Synthesis of chlorogenic acid and p-coumaroyl shikimate by expressing shikimate gene modules in Escherichia coli.

AIM: Chlorogenic acid and p-coumaroyl shikimate are hydroxycinnamic acid derivatives. These compounds are nutraceutical supplements due to their biological activities including prevention of cardiovascular disease and cancers. These two compounds were synthesized in Escherichia coli through two-culture system using two mutants, which are biochemically interdependent. The aim of this work was to improve the titres of their production in a single E. coli mutant in which all necessary genes were introduced. This was done by testing various shikimate gene combinations to determine the optimal gene combination for the synthesis of chlorogenic acid and p-coumaroyl shikimate. METHODS AND RESULTS: A series of gene modules harbouring shikimate pathway genes were constructs. Six gene module constructs for chlorogenic acid synthesis and eight constructs for p-coumaric acid synthesis were tested in order to find the best one. Chlorogenic acid synthesis showed highest with the gene module construct containing ydiB, aroB, aroGf , ppsA and tktA. Using the E. coli strain, 109.7 mg L-1 chlorogenic acid was synthesized. The best gene module construct for the p-coumaroyl shikimate synthesis contained aroD and aroGf . In addition, we used two E. coli deletion mutant strains (ΔaroK and ΔaroL) to increase the final titre. The E. coli ΔaroK mutant harbouring this gene module construct synthesized 713.4 mg L-1 of p-coumaroyl shikimate. CONCLUSION: The chlorogenic acid synthesis using the current system was approximately 35.4% higher of the titre than titres obtained with an alternative method that depends on co-cultivation of two mutants. At the same time, production of p-coumaroyl shikimate increased 5.8 times. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study's findings indicate that our selection of the shikimate gene module contributed to increases in the levels of the substrates and could be applied to synthesize other compounds whose synthesis requires intermediates of the shikimate pathway.

Synthesis of acridone derivatives via heterologous expression of a plant type III polyketide synthase in Escherichia coli.

BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.

Synthesis of avenanthramides using engineered Escherichia coli.

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.

Engineering of Escherichia coli for the synthesis of N-hydroxycinnamoyl tryptamine and serotonin.

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC-tryptamines and HC-serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 µM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 µM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Biosynthesis of two quercetin O-diglycosides in Escherichia coli.

Various flavonoid glycosides are found in nature, and their biological activities are as variable as their number. In some cases, the sugar moiety attached to the flavonoid modulates its biological activities. Flavonoid glycones are not easily synthesized chemically. Therefore, in this study, we attempted to synthesize quercetin 3-O-glucosyl (1→2) xyloside and quercetin 3-O-glucosyl (1→6) rhamnoside (also called rutin) using two uridine diphosphate-dependent glycosyltransferases (UGTs) in Escherichia coli. To synthesize quercetin 3-O-glucosyl (1→2) xyloside, sequential glycosylation was carried out by regulating the expression time of the two UGTs. AtUGT78D2 was subcloned into a vector controlled by a Tac promoter without a lacI operator, while AtUGT79B1 was subcloned into a vector controlled by a T7 promoter. UDP-xyloside was supplied by concomitantly expressing UDP-glucose dehydrogenase (ugd) and UDP-xyloside synthase (UXS) in the E. coli. Using these strategies, 65.0 mg/L of quercetin 3-O-glucosyl (1→2) xyloside was produced. For the synthesis of rutin, one UGT (BcGT1) was integrated into the E. coli chromosome and the other UGT (Fg2) was expressed in a plasmid along with RHM2 (rhamnose synthase gene 2). After optimization of the initial cell concentration and incubation temperature, 119.8 mg/L of rutin was produced. The strategies used in this study thus show promise for the synthesis of flavonoid diglucosides in E. coli.

Biological synthesis of coumarins in Escherichia coli.

BACKGROUND: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives. RESULTS: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway. CONCLUSIONS: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

Bacterial synthesis of N-hydroxycinnamoyl phenethylamines and tyramines.

