Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli

Flavonols are plant polyphenolic compounds that belong to the class of molecules collectively known as flavonoids. Because of their demonstrated health benefits towards a wide array of human pathological conditions, a great interest has emerged for their biosynthesis from well-characterized microbial hosts. We present the functional expression in Escherichia coli of a plant P450 flavonoid 3', 5'-hydroxylase (F3'5'H) as a fusion protein with a P450 reductase. This expression allowed metabolic engineering of E. coli to produce the flavonol kaempferol and the 3', 4' B-ring hydroxylated flavonol quercetin from the p-coumaric acid precursor by simultaneously co-expressing the fusion protein with 4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS). Biosynthesis of the B-ring tri-hydroxylated flavonol myricetin from the engineered strains was accomplished when flavanones rather than phenylpropanoid acids were used as precursor molecules. Cultivation of the recombinant strains in rich medium increased the synthesis of all flavonoids with the exception of myricetin. The present work opens the possibility of the future production of several other hydroxylated flavonoid molecules in E. coli.     

Thymidine diphosphate-6-deoxy-L-lyxo-4-hexulose reductase synthesizing dTDP-6-deoxy-L-talose from Actinobacillus actinomycetemcomitans

The serotype c-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans NCTC 9710 contains an unusual sugar, 6-deoxy-L-talose, which has been identified as a constituent of cell wall components in some bacteria. Two genes coding for thymidine diphosphate (dTDP)-6-deoxy-L-lyxo-4-hexulose reductases were identified in the gene cluster required for biosynthesis of serotype c-specific polysaccharide. Both dTDP-6-deoxy-L-lyxo-4-hexulose reductases were overproduced and purified from Escherichia coli transformed with the plasmids containing these genes. The sugar nucleotides converted by both reductases were purified by reversed-phase high performance liquid chromatography and identified by (1)H nuclear magnetic resonance and gas-liquid chromatography. The results indicated that one of two reductases produced dTDP-6-deoxy-L-talose and the other produced dTDP-L-rhamnose (dTDP-6-deoxy-L-mannose). The amino acid sequence of the dTDP-6-deoxy-L-lyxo-4-hexulose reductase forming dTDP-6-deoxy-L-talose shared only weak homology with that forming dTDP-L-rhamnose, despite the fact that these two enzymes catalyze the reduction of the same substrate and the products are determined by the stereospecificity of the reductase activity. Neither the gene for dTDP-6-deoxy-L-talose biosynthesis nor its corresponding protein product has been found in other bacteria; this biosynthetic pathway is identified here for the first time.     

Cloning and characterization of a putative UDP-rhamnose synthase 1 from Populus euramericana Guinier

L-Rhamnose is a constituent of plant primary cell wall polysaccharides including rhamnogalacturonan-I, rhamnogalacturonan-II, and other natural plant-based compounds. UDP-rhamnose serves as a rhamnose donor whose synthesis is catalyzed by UDP-rhamnose synthase (RHM). A RHM gene, PRHM was cloned from Populus euramericana Guinier. PRHM contains two domains: the NAD dependent epimerase/dehydratase family domain and the RmlD (dTDP-keto-rhamnose-4-keto-reductase) substrate-binding domain. Because the recombinant PRHM did not demonstrate any activity during an in vitro assay, complementation with an Escherichia coli mutant was carried out. The rfbD (dTDP-4-dehydrorhamnose reductase), which encodes an enzyme catalyzing the conversion of dTDP-4-keto-rhamnose to TDP-rhamnose, was mutated in E. coli. The mutant strain B-rfbD was transformed with PRHM gene and a flavonoid rhanmosyltransferase gene, AtUGT78D1. The resulting transformant was able to convert quercetin into quercetin 3-O-rhamnoside in a manner similar to that by the wild type E. coli strain harboring AtUGT78D1. This result indicated that PRHM catalyzed the conversion of UDP-glucose into UDP-rhamnose.     

A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis

l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.     

Regioselective synthesis of plant (iso)flavone glycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.     

Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae)

The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar range of non-acylated flavonoids.     

Biosynthesis of dTDP-6-deoxy-beta-D-allose, biochemical characterization of dTDP-4-keto-6-deoxyglucose reductase (GerKI) from Streptomyces sp. KCTC 0041BP

dTDP-6-deoxy-d-allose, an unusual deoxysugar, has been identified as an intermediate in the mycinose biosynthetic pathway of several macrolide antibiotics. In order to characterize the biosynthesis of this deoxysugar, we have cloned and heterologously overexpressed gerK1 in Escherichia coli BL21 (DE3) cells. This gene encodes for a protein with the putative function of a dTDP-4-keto-6-deoxyglucose reductase, which appears to be involved in the dihydrochalcomycin (GERI-155) biosynthesis evidenced by Streptomyces sp KCTC 0041BP. Our results revealed that GerK1 exhibited a specific reductive effect on the 4-keto carbon of dTDP-4-keto-6-deoxy-d-allose, with the hydroxyl group in an axial configuration at the C3 position only. The enzyme catalyzed the conversion of dTDP-4-keto-6-deoxyglucose to dTDP-6-deoxy-beta-D-allose, according to the results of an in vitro coupled enzyme assay, in the presence of GerF (dTDP-4-keto-6-deoxyglucose 3-epimerase). The product was isolated, and its stereochemistry was determined via nuclear magnetic resonance analysis.     

A new route to dTDP-6-deoxy-l-talose and dTDP-L-rhamnose: dTDP-L-rhamnose 4-epimerase in Burkholderia thailandensis

dTDP-l-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-l-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD_Bth (BTH_I1472) and wbiB_Bth (BTH_I1476), respectively. Incubation of the recombinant WbiB_Bth with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-l-talose (dTDP-6dTal) from α-d-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. ¹H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB_Bth and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB_Bth alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.     

New flavonoid glycosides from Aconitum naviculare (Brühl) Stapf, a medicinal herb from the trans-Himalayan region of Nepal

Three new flavonoid glycosides, 3-O-[beta-D-glucopyranosyl-(1-->3)-(4-O-trans-p-coumaroyl)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranosyl]-7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]kaempferol, 3-O-[beta-D-glucopyranosyl-(1-->3)-(4-O-trans-p-coumaroyl)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranosyl]-7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]quercetin and 7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]quercetin were isolated from the aqueous extract of the aerial parts of Aconitum naviculare. Their structures were elucidated by spectral analysis (HRAPI-TOF MS, 1H, 13C NMR, HMQC, HMBC, DFQ-COSY, ROESY and TOCSY).     

A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.

The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.     

Antioxidant flavonoids from leaves of Polygonum hydropiper L

Ten flavonoid compounds were isolated from the dried leaves of Polygonum hydropiper L. (Laksa leaves), and identified as 3-O-alpha-L-rhamnopyranosyloxy-3',4',5,7-tetrahydroxyflavone; 3-O-beta-D-glucopyranosyloxy-4',5,7-trihydroxyflavone; 6-hydroxyapigenin; 6"-O-(3,4,5-trihydroxybenzoyl) 3-O-beta-D-glucopyranosyloxy-3', 4', 5, 7-tetrahydroxyflavone; scutillarein; 6-hydroxyluteolin; 3',4',5,6,7-pentahydroxyflavone; 6-hydroxyluteolin-7-O-beta-D-glucopyranoside; quercetin 3-O-beta-D-glucuronide; 2"-O-(3,4,5-trihydroxybenzoyl) quercitrin; quercetin. Evaluation of the antioxidative activity, conducted in vitro, by using electron spin resonance (ESR) and ultraviolet visible (UV-vis) spectrophotometric assays, showed that these isolated flavonoids possess strong antioxidative capabilities. Measurement of the Trolox equivalent antioxidant capacity (TEAC) values, against ABTS (2,2'-azinobis(3-ethyl-benzo-thiazoline-6-sulphonic acid) radicals and phenyl-tert-butyl nitrone (PBN) azo initiator (AI) also showed strong anti-oxidative activity. The most powerful of the antioxidants was 2"-O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin). A combination of two flavonoid compounds was tested for synergistic anti-oxidative capacity, but no significant improvement was observed.     

