Production of a Novel Quercetin Glycoside through Metabolic Engineering of Escherichia coli

Yoon

et al. 2014

Appl Environ Microbiol

Self Cite

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-L-Ara4N formyltransferase/UDPGlcA C-4؆-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.

Section: Methodsmentioning

confidence: 99%

Synthesis of Flavonoid O -Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase

Han

Yoon

et al. 2014

Appl Environ Microbiol

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“…Moreover, it is also the first bacterial enzyme reported to transfer various dTDP-activated hexose sugars to polyphenols (see below), in contrast to the usually stringent donor specificities of Gtf-like enzymes such as GtfD (57). With respect to the notion that many NDP-sugars in prokaryotes are dTDP and not UDP activated, GtfC might be a promising biocatalyst in glyco-diversification approaches (58,62,63). GtfC is similar to predicted GTs from Cytophagaceae bacteria (64)(65)(66).…”

Section: Discussionmentioning

confidence: 99%

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

Rabausch

Jüergensen

Ilmberger

et al. 2013

Appl Environ Microbiol

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. For more than a decade, metagenome research has demonstrated that it is a powerful tool for the discovery of novel biocatalysts and other valuable biomolecules by using either function-or sequence-based screening technologies (1-3). Sequencebased approaches allow the identification of candidate genes. In particular, the development of next-generation sequencing (NGS) technology and improved bioinformatic tools have significantly advanced this methodology (4). However, a major drawback of sequence-based screening technologies is that they do not allow direct conclusions about the functionality and biochemical parameters of the encoded enzymes. Furthermore, sequence-based searches are limited to the identification of homologs of already known motifs (5). Yet another problem associated with the sequence-based approach is that it often reveals only partial genes, which make subsequent expression and detailed biochemical analysis of the gene products difficult if not impossible. In contrast, the function-driven approach is usually much slower and more labor-intensive and costly but results in the detection of complete and active enzyme clones. It is of course well known that function-driven metagenomics is hampered due to the problems of expressi...

“…For the synthesis of TDP-6-deoxytalose, additional enzyme, dTDP-6-deoxy-L-lyxo-4-hexulosereductase (tll), was needed. Yoon et al (2012) synthesized the tll gene based on the sequence from Actinobacillus actinomycetemcomitans and introduced it into E. coli. Moreover, they screened 50 UGTs to find the UGT that uses TDP-6-deoxytalose as a sugar donor.…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis and production of glycosylated flavonoids in Escherichia coli: current state and perspectives

Yang

Appl Microbiol Biotechnol

et al. 2015

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Flavonoids are plant secondary metabolites containing several hydroxyl groups that are targets for modification reactions such as methylation and glycosylation. In plants, flavonoids are present as glycones. Although glucose is the most common sugar attached to flavonoids, arabinose, galactose, glucuronic acid, rhamnose, and xylose are also linked to flavonoids. Depending on the kind and the position of the attached sugar, flavonoid glycones show different biological properties. Flavonoid-O-glycosides are synthesized by uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugar as a sugar donor and a diverse compound as a sugar acceptor. Recently, diverse flavonoid-O-glycosides have been synthesized in Escherichia coli by introducing UGTs from plants and bacteria and modifying endogenous pathways. The nucleotide sugar biosynthesis pathway in E. coli has been engineered to provide the proper nucleotide sugar for flavonoid-O-glycoside biosynthesis. In this review, we will discuss recent advances in flavonoid-O-glycoside biosynthesis using engineered E. coli.