Arabidopsis thaliana (L.)

Guo, Shaobin, Wang, Mingdi, Xu, Wen, Zou, Fuxian, Lin, Jingjing, Peng, Qin, Xu, Wei, Xu, Shaohua & Shi, Xianai, 2022, Rapid screening of glycosyltransferases in plants using a linear DNA expression template based cell-free transcription-translation system, Phytochemistry (113007) 193, pp. 1-9 : 2-3

2.1. Expression of UGTs from A. thaliana using the LET-based-TX-TL system

In order to demonstrate that the LET-based-TX-TL system can be used to screen plant enzymes, we first expressed UGTs from the model plant A. thaliana , whose whole genome sequence is well-characterized. We selected the reviewed group of AtUGTs (See Table 1 ), which consists of 11 AtUGTs whose catalytic activities have been demonstrated in previous studies ( Lim et al., 2004).

As a positive control, we synthesized an AtUGT gene AT5G17050 (AT5G17050-synthesis) from the reviewed group, which encodes for UGT78D2 and has been shown to have the highest glycosyltransferase activity among all the AtUGTs tested ( Lim et al., 2004). We extracted RNA from A. thaliana and performed reverse transcription to get cDNA. Then we used this cDNA as a template to amplify the AT5G17050 genes of A. thaliana . After obtaining expressible linear DNAs of AT5G17050-synthesis (obtained by gene synthesis), AT5G17050-cDNA (amplified from cDNA), with either long primers or short primers followed by ligation, we mixed them with the TX-TL extract and buffer to make the reaction mixtures for expression at 29 ◦ C for 8 h and then directly used them to perform glycosylation reactions using quercetin as the substrate. UPLC-MS (Ultra Performance Liquid Chromatography tandem Mass Spectrometry) results showed that the positive control AT5G17050-synthesis and AT5G17050-cDNA exhibited significant glycosyltransferase activity which catalyzed the conversion of quercetin to isoquercitrin (See Supplementary Fig. S1 , S 2 ). This suggested that the TX-TL system can be used to screen active enzymes from plants in vitro. We then test the rest of the reviewed group consisting of 10 AtUGTs. To increase the ligation efficiency of the Golden Gate assembly, we picked the AT2G15490 gene to explore the optimal template concentration in polymerase chain reactions to get gene fragments with the highest quality for the assembly. We found that the cleanest band was obtained when the final template concentration was 10 ng /μL (See Supplementary Fig. S3 ). So a final template concentration of 10 ng /μL was used to amplify the remaining 10 AtUGTs. We successfully amplified 10 AtUGTs gene fragments using PCR and ligated the gene fragments with a constitutive promoter pOR2-OR1-Pr, a strong ribosome binding site UTR1, a strong terminator T500, and a backbone to generate circular DNAs. We then performed another PCR to get expressible linear DNAs.

In total, we obtained 10 different linear DNAs ready for the TX-TL reaction. Linear DNAs were added to TX-TL extract and buffer to make the reaction mixtures, which were incubated at 29 ◦ C for 8 h. After expressing all 10 AtUGTs in TX-TL, we used the reaction mixtures directly to catalyze the glycosylation of quercetin. UPLC-MS results showed that all of the tested glycosyltransferases exhibited positive glycosylation activity (See Fig. 2 , Supplementary Table S1 ).

Figures

Fig. 1. Rapid screening of plant glycosyltransferases using the LET-based-TX-TL system. We can either use long primers which contain a promoter, a ribosome binding site, and a terminator to generate expressible linear DNAs or use short primers to amplify the targeted gene fragments and then ligate them with a promoter, a ribosome binding site, a terminator, and a backbone; then another pair of primers is used to generate expressible linear DNAs. Afterward, combine TX-TL extracts, buffers, and expressible linear DNAs to start protein expression. Then this TX-TL mixture is directly added with substrates (such as quercetin) to start glycosylation reactions. Finally, UPLC-MS is used to analyze the reaction mixture to examine whether targeted products (such as isoquercitrin) are generated.

Fig. 2. UPLC-MS analysis of isoquercitrin converted from quercetin by AtUGTs expressed in TX-TL.(A) A glycosylation reaction catalyzed by a UDP-glucose glycosyltransferase. (B) The chromatogram of the blank sample, which only has 50% methanol solvent. (C) The chromatogram of the negative group, which has TX-TL, quercetin, UDPglucose but no additional DNA. (D) The chromatogram of the quercetin standard. (E) The chromatogram of the isoquercitrin standard. (F–O) Chromatograms of products from the catalysis of quercetin by different AtUGTs (the final concentrations of the linear DNAs used for each AtUGTs are listed below in brackets): (F) AT1G07250 (32.25 nM), (G) AT1G07260 (29.24 nM), (H) AT2G36790 (20.64 nM), (I) AT2G15480 (29.98 nM), (J) AT2G15490 (29.50 nM), (K) AT3G16520 (25.03 nM), (L) AT3G21750 (23.7 nM), (M) AT3G46660 (24.14 nM), (N) AT4G15280 (24.47 nM), and (O) AT4G34138 (22.52 nM), see Supplementary Fig. S4 for the duplicate group. The y axis indicates ion signal abundance relative to the highest signal in each chromatogram (%). The numbers in the top right of each chromatogram (for instance 3.38e7 in [E]) indicate the ion counts represented by “100” relative abundance. The retention time of the isoquercitrin standard is 4.0 min, and the red arrow in each figure indicates the retention time of the product isoquercitrin. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. UPLC-MS analysis of isoquercitrin converted from quercetin by ArUGTs expressed in TX-TL. (A) A glycosylation reaction catalyzed by a UDP-glucose glycosyltransferase. (B) The chromatogram of the blank sample is 50% methanol solvent. (C) The chromatogram of the negative group, which has TX- TL, quercetin, UDP-glucose but no additional DNA. (D) The chromatogram of quercetin standard. (E) The chromatogram of isoquercitrin standard. (F–K) Chromatograms of products from the catalysis of quercetin by different ArUGTs (the final concentrations of the linear DNAs used for each ArUGTs are 30 nM): (F) AR14572, (G) AR11662, (H) AR43718, (I) AR06047, (J) AR06981, and (K) AR07558, see Supplementary Fig. S5 for the duplicate group. The y axis indicates ion signal abundance relative to the highest signal in each chromatogram (%). The numbers in the top right of each chromatogram (for instance 2.21e7 in [E]) indicate the ion counts represented by “100” relative abundance. The retention time of the isoquercitrin standard is 4.08 min, and the red arrow in each figure indicates the retention time of the product isoquercitrin. The numbers under the protein names are the peak intensities of the product. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)