Difference between revisions of "Part:BBa K4805006"
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| − | <p style="font-size: smaller; margin-top: 10px;">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. | + | <p style="font-size: smaller; margin-top: 10px;"> 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. </p > |
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Revision as of 13:41, 12 October 2023
SaCPR2
Description
SaCPR2 is a cytochrome P450 reductase from S. album (GenBank Accession: KC842188) . When it is heterologously expressed in yeast, its primary function revolves around mediating electron transfer reactions in the context of diverse metabolic pathways. Thus, it assumes a critical role as an electron donor to cytochrome P450 enzymes, which, subsequently, employ these electrons to initiate a broad spectrum of chemical reactions.
Structurally, this enzyme is comprised of multiple domains, encompassing a flavin adenine dinucleotide (FAD)-binding region, a flavin mononucleotide (FMN)-binding region, and a nicotinamide adenine dinucleotide phosphate (NADPH)-binding region, thus enabling interactions with various partner proteins and coenzymes. In our constructed pathway, SaCPR2 collaborates with CYP736A167 to facilitate a reduction-oxidation reaction, converting santalene into santalol.
Our part can be used to inspire teams to build and perfect the pathway of producing santalol. 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
SaCPR2 is a reductase that is mainly acting as an electron donor in the oxidation reaction of CYP736A167 catalysing the formation of santalol from santalene. In 2020, the working efficiency of SaCPR2 was compared with the another CPR found in S. album. However, it is characterized that SaCPR2 have a better performance in santalol production than SaCPR1 (W. et al., 2020). This year, Our team used this part with CYP736A167 to convert Santalene into Santalol.
Source
S. album
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).
< img src="https://static.igem.wiki/teams/4805/wiki/engineering-success/es7.png" style="width: 50vw;">
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1245
Illegal NheI site found at 1345 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1687
- 1000COMPATIBLE WITH RFC[1000]