Results

Overview

After 6 weeks, we successfully completed our experimental part. We successfully constructed an $E. coli$ Rosetta cell factory capable of producing C-glycosyltransferase Gt6CGT. The expression of Gt6CGT protein and its activity as C-glycosyltransferase were successfully verified. In addition, we detected isoorientin production using luteolin as substrate and UDP-glucose as glycosyl donor.

Construction of expression vectors

We synthesized a sequence of the catalase gene Gt6CGT on a cloning vector at Genscript Biotech Corporation, 1431bp, and we need to amplify the fragment to ligate it into the expression vector pET-21a(+).

DNA amplification PCR

We transferred the synthetic plasmid containing Gt6CGT gene into TOP10 strains of $E. coli$ and extracted the plasmid as a PCR template.

M: DNA Marker, 1: Gt6CGT plasmid as the template with a band size of 1652bp, 2: pET-21a(+) plasmid as the template with a band size of 1755bp.

We will then validate the correct synthetic plasmid containing Gt6CGT gene as a template for PCR amplification, in this case using a high-fidelity enzyme. At the same time, We used the restriction enzymes BamHI to digest the vector pET-21a(+)（Figure 2）.

M: marker, 1-3 : PCR products of the amplification of the target gene Gt6CGT using a high-fidelity enzyme, 4-6: the products of pET-21a(+) carrier enzyme digestion with the restriction endonuclease BamHI.

Seamless cloning and transformation

The products amplified by Gt6CGT PCR and digested by pET-21a(+) were recovered as DNA glue, and then homologous recombination was performed using seamless cloning kit (see experimental records for details). We obtained a recombinant plasmid, and then transformed the recombinant plasmid into $E. coli$ TOP10, and coated them on solid LB medium, and the generation of colonies could be observed (Figure3) after inverted overnight culture at 37℃.

Colony PCR identification and sequencing

We selected 7 single colonies on the transformed solid LB medium as templates (lanes 1-7) for PCR verification, and the verification results were shown in Figure 4. Lane 8 was the positive control. PCR showed a target band (1652bp), indicating successful transformation. Two clones were selected and cultured at 220rpm overnight at 37℃, and the plasmid was extracted and sequenced. The sequencing results were shown in Figure 5, indicating that the Gt6CGT gene was successfully integrated into the pET-21a(+) vector.

Expression of protein in engineered bacteria

Recombinant plasmid transforms Rosetta expressing bacteria and verified by PCR

The plasmids correctly sequenced in the TOP10 transformation were transformed into Rosetta receptive cells, and 7 monoclones were selected for PCR verification (lane 1-7), lane 8 was the positive control. The results of agarose gel electrophoresis showed that the expression strains containing the target genes were successfully obtained.

Protein expression

We induced the expression of Gt6CGT protein by adding IPTG to Rosetta strains that successfully transformed the Gt6CGT_pET-21a(+) plasmid. The final concentration of IPTG used was 0.1mM, and the induction condition was 37℃, 200rpm, 15h. Then the induced bacterial solution was centrifuged, the bacteria were collected and re-suspended with Tris-HCl, and the protein expression was verified by SDS-PAGE. The expression results were shown in Figure 7. Lanes 1 and 4 were bacteria samples without IPTG added, while lanes 2-3 and lanes 5-7 were bacteria samples induced by 0.1mM IPTG at 37℃ for 15h. Our Gt6CGT protein is known to be 53.4kDa in size (the red arrow indicates the location). It can be concluded from the results in Figure 7 that we successfully expressed Gt6CGT protein.

Detection of Gt6CGT protein activity

After the successful expression of Gt6CGT protein, we detected the activity of the protein by enzymoscope. We used UDP-glucose as a glycosyl donor and 1-naphthol as a glycosyl acceptor. α-naphthol glucoside produced under the action of Gt6CGT protein was detected at excitation wavelength of 287nm and emission wavelength of 335nm, and slit width of 5nm to detect changes in the value of smart light intensity and then reflect the size of enzyme activity. The test results are shown in Figure 8. The CK group used Tris-HCl as the blank control, and the other two groups used the supernatant and inclusion body after ultrasonic crushing of $E. coli$ as the enzyme solution for reaction. Compared with CK group, the fluorescence values of supernatant and inclusion body group were significantly improved, and the fluorescence values of supernatant group were greater than those of inclusion body group. It can be concluded that the Gt6CGT protein extracted by us is active.

1: PB buffer; 2: supernatant protein; 3: inclusion body protein

Detection of isoorientin production

To further verify the production of isoorientin, we used luteolin as the substrate and UDP-glucose as the glycosyl donor in an enzymatic reaction conducted in 50 mM phosphate buffer (pH 7.5). The reaction mixture was incubated at 50°C for 30 minutes, and the reaction was terminated by the addition of methanol. The reaction products were then analyzed by high-performance liquid chromatography (HPLC). As shown in the figure, isoorientin was clearly detected in both Group C and Group D. This result confirms that the engineered recombinant strain possesses the ability to catalyze the production of isoorientin, further demonstrating its potential application in large-scale isoorientin production.

A: Isoorientin standard; B: CK-PBS replaces enzyme solution; C: the supernatant of induced bacterial solution after crushing; D: the induced bacterial solution.