Molecular cloning and expression analysis of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

A new putative gene encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (designated as SmHMGS, GenBank Accession No. FJ785326), which catalyses the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the mevalonic acid pathway, was isolated from young leaves of Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative SmHMGS was 1,655 bp containing a 1,381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids. Comparative and bioinformatic analyses revealed that SmHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated that SmHMGS belonged to the plant HMGS super family and had the closest relationship with HMGS from Hevea brasiliensis. Tissue expression pattern analysis revealed that the putative SmHMGS was constitutively expressed in all the tested tissues and strong in leaf, moderate in stem, weak in root, which was in contrast to SmHMGR reported before. The putative SmHMGS was found to be an elicitor-responsive gene, which could be induced by exogenous elicitors, including salicylic acid (SA) and methyl jasmonate (MJ). These results will help in understanding the role of HMGS in tanshinones biosynthesis in S. miltiorrhiza.     

The <Emphasis Type="Italic">Arxula adeninivorans</Emphasis><Emphasis Type="Italic">ATAL</Emphasis> gene encoding transaldolase-gene characterization and biotechnological exploitation

The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.     

Identification and characterization of class 1 <Emphasis Type="Italic">DXS</Emphasis> gene encoding 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate synthase,the first committed enzyme of the MEP pathway from soybean

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) catalyses the first committed step of the 2C-methyl-d-erythritol-4-phosphate (MEP) pathway, which is an alternative isoprenoids biosynthetic route that has been recently discovered. In this work, a DXS1-like cDNA (GmDXS1) was isolated from soybean. The full-length cDNA of GmDXS1 encoded 708 amino acid residues with a predicted molecular mass of 76.4 KD. Sequence alignment showed that GmDXS1 had high homology to known DXS proteins from other plant species and contained the conserved N-terminal plastid transit peptide, the N-terminal thiamine binding domain and pyridine binding DRAG domain. Phylogenetic analysis indicated that GmDXS1 belonged to the plant DXS1 cluster. Southern blot analysis indicated that a single copy of GmDXS1 gene existed in soybean genome. Tissue expression analysis revealed that GmDXS1 expressed in all photosynthetic tissues except pod walls and roots. Green fluorescence analysis with the fusion protein 35S:GmDXS1:GFP suggested that GmDXS1 was localized in plastid. The relatively higher photosynthetic pigment content in transgenic tobacco leaves compared to the control implied that GmDXS1 catalyzed the first potential regulatory step in photosynthetic pigment biosynthesis via the MEP pathway.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

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.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Fungal arabinan and <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolism

l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases, including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose.     

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.     

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.     

Methyl jasmonate- and salicylic acid-induced <Emphasis Type="SmallCaps">d</Emphasis>-chiro-inositol production in suspension cultures of buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>)

The aim of this work is to investigate the effects of methyl jasmonate (MeJ) and salicylic acid (SA) on d-chiro-inositol (DCI) production in buckwheat (Fagopyrum esculentum) suspension cultures. In this study, adding optimal concentrations of MeJ and SA at an appropriate time markedly increased DCI production (yield 6.141 and 5.521 mg/g DW, respectively). In addition, treatment of buckwheat cultures with a combination of 0.2 mM MeJ and 0.6 mM SA on days 0 and 9 increased the DCI yield to 7.579 mg/g DW, which was 3.726 times higher than that in the control; furthermore, the former yield was higher than that achieved by the addition of either elicitor alone. Moreover, unlike MeJ, SA did not exert a negative effect on cell growth.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

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.     

Effect of wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis>

Abstract-Effects of mechanical wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua leaves were investigated. HPLC-ELSD analysis indicated that there was a remarkable enhancement of the artemisinin content in 2 h after wounding treatment, and the content reached the maximum value at 4 h (nearly 50% higher than that in the control plants). The expression profile analysis showed that many important genes (HMGR, ADS, CPR, and CYP71AV1) involved in the artemisinin biosynthetic pathway were induced in a short time after wounding treatment. This study indicates that the artemisinin biosynthesis is affected by mechanical wounding. The possible mechanism of the control of gene expression during wounding is discussed.     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.