Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Identification and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Anoxybacillus flavithermus</Emphasis> useful in <Emphasis Type="SmallCaps">d</Emphasis>-tagatose production

d-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert d-galactose into the valuable d-tagatose using l-arabinose isomerase (l-AI). In this study, a thermophilic strain possessing l-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding l-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). l-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more d-tagatose from d-galactose by raising the reaction temperatures and adding borate. A 60% conversion of d-galactose to d-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k _cat /K _m) for d-galactose with borate was 9.47 mM⁻¹ min⁻¹, twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for d-galactose, suggesting its great potential for producing d-tagatose.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

<Emphasis Type="Italic">Arthroderma benhamiae</Emphasis> (The Teleomorph of <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis>) Mating Type-Specific Genes

This study first report to identify the mating type (−)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG).     

Functional Characterization of Trehalose Biosynthesis Genes from <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis>: An Osmolyte Involved in Stress Tolerance

Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

Signal peptide of <Emphasis Type="Italic">Aureobasidium</Emphasis><Emphasis Type="Italic">pullulans</Emphasis> xylanase: use for extracellular production of a fungal xylanase by <Emphasis Type="Italic">Escherichia coli</Emphasis>

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.     

Cloning,Purification and Characterization of an NAD-Dependent <Emphasis Type="SmallCaps">d</Emphasis>-Arabitol Dehydrogenase from Acetic Acid Bacterium, <Emphasis Type="Italic">Acetobacter suboxydans</Emphasis>

d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K _m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.     

Molecular cloning,characterization and expression of a chalcone reductase gene from <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> Bge. var. <Emphasis Type="Italic">mongholicus</Emphasis> (Bge.) Hsiao

A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38% extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa, which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus.     

Uptake and conversion of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The d-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of d-amino acids (d-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of d-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their d-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey’s reagent and separated by HPLC–MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied d-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of d-AAs. The addition of particular amino acids (d-Trp, d-Phe, d-Met and d-His) led to the accumulation of the corresponding l-amino acid. In almost all cases, the application of a d-AA resulted in the accumulation of d-Ala and d-Glu. The presented results indicate that soil borne d-AAs can actively be taken up and metabolized via central metabolic routes.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

Molecular chaperones facilitate the soluble expression of <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolases in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone.     

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.