Part:BBa_K4805007

pTDH3-tHMG1-tTDH1-pPGK1-IDI1-tPGK1

Description

tHMG1 is a gene encoding 3-hydroxy-3-methyllutaryl coenzyme A reductase that boost the supply of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IDI1 encodes an IPP isomerase that catalyzes the conversion of the relatively un-reactive isopentenyl pyrophosphate (IPP) to the more reactive electrophile dimethylallyl pyrophosphate (DMAPP). The integration of this composite part in yeast genome can optimize MVA pathway, increase the production yield of MVA pathway’s products- IPP and DMAPP.
Terpenoids are a class of naturally occurring organic chemicals derived from the IPP and DMAPP. Spotting on the biosyhthetic pathway for santalol and ambrein, it is clear that both our target products belong to the terpenoids class. And the overexpression of tHMG1 and IDI1 can help construct a terpenoids producing yeast strain, which is suitable for the production of valuable terpenoids, including santalol and ambrein. We create a terpenoids producing collection for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012. This collection can provide inspiration and efficient methods to utilize the MVA pathway in S.cerevisiae for other teams.

Usage and Biology

From previous studies, we found that biggest rate-limiting step is mediated by the enzyme HMGR, which catalyzes HMG-CoA into mevalonate . According to Liu's research, the knock-in of tHMG1 and IDI1 can improve improving the speed limiting effect of enzymes and the production of FPP (Liu et al., 2013). Therefore, this composite part can be used to overexpress tHMG1 and IDI1 in S. cerevisiae to boost the supply of IPF and DMAPP and thus enhance the MVA pathway.

Characterization

We optimized the tHMG1 and IDI1 codons, making them compatible with S. cerevisiae translation system and enabling them to be expressed under the control of pTDH3 and pPGK1, eventually knocking them into the HIS3 site through CRISPR/Cas9 system for further modification (Figure 1).

Figure 1. (A) Schematic strategy of tHMG1 and IDI1 integration into site HIS3. (B) After transformation, colony PCR was performed to screen strain Lv1. (C) Strain 1, 4 and 5 has been confirmed positive by sequencing.

As the quantitative detection of farnesol in yeast fermentation broth can indirectly reflect the synthesis and accumulation of FPP in the strain, we found a significant difference in the concentrations of farnesol and squalene in the extract of wildtype and strain Lv1 (Figure 2A-B). Finally, 2.21 mg/L of farnesol and 0.945 mg/L of squalene were detected in the fermentation broth of strain Lv1. However, both farnesol and squalene were not detected in the wildtype strain (Figure 4 C-E). Therefore, the insertion of tHMG1 and IDI1 helps to increase the substrate required for terpenoid synthesis, such as the supply of FPP and squalene, and may help to increase the production of terpenoid products.

Figure 2. Analysis of FOH and squalene accumulated in wildtype and strain Lv1 by GC-MS. (A) GC results of farnesol in different samples and standard. (B) GC results of squalene in different samples and standard. (C) Standard curve of farnesol. (D) Standard curve of squalene. (E) The quantitative results of farnesol and squalene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1998
  Illegal PstI site found at 692
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 692
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1372
  Illegal BglII site found at 3643
  Illegal XhoI site found at 2287
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1998
  Illegal PstI site found at 692
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1998
  Illegal PstI site found at 692
  Illegal NgoMIV site found at 2143
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2541
  Illegal BsaI.rc site found at 3653
  

References

Liu, J., Zhang, W., Du, G., & Chen, J. (2013). Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae. Journal of Biotechnology, 168(4), 446–451. https://doi.org/10.1016/j.jbiotec.2013.10.017 Rubat, S., Varas, I., Sepúlveda, R., Almonacid, D., González-Nilo, F., & Agosin, E. (2017). Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase. FEMS Yeast Research, 17(4). https://doi.org/10.1093/femsyr/fox032