Part:BBa_K4805011

pTEF2-ERG20_F96W-tSSA1-pPGK1-ClSS_ S532A,F441V-tPGK1

description

ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. ClSS_S532A,F441V encodes for a variant of ClSS, a santalene synthase from Clausena lansium(Genbank Accession: ADR71055.1), which possesses two site direct mutagenesis of S532A and F441V. Our part can be a reference for other teams to perfect the pathway producing santalene from FPP. It belongs to the terpenoid part collection we have established for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012.

usage and biology

ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). So far, the S532A mutation has been found to enable ClSS to produce approximately 80% more santalene in E. coli. And ClSS with mutation of F441V can produce different structures of santalene with similar composition to sandalwood oil. In our project we inserted it in the genome of S. cerevisiae in order to yield santalene. Therefore, out of the demand for increased production of santalene, we instructed a yeast strain with ClSS_F441V, S5321 gene inserted. And we make a comparison of catalyzing ability between ClSS wildtype and ClSS_F441V, S532A.

sequencing and features

Assembly Compatibility:

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

  Illegal BglII site found at 1302
  Illegal BglII site found at 1363
  Illegal BglII site found at 2828
  Illegal BglII site found at 3917
  Illegal XhoI site found at 1479
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1018
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1465
  Illegal BsaI site found at 1726
  Illegal BsaI.rc site found at 1715
  Illegal BsaI.rc site found at 2838
  

characterization

2023 LINKS-China chose a commonly used 106 site to insert ERG20_F96W and ClSS at the same time(Figure 1A). It is obvious that we successfully screened one positive strain out of 10 strains, which is named as Lv2s-1 (Figure 1B). Same as ClSS, ClSS_F441V, S532A was also inserted into 106 site.

Figure 5. (A) Schematic strategy of ERG20_F96W and ClSS and ClSS_F441V, S532A the integration into site 106. (B) Colony PCR results performed to screen strain Lv2s-1 and Lv2s-2.

Then we obtained strain Lv2s-1 and Lv2s-2 which possesses ClSS and ClSS_F441V, S532A respectively. According to the GC results, it is apparent that both strains can produce santalene(Figure 2A). Using humulene as an internal standard, the yield of a-santalene in strains Lv2s-1 and Lv2s-2 was quantified as 2.513 mg/L and 1.148 mg/L, respectively. The double site mutation of ClSS not only failed to achieve the desired increase in yield, but also decreased the yield of a-santalene by about 54%. Therefore, it is more suitable to choose Lv2s-1 as our chassis cell for modification towards a santalol producer.

Figure 1. (A) GC results of santalene accumulated in strain Lv2s-1 and Lv2s-2 by GC-MS. (B) MS results at 10.13 s and 10.549 s. (C) The yield comparison of santalene in different strains.

reference

Hua G; Hu Y; Yang C; Liu D; Mao Z; Zhang L; Zhang Y; (2018) Characterization of santalene synthases using an inorganic pyrophosphatase coupled colorimetric assay, Analytical biochemistry. Available at: https://pubmed.ncbi.nlm.nih.gov/29438678/ Zha, W., Zhang, F., Shao, J. et al. Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil. Nat Commun 13, 2508 (2022). https://doi.org/10.1038/s41467-022-30294-8 Zhang, Jia, et al. “Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia Coli.” Journal of Agricultural and Food Chemistry, vol. 70, no. 17, 24 Apr. 2022, pp. 5377–5385, https://doi.org/10.1021/acs.jafc.2c00754.ed.ncbi.nlm.nih.gov/31940436/