Elsevier

Plant Science

Volume 299, October 2020, 110577
Plant Science

Functional characterization of UDP-glycosyltransferases from the liverwort Plagiochasma appendiculatum and their potential for biosynthesizing flavonoid 7-O-glucosides

https://doi.org/10.1016/j.plantsci.2020.110577Get rights and content

Highlights

Abstract

Flavonoid glucosides, typically generated from aglycones via the action of uridine diphosphate-dependent glycosyltransferases (UGTs), both contribute to plant viability and are pharmacologically active. The properties of UGTs produced by liverworts, one of the basal groups of non-vascular land plants, have not been systematically explored. Here, two UGTs potentially involved in flavonoids synthesis were identified from the transcriptome of Plagiochasma appendiculatum. Enzymatic analysis showed that PaUGT1 and PaUGT2 accepted various flavones, flavonols, flavanones and dihydrochalcones as substrates. A mutated form PaUGT1-Q19A exhibited a higher catalytic efficiency than did the wild type enzyme. When expressed in Escherichia coli, the yield of flavonol 7-O-glucosides reached to over 70 %. Co-expression of PaUGT1-Q19A with the upstream flavone synthase I PaFNS I-1 proved able to convert the flavanone aglycones naringenin and eriodictyol into the higher-yield apigenin 7-O-glucoside (A7G) and luteolin 7-O-glucoside (L7G). The maximum concentration of 81.0 μM A7G and 88.6 μM L7G was achieved upon supplementation with 100 μM naringenin and 100 μM eriodictyol under optimized conditions. This is the first time that flavonoids UGTs have been characterized from liverworts and co-expression of UGTs and FNS Is from the same species serves as an effective strategy to synthesize flavone 7-O-glucosides in E. coli.

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).

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