Part:BBa_K5223015

Resveratrol Synthesis System in Nicotiana benthamiana

Biology and Usage

In the design of resveratrol system, we integrated VvSTS and YIARO3 genes into Agrobacterium and then transiently transfected into Nicotiana benthamiana to produce resveratrol efficiently. According to available literature, grape-derived stilbene synthase (VvSTS) is an efficient key enzyme for the synthesis of resveratrol. And the YlARO3 gene is responsible for encoding 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (DAHPS) [3], which retains the activity of the wild-type enzyme but deregulates the inhibitory effect of L-Phe in the upstream pathway of resveratrol synthesis [1]. We optimized the design of these two gene sequences and transiently transfected tobacco separately or simultaneously for comparison, and we finally obtained tobacco plants capable of efficient resveratrol production.

Design and Properties:

We placed the VvSTS gene and YIARO3 gene downstream of the CaMV 35S promoter and constructed the target plasmid using Gateway technology (Figure 1). Subsequently, the two plasmids were transiently transfected into Nicotiana benthamiana, either separately or simultaneously. The results showed that co-transfecting both genes into tobacco could produce more resveratrol(Figure 2).

Fig1: The constructed YIARO3(left) and VvSTS(right) expression plasmids

Fig.2: The resveratrol yield after the integration of YIARO3 and VvSTS

Experimental Approach:

(1) Transiently transfection
1. Preparation of resuspension : 90mL ddH2O, 10mL MES mother liquor and1mL MaClz were added to100mL resuspension. Mother liquor and 100μL AS mother liquor composition.
2. The centrifuge was pre-cooled and centrifuged at 4000 r for 10 min at 4°C.
3. Pour the medium, and add 25mLddH2O and vortex oscillation evenly.
4. Centrifuge at 4000 r for 10 min at 4 °C, and pour ddH2O.
5. Add the resuspension and incubate in the dark for 2h.
6. The bacterial liquid was removed, and the OD6oo was adjusted to 0.8-1.0.
7. The bacterial liquid was injected into tobacco leaves with a 2mL syringe.
8. Cultured in the dark for 72h.

(2) Tobacco metabolome extraction
1: The tobacco samples were freeze-dried and ground.
2: Weigh the treated samples. According to the sample: methanol ( w / mg: v / ml ) = 1: 10 preparation, add methanol after blowing and mixing. After the preparation, it was taken out by ultrasound at 4 °C for 15 min.
3: 4 °C 20000 × g centrifuged for 10min, the supernatant was added to a clean centrifuge tube, again 4 °C 20000 × g centrifuged for 10min, the supernatant was taken.
4: The diluted sample was sucked: 500ul was added to the sample bottle, and a 20ul sample was added to Qc in each tube ( two tubes were prepared ). Then, 10ul 2-chlorophenylalanine ( 1 mg/ml) was added to each tube.
5: The content of resveratrol compounds was detected by LC-MS/MS ( AB SCIEX Triple Quad TM QqQ 5500 ) and analyzed by Analyst 1.6.3 software. The detection source was electrospray ion source ( ESI ), negative ion mode, and the chromatographic column was C18 chromatographic column ( Thermo Fisher Scientific )—mobile phase A: 0.1 % formic acid water; B: Pure acetonitrile.
The gradient elution procedure and the detection mass spectrometry conditions are as follows [2]

Table 1 Gradient elution procedure

Table 2 Mass spectrometry conditions for resveratrol detection

Reference:

[1] Liu H, Xiao Q, Wu X, Ma H, Li J, Guo X, Liu Z, Zhang Y, Luo Y. Mechanistic investigation of a D to N mutation in DAHP synthase that dictates carbon flux into the shikimate pathway in yeast. Commun Chem. 2023 Jul 15;6(1):152. doi: 10.1038/s42004-023-00946-x. PMID: 37454208; PMCID: PMC10349828.
[2] Yuan Y, Ren S, Liu X, Su L, Wu Y, Zhang W, Li Y, Jiang Y, Wang H, Fu R, Bouzayen M, Liu M, Zhang Y. SlWRKY35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit. New Phytol. 2022 Apr;234(1):164-178. doi: 10.1111/nph.17977. Epub 2022 Feb 15. PMID: 35048386.
[3] Jeong YJ, An CH, Woo SG, Park JH, Lee KW, Lee SH, Rim Y, Jeong HJ, Ryu YB, Kim CY. Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway. Plant Mol Biol. 2016 Sep;92(1-2):117-29. doi: 10.1007/s11103-016-0497-0. Epub 2016 Jun 23. PMID: 27338256.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1444
  Illegal XhoI site found at 2994
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1168
  Illegal BsaI site found at 2718
  Illegal BsaI.rc site found at 1447
  Illegal BsaI.rc site found at 2997
  Illegal SapI.rc site found at 964
  Illegal SapI.rc site found at 1744
  Illegal SapI.rc site found at 2565