Production of 3-O-xylosyl quercetin in Escherichia coli

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: Discussionmentioning

confidence: 99%

“…However, other flavonoid O-glycosides, such as flavonoid O-rhamnoside and flavonoid O-xyloside, have been synthesized using E. coli by expressing plant UGTs along with the corresponding nucleotide biosynthesis genes (5,(17)(18)(19). The biological activities of a variety of flavonoid O-glycosides have been elucidated (20)(21)(22)(23).…”

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confidence: 99%

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

Han

Kim

Yoon

et al. 2014

“…Therefore, chemical synthesis is not suitable for the production of flavonoids to be incorporated into food ingredients or cosmetics. Among the microorganisms used for the biosynthesis of natural products, Escherichia coli has been the most intensively investigated host because of its genetic tractability and favorable fermentation properties (12).…”

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confidence: 99%

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Zhu

et al. 2014

The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3=-hydroxylase (tF3=H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from L-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from L-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from L-tyrosine or even glucose in E. coli.

“…4A), as described in materials and methods in the supplemental material. The optimal growth conditions described in our previous report (21) were used for the bioconversion. The HPLC-PDA analysis of in vivo reactions showed the production of four phloretin glucosides with the same retention time as in in vitro reactions ( Fig.…”

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confidence: 99%

Enzymatic Synthesis of Novel Phloretin Glucosides

Pandey

Kim

et al. 2013

Self Cite

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli. P hloretin is a dihydrochalcone, an intermediate of the biosynthetic pathway of flavonoids in plants, which is abundantly present in the peel of apple (1, 2) and in strawberries (3). They occur in different glycosidic forms, such as naringin dihydrochalcone, phlorizin, and phloretin-4=-O-glucoside, in the different parts of the plants, contributing to various physiological properties of the plants, as well as to their color. Phloretin and its glycosides have been determined to have beneficial biological activities. Studies have uncovered that phloretin has inhibitory activity against glucose cotransporter 1 (4, 5), antioxidant activity (6), and activity to suppress the tumor necrosis factor alpha-induced inflammatory response, ameliorate inflammation of the colon, positively affect body weight loss (7), modulate Ca 2ϩ -activated K ϩ channels, and increase endothelial nitric oxide production, which might help to protect against atherosclerosis (8). Importantly, phloretin has other biological functions, like anticarcinogenic (9) and estrogenic activities (10) and inhibition of cardiovascular disease (11, 12). Irrespective of their diverse physiological and pharmacological activities, the use of most of the natural polyphenols as drugs and food additives has been limited because of their water insolubility and low absorbability. Glycosylation enhances the bioavailability and pharmacological properties of compounds by increasing their solubility and stability (13,14). Importantly, the sugar moieties of the glycosides often participate in the specific recognition of their biological targets and help to determine their efficacy in drug development (14, 15). According to the CAZy database (http://www .cazy.org/) (16,17,18), glycosyltransferase family 1 (GT1) proteins contain the UDP-glycosyltransferases that are common in all domains of life (19) and predominantly recognize small molecules as the sugar acceptors. A recent report showed that YjiC, a Bacillus licheniformis UDP-glycosyltransferase that falls in the GT1 family of proteins, can glycosylate at different hydroxyl positions of geldanamycin analogs (20). Here, we report the use of this glycosyltransferase for the biosynthesis of diverse phloretin glucosides in vitro and the subsequent application of YjiC for in vivo production of phloretin glucosides in an Escherichia coli mutant generating a cytoplasmic pool of UDP-glucose, since the YjiC-homologous glycosyltransferases from other Bacillus species were found to have flexible glycosyltransferase activities toward different flavonoid groups of compounds. Moreover, we found that by reversing the glycosylation reaction, the enz...