Part:BBa_K3998000

flr

flr

Usage and Biology

In this project, we put the FLR gene into E. coli to produce a strain secreting FLR enzyme efficiently. This strain can better express the FLR gene, improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

Figure1 Principles of the degradation of intestinal bacteria flavonoids: Flavonoids - > dihydroflavones - > charone - > dihydrocarboncarbonone.

Construct design

The target protein expression fragment（BBa_K3998000） is constructed into a vector of pET28a. According to Yang, G., et al. (2021), flavone reductase (FLR) discovered from Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740) plays a key step in catalyzing flavonoid. Thus, we plan to over-express FLR in Eco. li (BL21) and test its function in degrading flavonoid. The composite part is transcripted by the T7 promoter and stopped by the T7 terminator. Meanwhile, a His protein tag is inserted for future protein purification. We carried out molecular biology experiments and successfully constructed the composite part above in the vector of Eco. Li (BL21).

Figure2 protein expression box..

Figure3 Schematic map of pET 28a and Flr expression plasmids..

Proof of function

Enzyme Activity Test of FLR

After obtaining the purified protein containing FLR enzyme, we tested its effectiveness in degrading flavonoid. We conducted enzyme activity tests by using 4 kinds of flavonoid samples: apigenin, chrysin, luteolin, diosmetin with the initial concentration 10mg/L and the concentration of FLR enzyme was 1mM/L. Each sample was guaranteed three replicates of the enzyme activity test in order to to ensure the credibility of the test results. The test results are listed as follows:

Figure4 Bar graph of the enzyme activity test results..

Results show that the concentration of these 4 flavonoids remarkably decreases after 2 hours which were degraded by the enzyme, even there were only 0.3-5% left after 6 hours. All these results indicate that our enzyme has high activity and is capable of degrading multiple flavonoids with a certain universality.

Bacteria Activity Test

Figure5 Bar graph of the bacteria activity test results ..

All these flavonoids were almost degraded after 2 hours with only 1-7% left, which indicates the strong ability of our engineered E. coli to degrade flavonoids as we expected. Above all, we could come to the conclusion that the FLR enzyme, as well as the engineered E. coli, is capable of degrading various flavonoids with a certain universality and practicability. In comparison, the degradability of FLR enzyme to these flavonoids could be ranked from greatest to least as apigenin > chrysin > luteolin > diosmetin.

References

4. Ravishankar D, Rajora A K, Greco F, et al. Flavonoids as prospective compounds for anti-cancer therapy[J].Int J Bio- chem Cell Biol, 2013, 45(12):2821-2831

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 469
  Illegal NgoMIV site found at 637
  
• 1000
  COMPATIBLE WITH RFC[1000]