Part:BBa_K3570002

Provitamin A synthesis from GGPP in S. cerevisiae

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2012
  Illegal NheI site found at 5006
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 738
  Illegal BglII site found at 3364
  Illegal BglII site found at 5489
  Illegal BglII site found at 6585
  Illegal BamHI site found at 3157
  Illegal XhoI site found at 4943
  Illegal XhoI site found at 4984
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  Illegal NgoMIV site found at 2018
  
• 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. 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 CYC1 and PGK1 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.

CYC1 and PGK1 terminators are chosen because of their large usage in yeast biotechnological manipulations[7] and from the personal communication with Dr. Anthony Henras.

DPP1 upstream and downstream homology arms (BBa_K3570006 and BBa_K3570007 are used target a functional yeast integration locus. This will result in homologous recombination within the Diacylglycerol pyrophosphate phosphatase 1 (DPP1) gene and thus integration in into the S. cerevisiae's genome[8]. The sequence was identified from personal communication with Dr. Gilles Truan.

Finally, HIS3 selection marker (BBa_K3570008) is a gene that is commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and HIS3 gene in it) would be able to grow without histidine addition in the medium.