BACKGROUND: Hydroxycinnamic acids (HCAs) including cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, are C6-C3 phenolic compounds that are synthesized via the phenylpropanoid pathway. HCAs serve as precursors for the synthesis of lignins, flavonoids, anthocyanins, stilbenes and other phenolic compounds. HCAs can also be conjugated with diverse compounds including quinic acid, hydroxyl acids, and amines. Hydroxycinnamoyl (HC) amine conjugates such as N-HC tyramines and N-HC phenethylamines have been considered as potential starting materials to develop antiviral and anticancer drugs. RESULTS: We synthesized N-HC tyramines and N-HC phenethylamines using three different approaches in Escherichia coli. Five N-HC phenethylamines and eight N-HC tyramines were synthesized by feeding HCAs and phenethylamine or tyramine to E. coli harboring 4CL (encoding 4-coumarate CoA:ligase) and either SHT (encoding phenethylamine N-HC transferase) or THT (encoding tyramine N-HC transferase). Also, N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid using E. coli harboring an additional gene, PDC (encoding phenylalanine decarboxylase) or TDC (encoding tyrosine decarboxylase). Finally, we synthesized N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine from glucose by reconstructing the metabolic pathways for their synthesis in E. coli. Productivity was maximized by optimizing the cell concentration and incubation temperature. CONCLUSIONS: We reconstructed the metabolic pathways for synthesis of N-HC tyramines and N-HC phenethylamines by expressing several genes including 4CL, TST or SHT, PDC or TDC, and TAL (encoding tyrosine ammonia lyase) and engineering the shikimate metabolic pathway to increase endogenous tyrosine concentration in E. coli. Approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine and 495.4 mg/L N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid. Furthermore, 152.5 mg/L N-(p-coumaroyl) phenethylamine and 94.7 mg/L N-(p-coumaroyl) tyramine were synthesized from glucose.

Metabolic engineering of Escherichia coli for the biosynthesis of flavonoid-O-glucuronides and flavonoid-O-galactoside.

Most flavonoids are glycosylated and the nature of the attached sugar can strongly affect their physiological properties. Although many flavonoid glycosides have been synthesized in Escherichia coli, most of them are glucosylated. In order to synthesize flavonoids attached to alternate sugars such as glucuronic acid and galactoside, E. coli was genetically modified to express a uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) specific for UDP-glucuronic acid (AmUGT10 from Antirrhinum majus or VvUGT from Vitis vinifera) and UDP-galactoside (PhUGT from Petunia hybrid) along with the appropriate nucleotide biosynthetic genes to enable simultaneous production of their substrates, UDP-glucuronic acid and UDP-galactose. To engineer UDP-glucuronic acid biosynthesis, the araA gene encoding UDP-4-deoxy-4-formamido-L-arabinose formyltransferase/UDP-glucuronic acid C-4″ decarboxylase, which also used UDP-glucuronic acid as a substrate, was deleted in E. coli, and UDP-glucose dehydrogenase (ugd) gene was overexpressed to increase biosynthesis of UDP-glucuronic acid. Using these strategies, luteolin-7-O-glucuronide and quercetin-3-O-glucuronide were biosynthesized to levels of 300 and 687 mg/L, respectively. For the synthesis of quercetin 3-O-galactoside, UGE (encoding UDP-glucose epimerase from Oryza sativa) was overexpressed along with a glycosyltransferase specific for quercetin and UDP-galactose. Using this approach, quercetin 3-O-galactoside was successfully synthesized to a level of 280 mg/L.

Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

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

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.

Synthesis of flavonoid O-pentosides by Escherichia coli through engineering of nucleotide sugar pathways and glycosyltransferase.

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/UDP-GlcA 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.

Production of kaempferol 3-O-rhamnoside from glucose using engineered Escherichia coli.

Flavonoids are ubiquitous phenolic compounds and at least 9,000 have been isolated from plants. Most flavonoids have been isolated and assessed in terms of their biological activities. Microorganisms such as Escherichia coli and Saccharomyces cerevisiae are efficient systems for the synthesis of flavonoids. Kaempferol 3-O-rhamnoside has notable biological activities such as the inhibition of the proliferation of breast cancer cells, the absorption of glucose in the intestines, and the inhibition of the self-assembly of beta amyloids. We attempted to synthesize kaempferol 3-O-rhamnoside from glucose in E. coli. Five flavonoid biosynthetic genes [tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), and flavonol 3-O-rhamnosyltransferase (UGT78D1)] from tyrosine were introduced into E. coli that was engineered to increase tyrosine production. By using this approach, the production of kaempferol 3-O-rhamnoside increased to 57 mg/L.

