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Biosynthesis of three N-acetylaminosugar-conjugated flavonoids using engineered Escherichia coli.

ABSTRACT: Nucleotide sugars serve as sugar donors for the synthesis of various glycones. The biological and chemical properties of glycones can be altered depending which sugar is attached. Bacteria synthesize unusual nucleotide sugars. A novel nucleotide sugar can be synthesized in Escherichia coli by introducing nucleotide biosynthetic genes from other microorganisms into E. coli. The engineered E. coli strains can be used as a platform for the synthesis of novel glycones.Four genes, Pdeg (UDP-N-acetylglucosamine C4,6-dehydratase), Preq (UDP-4-reductase), UDP-GlcNAc 6-DH (UDP-N-acetylglucosamine 6-dehydrogenase), and UXNAcS (UDP-N-acetylxylosamine synthase), were employed to synthesize UDP-quinovosamine, UDP-N-acetylglucosaminuronic acid, and UDP-N-acetylxylosamine in E. coli. We engineered an E. coli nucleotide sugar biosynthetic pathway to increase the pool of substrate for the target nucleotide sugars. Uridine diphosphate dependent glycosyltransferase (UGT) was also selected and introduced into E. coli. Using engineered E. coli, high levels of three novel flavonoid glycosides were obtained; 158.3 mg/L quercetin 3-O-(N-acetyl)quinovosamine, 172.5 mg/L luteolin 7-O-(N-acetyl)glucosaminuronic acid, and 160.8 mg/L quercetin 3-O-(N-acetyl)xylosamine.We reconstructed an E. coli nucleotide pathway for the synthesis of UDP-quinovosamine, UDP-N-acetylglucosaminuronic acid and UDP-N-acetylxylosamine in an E. coli galU (UDP-glucose 1-phosphate uridylyltransferase) or pgm (phosphoglucomutase) deletion mutant. Using engineered E. coli strains harboring a specific UGT, three novel flavonoids glycones were synthesized. The E. coli strains used in this study can be used for the synthesis of diverse glycones.

SUBMITTER: Cho AR

PROVIDER: S-EPMC5078965 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Biosynthesis of three N-acetylaminosugar-conjugated flavonoids using engineered Escherichia coli.

Cho A Ra AR Lee Su Jin SJ Kim Bong Gyu BG Ahn Joong-Hoon JH

Microbial cell factories 20161024 1

<h4>Background</h4>Nucleotide sugars serve as sugar donors for the synthesis of various glycones. The biological and chemical properties of glycones can be altered depending which sugar is attached. Bacteria synthesize unusual nucleotide sugars. A novel nucleotide sugar can be synthesized in Escherichia coli by introducing nucleotide biosynthetic genes from other microorganisms into E. coli. The engineered E. coli strains can be used as a platform for the synthesis of novel glycones.<h4>Results< ...[more]

PMID: 27776529

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Project description:BACKGROUND:Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource. RESULTS:We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D. CONCLUSIONS:Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.

Project description:Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

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Biosynthesis of three N-acetylaminosugar-conjugated flavonoids using engineered Escherichia coli.

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Biosynthesis of three N-acetylaminosugar-conjugated flavonoids using engineered Escherichia coli.

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