Difference between revisions of "Part:BBa K3570000"

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GGPP production enhancement in S. cerevisiae

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 choice of a couple of promoters was essential for the optimal functioning of our construct since tHMG1 and CrtE needed to be expressed at a constant level under different conditions (such as carbon source, for example). TDH3 and TEF1 promoters proved themselves to have a non-significant difference in the expression level of the downstream gene, and to be quite versatile under different carbon sources for yeast[4]. TDH3 promoter is a gene-specific promoter from the yeast TDH3 gene[5], in parallel, TEF1 promoter is a gene-specific promoter from the yeast TEF1 gene[6]. The bidirectional TDH3-TEF1 promoter was designed for this construction. The sequence was identified from personal communication with Dr. Gilles Truan.

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]- Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., & Vickers, C. E. (2015). Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microbial Cell Factories, 14(1). https://doi.org/10.1186/s12934-015-0278-5
• [5]- SGD:S000003424
• [6]- SGD:S000006284