Part:BBa_K5501008

Gentiana triflora glycosyltransferase Gt6CGT

This piece is the DNA sequence that encodes Gentiana triflora C-glycosyltransferase Gt6CGT. We attached it to the pET21a vector and enabled E. coli to produce C-glycosyltransferase Gt6CGT. This protein was subsequently used to produce isoorientin from luteolin.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 454
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 346
  Illegal AgeI site found at 354
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Gt6CGT, derived from Gentiana triflora, is a type of C-glycosyltransferase that catalyzes the glycosylation of luteolin to synthesize isoorientin by transferring a sugar molecule from UDP-glucose to the acceptor. Our goal is to produce isoorientin, for which we constructed a biological part (BBa_K5501008) that catalyzes the glycosidic bond of luteolin (Figure 1).

Figure 1. Constitution of Gt6CGT gene circuits.

Characterization

Protein expression

We used homologous recombination to construct the expression vector Gt6CGT_pET-21a(+) and transformed it into the Rosetta strain. After IPTG induction, protein expression was detected using SDS-PAGE. As shown in Figure 2, compared to the uninduced samples (lanes 1 and 4), the IPTG-induced strains (lanes 2, 3, 5, 6, and 7) displayed protein bands between 45 kDa and 66.2 kDa, indicating that the Gt6CGT protein was successfully expressed in the Rosetta strain.

Figure 2. SDS-PAGE results of Gt6CGT_pET-21a(+) 1,4: 0mM IPTG, 2,3,5,6,7: 0.1mM IPTG

Gt6CGT catalyzes glycosyl transfer

We identified a method for measuring glycosyltransferase activity through literature review. We used α-naphthol as the substrate and UDP-glucose as the glycosyl donor. Under the action of glycosyltransferase, α-naphthol glucoside is produced, which emits fluorescence with an excitation wavelength of 287 nm and an emission wavelength of 335 nm. The change in fluorescence intensity reflects the enzyme's activity. As shown in Figure 3, using the supernatant and inclusion bodies obtained after ultrasonic disruption as the enzyme solution, the fluorescence value significantly increased after reacting at 37°C for 1 minute, compared to the control group treated with PB buffer. This indicates that Gt6CGT successfully performed the glycosylation function.

Figure 3. Enzyme activity of Gt6CGT. (A):Enzymatic reaction equation; (B)：Enzyme activity test results, 1:PB buffer;2:supernatant protein; 3:inclusion body protein

Isoorientin was produced by Gt6CGT

To further verify the catalytic ability of Gt6CGT in producing isoorientin, we conducted high-performance liquid chromatography (HPLC) analysis. The reaction mixture consisted of 1 mM UDP-glucose as the glycosyl donor, 1 mM luteolin as the glycosyl acceptor, 50 mM phosphate buffer (pH 7.5), 1% (v/v) DMSO, and 100 μL of Gt6CGT enzyme solution. The reaction was incubated at 50°C for 30 minutes and then terminated by adding 400 μL of methanol. As shown in Figure 4, the HPLC results indicated the production of isoorientin in the experimental group(B and C) compared to the control（A）, further confirming the catalytic activity of Gt6CGT in this reaction.

Figure 4. The synthesis of isoorientin was determined by HPLC A: Isoorientin standard; B: CK-PBS replaces enzyme solution; C: the supernatant of induced bacterial solution after crushing; D: the induced bacterial solution.

Conclusion

Based on the above results, we successfully constructed the prokaryotic expression system for Gt6CGT, and the Gt6CGT protein was successfully obtained through IPTG induction. We also measured the glycosyltransferase activity of Gt6CGT using a microplate reader and confirmed the production of isoorientin through HPLC analysis.

References

[1] Pei J, Sun Q, Gu N, Zhao L, Fang X, Tang F, Cao F. Production of isoorientin and isovitexin from luteolin and apigenin using coupled catalysis of glycosyltransferase and sucrose synthase. Appl Biochem Biotechnol. 2020 Feb;190(2):601-615.

[2] Pei J, Dong P, Wu T, Zhao L, Fang X, Cao F, Tang F, Yue Y. Metabolic Engineering of Escherichia coli for Astragalin Biosynthesis. J Agric Food Chem. 2016 Oct 26;64(42):7966-7972.

[3] Pei J, Sun Q, Zhao L, Shi H, Tang F, Cao F. Efficient Biotransformation of Luteolin to Isoorientin through Adjusting Induction Strategy, Controlling Acetic Acid, and Increasing UDP-Glucose Supply in Escherichia coli. J Agric Food Chem. 2019 Jan 9;67(1):331-340.