Part:BBa_K5207020

TS-HydroxySugiol

This composite part consists of six expression cassettes, expressing SmGGPPS, SmCPS1, SmKSL, SmCPR, CYP76AH1, and CYP76AH3. This plasmid is engineered to synthesize tanshinone derivatives, which are key compounds derived from Salvia miltiorrhiza (Danshen) and are known for their potential therapeutic properties, particularly in cardiovascular treatments.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 4456
  Illegal NheI site found at 11899
  
• 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 NgoMIV site found at 13298
  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
  Illegal SapI.rc site found at 13326
  Illegal SapI.rc site found at 14553
  Illegal SapI.rc site found at 15303
  

Usage and Biology

To realize the production of tanshinones precursor, the active ingredients of Salvia plants, on the yeast platform, we planned to integrate various key enzyme genes, SmGGPPS, SmCPS, SmKSL, SmCPR, CYP76AH1, CYP76AH3, 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 TY9 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.

Figure 1 Flowchart for Level-0 to Level-2 assembly

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.

Figure 2 (a) Fluorescent labeling screening of pYTK095, (b) Validation plot of pYTK096 gel electrophoresis

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.

Figure 3 (a) Graph of the results of the screening of URA-deficient media of TY 9, (b) Gel validation of TY9 and TY10 recombinant plasmids in yeast cells

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.

Figure 4 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:

Figure 5 Chromatogram and mass spectrogram of S. c BY4742/TY9 extract.

As shown in Figure 5, the product of TY9 is mainly composed of two compounds 1,2 which appeared near 6 and 9 minutes, with molecular weights of 316 and 298 under positive mode, respectively. Through literature analysis and discussed with our instructor, we confirmed that the compound MW of 316 is 11-hydroxy-sugiol, and MW 298 has not been found to point to any specific substance at this point in time.

So, the 11-hydroxy-sugiol 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 11-hydroxy-sugiol is more predominant in the yeast cell fermentation broth extracts. In summary, we successfully synthesized key bioactive compounds, tanshinone derivatives using Saccharomyces cerevisiae as a platform.

Reference: Guo et al., 2016, New Phytologist，210: 525–534