Part:BBa_K5207021
DS-CarnosicAcid
This composite part consists of six expression cassettes, expressing SmGGPPS, SmCPS1, SmKSL, SmCPR, CYP76AH22, and CYP76AK6.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4456
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 685
Illegal BglII site found at 3217
Illegal BglII site found at 6298
Illegal BglII site found at 9089
Illegal BamHI site found at 2815
Illegal XhoI site found at 2951
Illegal XhoI site found at 2975
Illegal XhoI site found at 4268
Illegal XhoI site found at 4478
Illegal XhoI site found at 4950
Illegal XhoI site found at 8131
Illegal XhoI site found at 11232 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2620
Illegal NgoMIV site found at 4037
Illegal NgoMIV site found at 4601
Illegal NgoMIV site found at 8578
Illegal AgeI site found at 861
Illegal AgeI site found at 11195 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5196
Illegal BsaI site found at 8117
Illegal BsaI site found at 8378
Illegal BsaI.rc site found at 6308
Illegal BsaI.rc site found at 8367
Illegal BsaI.rc site found at 9099
Illegal BsaI.rc site found at 11477
Illegal SapI.rc site found at 12576
Usage and Biology
To realize the production of carnosic acid, the active ingredients of Salvia plants, on the yeast platform, we planned to integrate various key enzyme genes, SmGGPPS, SmCPS, SmKSL, SmCPR, CYP76AH22, and CYP76AK6, respectively, into pYTK096 vector, and to select the corresponding genes for transcription and termination to control the expression of these enzymes in Saccharomyces cerevisiae.
As shown in the following figure: we constructed the plasmid vectors of TY10 by three steps (1) Level 0: cloning the sequences containing the target genes by PCR reaction, and inserting them into vector pYTK001 by BsmBI respectively, (2) Level 1: combining the modules of each Level 0, constructing them into vector pYTK095 after ligating them by BsaI zymography, (3) Level 2: Combination of individual Level 1 vectors, constructed into vector pYTK096 after ligation by BsmBI digestion.
We combined the Level 1 vectors and constructed them into pYTK096 vector by BsmBI digestion and ligation, and successfully constructed the Level 2 vector. After transformation in E. coli DH10B identified positive monoclones by resistance, fluorescent labeling screening, and colony PCR. We can see that the one that does not show green fluorescence is the recombinant plasmid we want, which corresponds to the colony PCR agar gel image, and the recombinant plasmid is successfully constructed. Next, we extracted the corresponding plasmid DNA after amplification and culture, and used it for transformation in yeast cells.
The constructed vector was linearized by NotI endonuclease and then transferred into BY4742 yeast receptor cells, which were screened by ura-deficient medium, and the yeast genomic DNA was extracted for PCR identification to determine the positive single clones. The validation results were shown in Figure 3, which worked as expected.
Product Analysis
The successful monoclonal plasmid was expanded in culture, shaken small overnight and preserved, and the strain was further fermented and cultured using liquid YPDA medium for 5 days and then centrifuged to collect the organisms.
Saccharomyces cerevisiae cells were extracted with methanol by Ultrasonic Cell Disruption for 1 h. After centrifugation at low temperature, the supernatant extract was filtered with a filter membrane, and the filtered samples were transferred to liquid phase vials with lined tubes for Q-Exactive analysis, and the results were as follows:
The successful monoclonal plasmid was expanded in culture, shaken small overnight and preserved, and the strain was further fermented and cultured using liquid YPDA medium for 5 days and then centrifuged to collect the organisms.
As can be seen in Figure 5(A-C), CV and CA confirmed the correct synthesis of the products by the mass-to-charge ratios and intensities of the absorption peaks where they are located and the molecular masses compared to the standards. In Figure 5 (D-E), we used CV and CA standards and their corresponding peak areas to derive the corresponding equations: Equation for CA: y=2E+0.6x+1356.6 (R^2=0.9997) Equation for CV: 1E+0.6x+1252.5 (R^2=0.9993)
The yield of CA in the test was calculated to be 0.268 mg/ml, and similarly, the yield of CV in the test can be obtained to be 0.857 mg/ml. Further, the content of the extracted CA in 3 L of fermentation broth in the experiment was obtained to be 0.178 mg/L, and the content of CV was obtained to be 0.571 mg/L.
In summary, we successfully synthesized key bioactive compounds, carnosic acid, using Saccharomyces cerevisiae as a platform.
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