Part:BBa_K4274002

ERG20_F96W

Erg20 (Part Number: BBa_K849001), a bifunctional enzyme, catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). The residues F96 of Erg20 is essential for FPP synthesis due to the fact that they assist the production of monoterpene which eventually gives rise to FPP [1]. In previous research, it was noted that Erg20 mutant F96W was conducive to FPP generation in E. coli [2].

This year we express ERG20_F96W in E. coli DH5a (tnaA-) to catalyze the formation of GPP and FPP with higher efficiency to heterologously produce santalene. In the future, other teams could possibly use this part to increase GPP and FPP catalytic activity in E. coli.

Usage and Biology

Erg20(Part Number: BBa_K849001), a bifunctional enzyme, catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Research have shown that while one fundamental residue of ERG20, F96, was mutated to W, the production of monoterpene, which is produced after the production of GPP, can have a 3-fold increase (Ignea et al. 2014).

Naturally, Erg20 is an enzyme in S. cerevisiae specific for terpenoid production. However, the application of Erg20 heterologously was not quite successfully due to the fact that it shows relatively low capacity as GPP and FPP catalyst in E. coli. To cope with this problem, F96 is changed to W in Erg20 (F96W), providing enhancing activity of FPP synthase, in this way increase the production of FPP in E. coli. (Wang et al. 2020).

Taking the research Wang mentioned above into consideration, we decided to express Erg20 F96W along with other genes on a kanamycin resistant vector as a mean to catalyze the formation of GPP and FPP to produce santalene in E. coli with higher efficiency. Part BBa_K4274002 can also be used along with BBa_K4274001 to increase the production of santalene all the way from the formation of GPP and FPP from their building blocks, IPP and DMAPP, to the synthesis of santalene from FPP.

Source

S. cerevisiae

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 899
  Illegal BglII site found at 960
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 615
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Ignea, C., Pontini, M., Maffei, M. E., Makris, A. M., & Kampranis, S. C. Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synthetic Biology, 3(5), 298-306 (2014). https://doi.org/10.1021/sb400115e
[2]Wang, X., Wu, J., Chen, J., Xiao, L., Zhang, Y., Wang, F., & Li, X. Efficient biosynthesis of R-Linalool through adjusting the expression strategy and increasing GPP supply in Escherichia coli. Journal of Agricultural and Food Chemistry, 68(31), 8381-8390(2020). https://doi.org/10.1021/acs.jafc.0c03664
[3] Jia Z., Xun W., Xinyi Z., et al. Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia coli. J. Agric. Food Chem. 70 (17), 5377-5385 (2022). https://doi.org/10.1021/acs.jafc.2c00754