Functional characterization of UDP-glycosyltransferases from the liverwort Plagiochasma appendiculatum and their potential for biosynthesizing flavonoid 7-O-glucosides
Introduction
Flavonoids are plant secondary metabolites that are thought to have evolved during land colonization [1,2]. They are responsible for a wide range of physiological processes, such as UV protectants, pigments, chemical messengers, physiological regulators and cell cycle inhibitors [3]. Some of these compounds have been shown to inhibit the growth of fungal pathogens [4]. Many of the flavonoid glucosides characterized to date have been shown to be biologically active. Besides some well-known flavonol glucosides such as quercetin and kaempferol glucosides of biological, nutritional, and food chemical significance [5,6], a number of flavone glucosides have been reported to show good pharmacological properties. Apigenin 7-O-glucoside (A7G) has anti-inflammatory, antioxidant and anticandidal activity [7,8], while luteolin 7-O-glucoside (L7G) is protective against both doxorubicin-induced and starvation-induced cardiotoxicity, as well as inhibiting the growth of breast cancer cells [9,10]. Despite plants being the natural sources of flavonoid glucosides, large-scale production of these compounds by plant extraction is tedious and inefficient, which largely limits the in-depth exploration of their pharmacological activity.
The glycosylation of flavonoids in plants is typically catalyzed by uridine diphosphate-dependent glycosyltransferase (UGT) of family 1 glycosyltransferases [11,12], which are encoded by a large family of genes and catalyze the transfer of glycosyl moieties from UDP sugars to a wide range of acceptor molecules including hormones and plant secondary metabolites [13]. UGTs harbor a 44-residue motif at their C terminus, known to be required for substrate recognition [14]. Flavonoid UGTs have been discovered in a wide range of plant species, including Arabidopsis, maize, soybean and tea [[15], [16], [17], [18]]. The potential application of UGTs in glycoconjugate synthesis has attracted considerable interest in recent years [19,20]. Using the in vitro enzymatic activity of UGTs to produce glucosides require expensive sugar donor (UDP-sugar) as a co-substrate [21]. To tackle this problem, metabolic engineering and synthetic biology have been applied to improve the effective productivity of flavonoid glucosides. E. coli is one of the most commonly used microbes and previous studies have already made significant gains in demonstrating the feasibility of producing flavonoid glucosides [[22], [23], [24]]. However, the efficiency of plant UGTs remains a limiting factor for their exploration to produce flavonoid 7-O-glucosides in E. coli [[25], [26], [27]].
The liverwort species Plagiochasma appendiculatum synthesizes an abundance of metabolites including flavonoids (Our unpublished data). The genes encoding the key enzymes involved in flavonoids synthesis including 4-coumarate CoA ligase (4CL) [28], chalcone synthase (CHS) [29], chalcone flavanone isomerase (CHI) [30] and flavone synthase I (FNS I) [31] in P. appendiculatum have been isolated and characterized over the past few years, but those encoding the enzymes responsible for glycosylating flavonoids have yet to be identified. In the present investigation, we mined the transcriptome data of P. appendiculatum and two UGTs were characterized to catalyze the formation of flavonoid glucosides in vitro. A flavonoid 7-O-glycosyltransferase PaUGT1 mutant PaUGT1-Q19A served as a better UGT gene for 7-O-glycosylation of flavonols and flavones. In addition, PaUGT1-Q19A co-expressing with the upstream PaFNS I-1 offered a useful system to synthesize flavone glucosides in E. coli.
Section snippets
Plant materials and reagents
P. appendiculatum plants were grown in growth chambor held at 25 °C under a 12 h photoperiod. Total RNA was extracted from the four-week old thallus using cetyltrimethylammonium bromide (CTAB) method [32]. All available commercial chemicals used in this study were obtained from either Chengdu Must Bio-technology (Chengdu, China) or Sigma-Aldrich (St. Louis, MO, USA). All solvents used for HPLC–MS were of analytical grade.
