Part:BBa_K5207021

DS-CarnosicAcid

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4456
  
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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

We combined the Level 1 vectors and constructed them into pYTK096 vector by BsmBI digestion and ligation, and successfully constructed the Level 2 vector.

Figure 1. Snapgene diagrams of pYTK096

Similarly, after transformation in E. coli DH10B identified positive monoclones by resistance, fluorescent labeling screening, and colony PCR.

Figure 2. Fluorescent labeling screening of pYTK095

Figure 3. Validation plot of pYTK096 gel electrophoresis

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.

Figure 4. Graph of the results of the screening of URA-deficient media

Insert fragment PCR assay results:

Figure 5. Gel validation recombinant plasmids in yeast cells

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.

Figure 6. Schematic diagram of the fermented bacterial liquid

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:

TY10: as can be seen in Figure 18, where Figure 18-A is the result of the TY10 mass spectrometry test, showing that it contains two main compounds 3,4, Figure 18-B is the chromatogram of the carnosic acid (CA) standard, and Figure 18-C is the chromatogram of the carnosol (CV) standard.

Figure 7. Chromatogram of S. c BY4742/TY10 extract (A), carnosic acid (CA) standard (B), and carnosol (CV) standard (C); (D) CA and (E) CV standard curves.

The 11-hydroxy-sugiol, 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, and the 11-hydroxy-sugiol is more predominant in the yeast cell fermentation broth extracts. In Figure 18(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.