Difference between revisions of "Part:BBa K5207021"

Latest revision as of 13:11, 1 October 2024

DS-CarnosicAcid

This composite part consists of six expression cassettes, expressing SmGGPPS, SmCPS1, SmKSL, SmCPR, CYP76AH22, and CYP76AK6.

Sequence and Features

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.