Cloning and Expression Analysis of Glycosyltransferase Genes Involved in Gastrodin Biosynthesis in Pholidota chinensis Lindl.

doi:10.13560/j.cnki.biotech.bull.1985.2025-1297

Abstract

Abstract:

Objective This study aimed to discover the glycosyltransferase gene involved in the final key step of gastrodin biosynthesis in Pholidota chinensis, providing a basis for the high-yield production of gastrodin using engineered microbial systems. Method The candidate genes transcript28360/f2p0/1592 (1592) and transcript17418/f3p0/2174 (2174) were cloned by double restriction enzyme digestion. Using AsUGT as a positive control, the three genes were expressed in Escherichia coli and yeast. Result Gene 2174 consists of 1 413 nucleotides, encoding 470 amino acids with a molecular weight of approximately 51.12 kD, and is primarily localized in the chloroplast. Gene 1592 consists of 1 395 nucleotides, encoding 464 amino acids with a molecular weight of approximately 50.85 kD, and is primarily localized in the cytoplasm. The active sites of all three enzymes catalyzing gastrodianin conversion contained residues such as leucine, phenylalanine, threonine, and glutamic acid, which may represent key active residues for the conversion of gastrodianin to gastrodin. Expression studies in E. coli and yeast revealed that gene 2174 exhibited higher catalytic activity in converting gastrodigenin to gastrodin, demonstrating 2.31 - to 3.39-fold higher activity than the control gene AsUGT. However, gene 1592 showed weaker catalytic activity. Conclusion The glycosyltransferase genes 2174 and 1592 from P. chinensis exhibit differential catalytic activities in the conversion of gastrodigenin to gastrodin. Gene 2174 is a promising candidate for use as a key enzyme in the engineered production of gastrodin.

Key words: Pholidota chinensis, gastrodin, glycosyltransferase, Escherichia coli, yeast

LIU Bao-cai, HU Xue-bo, ZHAO Yun-qing, HUANG Ying-zhen, ZHANG Wu-jun, CHEN Jing-ying. Cloning and Expression Analysis of Glycosyltransferase Genes Involved in Gastrodin Biosynthesis in Pholidota chinensis Lindl.[J]. Biotechnology Bulletin, 2026, 42(11): 164-173.

Figures/Tables 14

Table 1 Primer sequences of PCR for gene clone

引物名称 Primer name	序列 Sequence（5′-3′）	用途 Purpose
2174-SpeI-F	GGACTAGTATGGAAATCGCTGGAT	Gene clone
2174-BglII-R	GAAGATCTCTAAAGTGCTACGCTCTT
1592-SpeI-F	GGACTAGTATGGCTGATGGCAACCCA
1592-BglII-R	GAAGATCTTCACTTAGTAGCCGTTGAATCCTT
2174-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGAAATCGCCG	Seamless gene clone
2174-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTACAGTGCCACACTTTTCCACTTAT	Seamless gene clone
1592-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGCTGATGGTAATCC
1592-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTATTTGGTTGCGGTGCTATCTTTCAGA
AsUGT-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGAACATACCCCG
AsUGT-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTAGGTACTACTAATCTTATTTTCCCA

Fig. 1 Gel electrophoresis of RNA and cloned genes in P. chinensisA: Gel electrophoresis of RNA. B: Gel electrophoresis of cloned genes. M: DL2 000TM DNA marker; A1:gene 1592; A2: gene AsUGT; A3: gene 2174

Fig. 2 Dual enzyme digestion of pESC370 with target gene and bacterial culture validationA: Double enzyme digestion of 1592 and 2174 (A1: linearized pESC370; A2: unlinearized pESC370; A3: unenzymatically digested 1592; A4: enzymatically digested 1592; A5: enzymatically digested 2174; A6: unenzymatically digested 2174). B: The pESC370-1592-Ura validation in E. coli bacterial fluid (B1-B8: The pESC370-1592-Ura monoclonal). C: pESC370-AsUGT-Ura and pESC370-2174-Ura validation in E. coli bacterial suspension (C1-C6: pESC370-AsUGT-Ura monoclonal; C7-C12: pESC370-2174-Ura monoclonal). M: DL2 000TM DNA marker

Fig. 3 Enzymatic validation of expression vector pET30a

Fig. 4 Comparison of amino acid sequences of three genes

Fig. 5 Conserved motifs of three genes

Table 2 Physicochemical properties of three genes

基因Gene

氨基酸数Amino acid quantity （aa）

相对分子质量Relative molecular weight （kD）

等电点 PI

不稳定指数Instability index

脂肪族氨基酸指数Aliphatic index

亲水性平均系数GRAVY

α螺旋 Alpha-helix （%）

延长链Extended strand

（%）

β转角 Beta-turn （%）

无规则卷曲Random coil （%）

亚细胞定位Subcellular localization

2174

chlo： 6， mito： 2， extr： 2， E.R.： 2， nucl： 1

AsUGT

cyto： 7， chlo： 4， plas： 2

1592

cyto： 8， chlo： 3， nucl： 2

Fig. 6 Predicted spatial structure of the three genes and active centers docked to gastrodigeninA and D: Gene 2174; B and E: gene 1592; C and F: gene AsUGT

Fig. 7 SDS-PAGE analysis of three genes induced expression productsA: AsUGT; B: 1592; C: 2174. M: SDS-PAGE Protein marker; 0: control; 1: induced at 15 ℃ for 16 h; 2: induced at 37 ℃ for 16 h; 3: induced at 15 ℃ for supernatants; 4: induced at 15 ℃ for precipitations; 5: induced at 37 ℃ for supernatants; 6: induced at 37 ℃ for precipitations

Fig. 8 SDS-PAGE analysis of three genes isolated target proteinsA: AsUGT; B: 1592; C: 2174. M: SDS-PAGE Protein marker; 1: centrifugation supernatant after inclusion body lysis; 2: supernatant with Ni-IDA incubation effluent; 3-5: elution fractions of 50 mmol/L imidazole; 6-8: elution fractions of 100 mmol/L imidazole; 9-13: elution fractions of 500 mmol/L imidazole

Fig. 9 SDS-PAGE analysis of three purified recombinant target proteinsM: SDS-PAGE marker; 1: BSA 1.0 μg; 2: BSA 2.0 μg; 3: 2174 protein 1.0 μg; 4: 2174 protein 2.0 μg; 5: 2174 protein 3.0 μg; 6: 1592 protein 1.0 μg; 7: 1592 protein 2.0 μg; 8: 1592 protein 3.0 μg; 9: AsUGT protein 1.0 μg; 10: AsUGT protein 2.0 μg; 11: AsUGT protein 3.0 μg

Fig. 10 Peak diagram of recombinant protein catalyzing the GAS generation from HBA

Fig. 11 Catalytic activity of recombinant protein

Fig. 12 Differences in catalytic activity after adding substrates to yeast solutionsA: Substrate; B: product. The red dashed line indicates the regression line of gastrodin generation from gastrodigenin catalyzed by gene 2174 and AsUGT in 0-48 h. The equations were regression equations based on the gastrodin content produced from 0 to 40 h and 0 to 48 h, respectively

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