Identification of PaUGT sequences and derivation of their phylogeny
P. appendiculatum genes encoding UGT enzymes were identified using a key
Isolation of UGT genes from P. appendiculatum
Examining the Swissprot annotation of the transcriptomic sequences derived from the liverwort species P. appendiculatum, 44 unigenes annotated as glycosyltransferases were screened out. Two UGTs likely encoding flavonoid glycosyltransferases were designated PaUGT1 (MN648205) and PaUGT2 (MN648206). The length of the open reading frame (ORF) represented in the two genes was, respectively, 1410 and 1485 bp, generating predicted translation products of length 469 and 494 residues, and molecular
P. appendiculatum harbors UGT genes
UGT genes, which encode key enzymes involved in flavonoid synthesis, have been characterized from a range of higher plant species, including Glycine max [18,25], A. thaliana [15], Camellia sinensis [17] and Zea mays [16]. However, until now, no attempt has been made to extend this characterization to the liverworts, a group of plants which exemplify the transition from marine to terrestrial plants. Here, a scan of the transcriptome data of liverworts species P. appendiculatum identified two UGT-
Disclosures
The authors declare that there is no conflict of interest.
Funding
This work was supported by the National Natural Science Foundation of China (No. 31770330, 31370330).
CRediT authorship contribution statement
Ting-Ting Zhu: Investigation, Writing - original draft. Hui Liu: Data curation. Piao-Yi Wang: Software. Rong Ni: Formal analysis. Chun-Jing Sun: Visualization. Jing-Cong Yuan: Formal analysis. Meng Niu: Validation. Hong-Xiang Lou: Supervision. Ai-Xia Cheng: Conceptualization, Methodology, Writing - review & editing.
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgements
We thank Professor Linguo Zhao from Nanjing Forestry University of China for providing the recombinant plasmids (pACYCDuet-Pgm-GalU and pACYCDuet-cscB-Basp-UgpA).
References (51)
Phenylpropanoid biosynthesis
Mol. Plant
(2010)- et al.
Flavonoids from carnation (Dianthus caryophyllus) and their antifungal activity
Phytochem. Lett.
(2008) - et al.
Apigenin-7-O-glucoside oxidation catalyzed by P450-bioinspired systems
J. Inorg. Biochem.
(2017) - et al.
Assessing acceptor substrate promiscuity of YjiC-mediated glycosylation toward flavonoids
Carbohydr. Res.
(2014) - et al.
An efficient chemoenzymatic production of small molecule glucosides with in situ UDP-glucose recycling
FEBS Lett.
(2007) - et al.
Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum
Phytochemistry
(2015) - et al.
Functional characterization of a Plagiochasma appendiculatum flavone synthase i showing flavanone 2-hydroxylase activity
FEBS Lett.
(2014) - et al.
Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation
Arch. Biochem. Biophys.
(2004) - et al.
Cloning of parsley flavone synthase I
Phytochemistry
(2001) The evolutionary paths towards complexity: a metabolic perspective
New Phytol.
(2014)
Flavonoids: biosynthesis, biological functions, and biotechnological applications
Front. Plant Sci.
In vitro anti-HIV-1 activities of kaempferol and kaempferol-7-O-glucoside isolated from Securigera securidaca
Res. Pharm. Sci.
Antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol
PLoS One
Apigenin-7-O-glucoside versus apigenin: insight into the modes of anticandidal and cytotoxic actions
EXCLI J.
Protective effects of luteolin-7-O-glucoside against starvationinduced injury through upregulation of autophagy in H9c2 cells
Biosci. Trends
Cuminum cyminum fruits as source of luteolin-7-O-glucoside, potent cytotoxic flavonoid against breast cancer cell lines
Nat. Prod. Res.
Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers
Planta
A genome-wide phylogenetic reconstruction of family 1 UDP- glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land
Plant J.
An evolutionary view of functional diversity in family 1 glycosyltransferases
Plant J.
The UDP glycosyltransferase gene superfamily: rcommended nomenclature update based on evolutionary divergence
Pharmacogenetics
A multigene family of glycosyltransferases in a model plant, Arabidopsis thaliana
Biochem. Soc. Trans.