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.     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Guanosine diphosphate-4-keto-6-deoxy-d-mannose reductase in the pathway for the synthesis of GDP-6-deoxy-d-talose in Actinobacillus actinomycetemcomitans.

The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy-d-talose. Guanosine diphosphate (GDP)-6-deoxy-d-talose is the activated sugar nucleotide form of 6-deoxy-d-talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy-d-talose synthetic enzymes, GDP-alpha-d-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-alpha-d-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy-d-talose is produced from GDP-alpha-d-mannose. This paper is the first report on the GDP-6-deoxy-d-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-d-mannose reductase in the synthesis of GDP-6-deoxy-d-talose.     

Stereochemistry of the dTDP-glucose oxidoreductase reaction.

The stereochemical course of the dTDP-glucose oxidoreductase (EC 4.2.1.46) reaction was studied using enzyme partially purified from Escherichia coli and dTDP-(6R)- and (6S)-[4-2H, 6-3H]glucose as substrate. The latter was prepared enzymatically by reduction of (3R)- and (3S)-3-P-[3-3H]glycerate to the 1-deuterated 3-P-glyceraldehyde with (4S)-[4-2H]NADH, followed first by conversion to glucose-1-P with the glycolytic enzymes, and then by transformation into the dTDP derivative. The stereospecifically labeled dTDP-glucose samples were mixed with nonlabeled carrier material and converted to dTDP-4-keto-6-deoxyglucose, which contained a chiral methyl group as shown by chirality analysis of the acetic acid resulting from Kuhn-Roth oxidation of the sugar nucleotide. These results confirm that the hydrogen transfer from C4 to C6 is intramolecular and show that the migrating hydrogen replaces the 6-hydroxyl group with inversion of configuration. Assuming that the hydrogen transfer, since it is intramolecular, must be suprafacial, it follows that the elimination of water from C5 and C6 is formally syn, whereas the reduction of the resulting delta5,6-double bond formally involves an anti addition of H+ and H-.     

Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.     

Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle

In an effort to characterize fruit ripening-related genes functionally, two glucosyltransferases, FaGT6 and FaGT7, were cloned from a strawberry (Fragaria x ananassa) cDNA library and the full-length open reading frames were amplified by rapid amplification of cDNA ends. FaGT6 and FaGT7 were expressed heterologously as fusion proteins in Escherichia coli and target protein was purified using affinity chromatography. Both recombinant enzymes exhibited a broad substrate tolerance in vitro, accepting numerous flavonoids, hydroxycoumarins, and naphthols. FaGT6 formed 3-O-glucosides and minor amounts of 7-O-, 4'-O-, and 3'-O-monoglucosides and one diglucoside from flavonols such as quercetin. FaGT7 converted quercetin to the 3-O-glucoside and 4'-O-glucoside and minor levels of the 7- and 3'-isomers but formed no diglucoside. Gene expression studies showed that both genes are strongly expressed in achenes of small-sized green fruits, while the expression levels were generally lower in the receptacle. Significant levels of quercetin 3-O-, 7-O-, and 4'-O-glucosides, kaempferol 3-O- and 7-O-glucosides, as well as isorhamnetin 7-O-glucoside, were identified in achenes and the receptacle. In the receptacle, the expression of both genes is negatively controlled by auxin which correlates with the ripening-related gene expression in this tissue. Salicylic acid, a known signal molecule in plant defence, induces the expression of both genes. Thus, it appears that FaGT6 and FaGT7 are involved in the glucosylation of flavonols and may also participate in xenobiotic metabolism. The latter function is supported by the proven ability of strawberries to glucosylate selected unnatural substrates injected in ripe fruits. This report presents the first biochemical characterization of enzymes mainly expressed in strawberry achenes and provides the foundation of flavonoid metabolism in the seeds.