Potentiation of Antibiotic Activity of Aztreonam against Metallo-ß-Lactamase-Producing Multidrug-Resistant Pseudomonas aeruginosa by 3-O-Substituted Difluoroquercetin Derivatives.

The combination of aztreonam (ATM) and ceftazidime-avibactam (CAZ-AVI; CZA) has shown therapeutic potential against serine-ß-lactamase (SBL)- and metallo-ß-lactamase (MBL)-producing Enterobacterales. However, the ability of CZA to restore the antibiotic activity of ATM is severely limited in MBL-producing multidrug-resistant (MDR) Pseudomonas aeruginosa strains because of the myriad of intrinsic and acquired resistance mechanisms associated with this pathogen. We reasoned that the simultaneous inhibition of multiple targets associated with multidrug resistance mechanisms may potentiate the antibiotic activity of ATM against MBL-producing P. aeruginosa. During a search for the multitarget inhibitors through a molecular docking study, we discovered that di-F-Q, the previously reported efflux pump inhibitor of MDR P. aeruginosa, binds to the active sites of the efflux pump (MexB), as well as various ß-lactamases, and these sites are open to the 3-O-position of di-F-Q. The 3-O-substituted di-F-Q derivatives were thus synthesized and showed hereto unknown multitarget MDR inhibitory activity against various ATM-hydrolyzing ß-lactamases (AmpC, KPC, and New Delhi metallo-ß-lactamase (NDM)) and the efflux pump of P. aeruginosa, presumably by forming additional hydrophobic contacts with the targets. The multitarget MDR inhibitor 27 effectively potentiated the antimicrobial activity of ATM and reduced the MIC of ATM more than four-fold in 19 out of 21 MBL-producing P. aeruginosa clinical strains, including the NDM-producing strains which were highly resistant to various combinations of ATM with ß-lactamase inhibitors and/or efflux pump inhibitors. Our findings suggest that the simultaneous inhibition of multiple MDR targets might provide new avenues for the discovery of safe and efficient MDR reversal agents which can be used in combination with ATM against MBL-producing MDR P. aeruginosa.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Glycoside-metabolizing oxidoreductase D3dgpA from human gut bacterium.

The Gfo/Idh/MocA family enzyme DgpA was known to catalyze the regiospecific oxidation of puerarin to 3"-oxo-puerarin in the presence of 3-oxo-glucose. Here, we discovered that D3dgpA, dgpA cloned from the human gut bacterium Dorea sp. MRG-IFC3, catalyzed the regiospecific oxidation of various C-/O-glycosides, including puerarin, in the presence of methyl ß-D-3-oxo-glucopyranoside. While C-glycosides were converted to 3"- and 2"-oxo-products by D3dgpA, O-glycosides resulted in the formation of aglycones and hexose enediolone from the 3"-oxo-products. From DFT calculations, it was found that isomerization of 3"-oxo-puerarin to 2"-oxo-puerarin required a small activation energy of 9.86 kcal/mol, and the O-glycosidic bond cleavage of 3"-oxo-products was also thermodynamically favored with a small activation energy of 3.49 kcal/mol. In addition, the reaction mechanism of D3dgpA was discussed in comparison to those of Gfo/Idh/MocA and GMC family enzymes. The robust reactivity of D3dgpA was proposed as a new general route for derivatization of glycosides.

Structural and dynamic features of cold-shock proteins of Listeria monocytogenes, a psychrophilic bacterium.

