Part:BBa_K4805012

pPGK1-SaCPR2-tPGK1-pTDH3-CYP736A167-tTDH1

SaCPR2 is a cytochrome P450 reductase from S. album (GenBank Accession: KC842188) . CYP736A167 is a cytochrome P450 enzyme (GenBank Accession: KU169302.1) specifically produces the Z type santalols. And the oxidation of santalene to Z-santalol by CYP736A167 relies on the cooperation of SaCPR2(a cytochrome P450 reductase from S. album).
This part is composite of SaCPR2 and CYP736A167 under the control of pTDH3 and pPGK1. It can be used to inspire teams to build the pathway of catalyzing santalene into santalol. It belongs to the terpenoid part collection we have established for the terpenoids production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012.

Usage and Biology

In 2016 Celedon group tested the production of sesquiterpene alcohols, including (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol and (Z)-α-exo-bergamotol. Of those, (Z)-α-santalol and (Z)-β-santalol which are the important fragrance-defining components of sandalwood oil (Celedon et al. 2016).
In 2021, Wang's team researched the extraction of santalol and santalene from S. album. And the optimized gene of P450-CPR redox was inserted into the chromosome of the yeast strain producing santalenes, resulting in 24.6 mg/L santalols (Wang et al.2021). In 2023, team LINKS- China applied and expressed CYP736A167 in yeast cell (S.cerevisiae) to catalyze santalene through oxidation and reduction reaction composing into santalol, to realize the production of our target product - the fragrance of sandalwood.

Characterization

To produce the fragrance of sandalwood, we insert CYP736A167 and SaCPR2 into the chromosome of the yeast strain producing santalene. We chose two different site as our insertion site, one of which is a common used site, YPRCd15c. For the concern that LPP1 encodes the lipid phosphate phosphatase degrading FPP, gene LPP1 is also our target to knock-out while carrying out insertion (Figure 1A).
Finally, gene CYP736A167 and SaCPR2 successfully knocked in at the site YPRCd15c and LPP1, leading to successful construction of strain Lv3s-YP and Lv3s-LP respectively (Figure 1B).

Figure 1. (A) Schematic strategy of CYP736A167 and SaCPR2 integration into the site YPRCd15c or LPP1. (B) After transformation, strain 1-4, 7-8 of Lv3s-YP appeared the successful integration into site YPRCd15c. (C) These two genes were also confirmed to be inserted into site LPP1 in the strain 7 of Lv3s-LP.

As the GC resluts shown in Figure 2A, both strain Lv3s-YP and Lv3s-LP can produce a-santalol. Also, there are some santalene can be detected in the fermentation broth (Fugure 2A). As a result, refer to the standard curve of santalol (Figure 2B), the yields of santalol in Lv3s-YP and Lv3s-LP are 40.65 mg/L and 21.21 mg/L respectively. And the yields of santalene in Lv2s-1, Lv3s-YP and Lv3s-LP are 2.51 mg/L, 24.23 mg/L and 24.73 mg/L respectively.
Apparently, the insertion site has a significant impact on the yield of santalol. However, Lv3s-YP can produces 1.92 fold of santalol than Lv3s-LP, which is totally out of our expectation. So far, it seems like the knock-out of LPP1 can't help improve the yield of santalol. Besides, further modifications have made the strain more capable of producing santalene. After all, Lv3s-YP is our favorite strain to produce santalol.

Figure 2. (A) Analysis of santalol and santalene accumulated in strain Lv3s-YP and Lv3s-LP by GC-MS. (B) The standard curve of santalol. (C) The yield comparison of santalene and santalol in different strains based on GC-MS.

reference

Wang YuChen, Gong, X., Li, F., Zuo, S.-S., Li, M., Zhao, J., Han, X., & Wen, M. (2021). Optimized biosynthesis of santalenes and santalols in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 105(23), 8795–8804. https://doi.org/10.1007/s00253-021-11661-9