 (tHMG1) gene from S. Cerevisiae and the CrtE gene from X. Dendrorhous as on figure 2.

Fig. 2: HMG1-CrtE-pUC19. The integrative locus used is DPP. The selective locus used is HIS. The genes coding for the truncated version of HMG1 comes from S. cerevisiae. The genes coding for CrtE comes from X. dendrorhous. The bidirectional TDH3-TEF1 promoter and the terminators tCYC1 and tPGK are used.

The HMG1 (3-hydroxy-3-methylglutaryl coenzyme A) enzyme is considered as a rate-limiting step in the mevalonate pathway. To counteract this, authors [2] amplified it's catalytic domain and named it tHMG1. The overexpression of tHMG1 and CrtE (GGPP synthase) in S. Cerevisiae led to a significant improvement of carotenoid production because the direct precursor GGPP was increased[3].

The bidirectional TDH3-TEF1 promoter was used in this construction. This sequence was identified from personal communication with Dr. Gilles Truan. TDH3 promoter is a gene-specific promoter from the yeast TDH3 gene[4], as well as TEF1 promoter is a gene-specific promoter from the yeast TDH3 gene[5]. This choice

Experiments

Refernces

  • [1]- Hery Rabeharindranto, Sara Castaño-Cerezo, Thomas Lautier, Luis F. Garcia-Alles, Christian Treitz, Andreas Tholey, Gilles Truan, 2019. Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metab Eng Commun. 2019 Jun; 8: e00086.
  • [2]- Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology, 49(1), 66–71. https://doi.org/10.1007/s002530051138
  • [3]- Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350. https://doi.org/10.1128/aem.02759-06
  • [4]- SGD:S000003424
  • [5]- SGD:S000006284