Genome-wide identification and phylogenetic analysis of Family-1 UDP glycosyltransferases in maize (Zea mays)
Planta
Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis)
J. Exp. Bot.
Genome-wide identification and functional characterization of UDP-glucosyltransferase genes involved in flavonoid biosynthesis in glycine max
Plant Cell Physiol.
Glycosylation and subsequent malonylation of isoflavonoids in E. coli: strain development, production and insights into future metabolic perspectives
J. Ind. Microbiol. Biotechnol.
Cited by (13)
Engineered production of bioactive polyphenolic O-glycosides
2023, Biotechnology AdvancesUGTs-mediated metabolic interactions contribute to enhanced anti-inflammation activity of Jinhongtang
2023, Journal of EthnopharmacologyCitation Excerpt :The constituents possessing inhibitory effect were identified as Sargentodoxoside A, Chanitracin Ia, Quercetin and Luteolin (Fig. 6). The inhibition characteristic was consistent with the previous reports that flavonoids and polyphenols isolated from T. mongolicum and S. cuneata inhibited a variety of UGTs enzymes (Wang et al., 2017; Wu et al., 2011; Zhu et al., 2020). Furthermore, other UGT isoforms, like UGT1A3, UGT1A7, UGT1A8, UGT1A10 and UGT2B7, also participated in the metabolism of active ingredients in R. palmatum (Fig. 5).
Structure[sbnd]function relationships in plant UDP-glycosyltransferases
2022, Industrial Crops and ProductsCitation Excerpt :Mutated residues might lead to distinct interactions between the enzymes and substrates, thereby changing the binding state of each other and resulting in significantly improved catalytic activity (Fig. 3B). The mutant Q19A of PaUGT1 allowed for firmer binding between the acceptor and the donor and posed these two ligands into a more favorable orientation to enhance activities toward flavonoids (Zhu et al., 2020). For MtUGT85H2 (Modolo et al., 2009a), I305T might form a hydrogen bond with the 5-OH of kaempferol to enhance the catalytic activity.
Comparative cytology combined with transcriptomic and metabolomic analyses of Solanum nigrum L. in response to Cd toxicity
2022, Journal of Hazardous MaterialsCitation Excerpt :Previous studies reported that the overexpression of SUS can increase cellulose contents and xylem cell wall thickness, thus enhancing plant resistance to adversity (Li et al., 2020). Glucosyltransferase/glycosyltransferase (GT) are greatly important to plant because they are related to the synthesis of cell wall compositions, which also catalyze the glycosylation of a wide variety of molecules and participated in biological processes, such as signal transduction, defense, hormones synthesis, etc. (Zhu et al., 2020; Yang et al., 2016). Here, the number of DEGs (15 upregulated and 1 downregulated) encode multiple GT was the most (Fig. 9c and Table S11), indicating that S. nigrum may be regulated GT to alleviate the damage caused by Cd on the cell wall and root surface structures.
Cloning and functional characterization of three flavonoid O-glucosyltransferase genes from the liverworts Marchantia emarginata and Marchantia paleacea
2021, Plant Physiology and BiochemistryCitation Excerpt :Owing to the unique position in evolution, it is of great significance to study the biosynthetic pathway of flavonoids in liverworts. The genes encoding key enzymes (4CL, CHS, CHI and FNS I) involved in the biosynthesis of flavonoids in liverworts have been investigated in our group (Gao et al., 2015; Yu et al., 2015; Cheng et al., 2018; Han et al., 2014; Li et al., 2020), however, only two flavonoid glycosyltransferases have been identified from liverworts (Zhu et al., 2020). The liverwort species M. emarginata and M. paleacea synthesize an abundance of metabolites including flavonoid glycosides (Markham and Porter, 1974).
Biochemical Characterization of Parsley Glycosyltransferases Involved in the Biosynthesis of a Flavonoid Glycoside, Apiin
2023, International Journal of Molecular Sciences