Cold-shock proteins (Csps), proteins expressed when the ambient temperature drops below the growth-supporting temperature, bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Listeria monocytogenes is a psychrophilic food-borne pathogen that is problematic for the food industry. Structures of Csps from psychrophilic bacteria have not yet been studied. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share a high degree of primary sequence homology and a high degree of three-dimensional structural similarity. Here, we investigated the structural and dynamic features as well as the thermostability of L. monocytogenes CspA (Lm-CspA). Lm-CspA has a five-stranded ß-barrel structure with hydrophobic core packing and two salt bridges. When heptathymidine (dT(7)) binds, values for the heteronuclear nuclear Overhauser effect and order parameters of residues in surface loop regions near nucleic acid binding sites increase dramatically. Moreover, Carr-Purcell-Meiboom-Gill experiments showed that slow motions observed for the nucleic acid binding residues K7, W8, F15, F27, and R56 disappeared in Lm-CspA-dT(7). Lm-CspA is less thermostable than mesophilic and thermophilic Csps, with a lower melting temperature (40 °C). The structural flexibility that accompanies longer surface loops and less hydrophobic core packing and a number of salt bridges and unfavorable electrostatic repulsion are likely key factors in the low thermostability of Lm-CspA. This implies that the large conformational flexibility of psychrophilic Lm-CspA, which more easily accommodates nucleic acids at low temperature, is required for RNA chaperone function under cold-shock conditions and for the cold adaptation of L. monocytogenes.

Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity.

Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k(cat)/K(m) value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.

Production of hydroxycinnamoyl-shikimates and chlorogenic acid in Escherichia coli: production of hydroxycinnamic acid conjugates.

BACKGROUND: Hydroxycinnamates (HCs) are mainly produced in plants. Caffeic acid (CA), p-coumaric acid (PA), ferulic acid (FA) and sinapic acid (SA) are members of the HC family. The consumption of HC by human might prevent cardiovascular disease and some types of cancer. The solubility of HCs is increased through thioester conjugation to various compounds such as quinic acid, shikimic acid, malic acid, anthranilic acid, and glycerol. Although hydroxycinnamate conjugates can be obtained from diverse plant sources such as coffee, tomato, potato, apple, and sweet potato, some parts of the world have limited availability to these compounds. Thus, there is growing interest in producing HC conjugates as nutraceutical supplements. RESULTS: Hydroxycinnamoyl transferases (HCTs) including hydroxycinnamate-CoA shikimate transferase (HST) and hydroxycinnamate-CoA quinate transferase (HQT) were co-expressed with 4-coumarateCoA:ligase (4CL) in Escherichia coli cultured in media supplemented with HCs. Two hydroxycinnamoyl conjugates, p-coumaroyl shikimates and chlorogenic acid, were thereby synthesized. Total 29.1 mg/L of four different p-coumaroyl shikimates (3-p-coumaroyl shikimate, 4-p-coumaroyl shikimate, 3,4-di-p-coumaroyl shikimate, 3,5-di-p-coumaroyl shikimate, and 4,5-di-p-coumaroyl shikimate) was obtained and 16 mg/L of chlorogenic acid was synthesized in the wild type E. coli strain. To increase the concentration of endogenous acceptor substrates such as shikimate and quinate, the shikimate pathway in E. coli was engineered. A E. coli aroL and aroK gene were mutated and the resulting mutants were used for the production of p-coumaroyl shikimate. An E. coli aroD mutant was used for the production of chlorogenic acid. We also optimized the vector and cell concentration optimization. CONCLUSIONS: To produce p-coumaroyl-shikimates and chlorogenic acid in E. coli, several E. coli mutants (an aroD mutant for chlorogenic acid production; an aroL, aroK, and aroKL mutant for p-coumaroyl-shikimates production) were made and each mutant was tested using an optimized construct. Using this strategy, we produced 235 mg/L of p-coumaroyl-shikimates and 450 mg/L of chlorogenic acid.

Chromenylchalcones with inhibitory effects on monoamine oxidase B.

Structure-activity relationship (SAR) calculations were used to find monoamine oxidase-B (MAO-B) inhibitors by identifying pharmacophores exhibiting high inhibitory activities. Several such chromenylchalcones were designed and synthesized accordingly. Their inhibitory effects on MAO-B were determined using an HPLC-based method and an MAO-B enzyme assay kit. (E)-3-(6-Methoxy-2H-chromen-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one exhibited a half-maximal inhibitory concentration of 320 nM. Its molecular-level binding mode with the three-dimensional structure of MAO-B was elucidated using an in silico docking study. The chromenylchalcone scaffold, which is derived from natural products including isoflavonoids and chalcones, had not been previously reported as an MAO-B inhibitor.