Part:BBa_K4805010

pTEF2-ERG20_F96W-tSSA1-pPGK1-ClSS-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 is a santalene synthase from Clausena lansium (Genbank Accession: ADR71055.1), which can catalyze the conversion of FPP(farnesyl diphosphate) into santalene. Compared with other santalene synthase, ClSS is capable of producing the most a-santalene(Jiachen Z. et al, 2022). In our project we expressed these two heterologous genes in the genome of S. cerevisiae in order to yield santalene.
Our part can inspire other teams to produce santalene from FPP. It belongs to the terpenoids 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). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. CISS (Santalene synthase from Clausena lansium) is a terpene-synthesizing enzyme responsible for forming Santalene from FPP.
In 2018, a Zhang Y.'s research team studied the characterization of different santalene synthase through a comparison of the end products after catalyzation, including the ones from S. album, S. austrocaledonicum, S. spicatum, C. camphora and C. lansium. The team discovered that santalene synthase from C. lansium tends to produce predominantly α-santalene. Meanwhile, some researches are devoted to optimize the specificity of ClSS by site-directed mutation of enzymes to reach higher yields of santalene. 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 a ratio closer to that in sandalwood oil.
Therefore, in our project, ERG20_F96W and CISS were designed to be regulated by pTDH3 and pTEF2 respectively. And it was inserted into the yeast genome to screen the best choice for santalene production. So as its mutant CISS_S532A, F441V(Part: BBa_K4805011). And after CYP736A167 and SaCPR2 are inserted, the a-santalene supplied by ClSS can be oxidized to a-santalol.

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 1. (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 2. (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.

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 1308
  Illegal BglII site found at 1369
  Illegal BglII site found at 2850
  Illegal BglII site found at 3945
  Illegal XhoI site found at 1493
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1024
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1479
  Illegal BsaI site found at 1748
  Illegal BsaI.rc site found at 1729
  Illegal BsaI.rc site found at 2860