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Applications of BBa_K4824000

Introduction

Bioparts are the basic building blocks of the iGEM project. Here, we constructed a catalytic lignan glycoside (BBa_K4824000) biopart (Figure 1). Based on the reported transcriptomic data of I. indigotica, the sequences of genes with glycosyltransferase functions were screened and a phylogenetic tree analysis was conducted with the functionally characterized lignan UGTs, and UGT72B2 was selected as the candidate, which the main component of the biopart. We then constructed expression vectors using homologous recombination and ultimately verified protein function.

Figure 1. UGT72B2-pET-32a+ prokaryotic expression vector.

Clone UGT72B2 gene

PCR amplification was performed using the cDNA library of I. indigotica as the template, and the product was detected by 1% agarose gel electrophoresis, which showed a specific fragment at about 1500 bp, which was consistent with the expected result of the full length of the UGT72B2 gene (Figure 2). Sequencing of the cloning result confirmed that it was identical to the full length of the sequence obtained from the database. Information analysis of the sequence using the open reading frame (ORF) Finder (https://www.ncbi.nlm.nih.gov/orffinder) online software revealed that the full length of the ORF sequence of the UGT72B2 gene was 1455 bp, coding for 484 amino acids, with a relative molecular mass of 53,269.86, a theoretical isoelectric point of 5.90, and a total average protein hydrophobicity of -0.074, making it an unstable protein.

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Figure 2. PCR amplification of UGT72B2 gene from I. indigotica. M: marker, 1-4: UGT72B2 gene PCR amplification band.

Expression and purification of UGT72B2

Xho I and Nco I were chosen as the cleavage sites to design primers for the construction of UGT72B2-pET-32a+ prokaryotic expression vector, and the recombinant expression vector was transformed into E. coli Rosetta (DE3) competent recipient cells after sequencing correctly and used as the subsequent protein expression. pET-32a+ vector was transformed into Rosetta (DE3) recipient cells by the same treatment as the negative control. The recombinant protein expression was detected by SDS-PAGE after induction by IPTG. As shown in the Figure 3, the strain containing the recombinant plasmid UGT72B2-pET-32a+ (the relative molecular mass of the target protein of UGT72B2 is about 53 kDa) has a protein band near 70 kDa; whereas, the control strain with pET-32a+ empty vector has no protein expression at the same position and only expresses His-Tag tagged protein near 18 kDa, and the results indicate that UGT72B2 was successfully expressed in E. coli Rosetta (DE3). Western blotting was further utilized to detect whether the resulting protein was the target protein. The results are shown in Figure 3. Compared with the empty vector control, UGT72B2-pET-32a+ expression signal was detected with a single band and good purity. The His-tagged fusion proteins were purified by Ni-NTA affinity chromatography. The concentration of purified UGT72B2-pET-32a+ fusion protein was determined by BCA quasi-curve method, and finally the UGT72B2 recombinant protein at a mass concentration of 3.60 mg/mL was obtained to be used as the subsequent enzyme activity experiments.

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Figure 3. SDS-PAGE and western blot analysis of purified proteins.

Substrate promiscuity of UGT72B2

(+)-Pinoresinol, (+)-lariciresinol, secoisolariciresinol, and matairesinol were selected as potential substrates for the catalytic analysis of UGT72B2. The reaction mixture was in a total volume of 50 μL containing phosphate buffer saline (pH 7.4), 2 mM UDP-glycoside, 200 μM substrates, and 10 μg of purified proteins. The mixture was preincubated at 30℃ for 10 min without proteins and then incubated for 12 h at 30℃ with a supplement of proteins. The enzyme activity products were characterized using UPLC-Q-TOF/MS.

Figure 4. UPLC-Q-TOF/MS for productions of UGT72B2 with lignans as substrates. 1：(+)-lariciresinol, 2: (+)-pinoresinol, 3: secoisolariciresinol, 4: matairesinol, 5 and 6: lariciresinol-4-O-β-D-glucopyranoside and lariciresinol-4'-O-β-D-glucopyranoside, 7: pinoresinol-4-O-β-glucopyranoside, 8: secoisolariciresinol-O-β-D-glucopyranoside and 9: matairesinol-O-β-D-glucopyranoside.

File:Https://static.igem.wiki/teams/4824/wiki/bioplus-shanghai-part-table1.png

According to previous studies, UGTs have a broad range of promiscuity towards the substrates of sugar acceptors. In our research, we found that with UDP-glycoside as the sugar donor, UGT72B2 could catalyze all tested substrates including (+)-pinoresinol, (+)-lariciresinol, secoisolariciresinol, and matairesinol (Figure 4, Table 1). So UGT72B2 is a catalytic enzyme with relatively heterogeneous substrates. However, the catalyzed products were only monoglycosides, and the formation of diglycosides was not detected. Taken together, UGT2B2 catalyzes the monoglycosylation of lignans with substrate promiscuity.

Enzyme Kinetic Parameters of UGT72B2

Considering that lariciresinol has much more pharmaceutical properties than others, we focused on the kinetics of UGT72B2 with lariciresinol as the substrate. The reaction mixture was the same as mentioned above, but the reaction time was shortened to 20 minutes. The enzyme activity products were characterized using LC-MS/MS. The kinetic parameters were analyzed through the Lineweaver-Burk plot. As a result, the catalytic efficiency of UGT72B2 for pinoresinol was as follow: Km 189 µM, Vmax 0.1079 µM/min, [E] 3.75 µM, Kcat 0.028min-1 (Figure 5).

Figure 5. The double reciprocal equations of substrate concentration [S] and the initial rate of enzyme catalysis [v].

Conclusion

In this study, we cloned and obtained the UGT72B2 gene, and for the first time, we comprehensively examined and validated the catalytic features of this gene. The constructed part provides a basis for realizing the heterologous and efficient synthesis of lignan or lignan-derivate glycoside components.

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