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Recently, production of D-mannose becomes a hotspot owing to it exhibiting many physiological functions on people's health and wide applications in food and pharmaceutical field. The use of biological enzymes to production of D-mannose is of particular receiving considerable concerns due to it possessing many merits over chemical synthesis and plant extraction strategies. D-Lyxose isomerase (D-LIase) plays a pivotal role in preparation of D-mannose from d-fructose through isomerization reaction. Thus, a novel putative D-LIase from thermophiles strain Thermoprotei archaeon which was expressed in E. coli BL21(DE3) was first identified and biochemically characterized. The recombinant D-LIase showed an optimal temperature of 80 and 85 °C and pH of 6.5. It was highly thermostable at 70 °C and 80 °C after incubating for 48 h and 33 h, respectively, with retaining over 50% of the initial activity. A lower concentration of Ni 2+ (0.5 mM) could greatly increase the activity by 25-fold, which was rare reported in other D-LIases. It was a dimer structure with melting temperature of 88.3 °C. Under the optimal conditions, 15.8 g L -1 of D-mannose and 33.8 g L -1 of D-xylulose were produced from 80 g L -1 of d-fructose and D-lyxose, respectively. This work provided a promising candidate sugar isomerase T. archaeon D-LIase for the production of D-mannose and D-xylulose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7 H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.05, designed with one pulse feed (t = 7 H) followed by a continuous feed and (ii) 2PM F -0.05, designed with two consecutive pulse feeds (t = 7 and 10 H) followed by a continuous feed. The continuous feeding of molasses for both fermentation strategies employed the same precalculated feeding rate, μ o = 0.05 H -1 . The maximum r-GI activities exhibited by PM-0.05 and 2PM F -0.05 were 29,050 and 30,642 U dm -3 , respectively. On the one hand, compared to PM-0.05 r-GI activity reached its maximum within a shorter cultivation time (∆t max = 2 H) at 2PM F -0.05, which could be preferable in terms of manufacturing costs and possible risks; on the other hand, PM-0.05 is a simpler fermentation regime compared to 2PM F -0.05 with respect to manipulations that should be considered in large-scale production., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.

d-Mannose isomerase (MIase) catalyzes the conversion of d-fructose to d-mannose. In this study, the MIase encoding gene (yihS) from Escherichia coli BL21 contains an ORF of 1242bp, was cloned and expressed in Bacillus subtilis WB800. This heterologous expression resulted in a hexamer with a molecular weight of 274.5kDa and Tm of 61.4°C. Efficient MIase secretory expression by the robust recombinant B. subtilis was achieved with activity of 51.2U/ml (d-mannose forming). Its optimal temperature and pH were 45.0°C and 7.0, respectively. Using d-fructose as the substrate, Km, kcat and catalytic efficiency value of kinetic reaction were 203.7±6.7mM, 27.7±0.7s(-1) and 136.0±2.9M(-1)s(-1), respectively. The production of d-mannose reached about 150g/l with approximately 25% turnover yield under the optimum conditions. These results demonstrated that B. subtilis was a promising candidate of MIase expression system for d-mannose production., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Saccharomyces cerevisiae strains with xylose isomerase (XI) pathway were constructed using a flocculating industrial strain (YC-8) as the host. Both strains expressing wild-type xylA (coding XI) from the fungus Orpinomyces sp. and the bacterium Prevotella ruminicola, respectively, showed better growth ability and fermentation capacity when using xylose as the sole sugar than most of the reported strains expressing XI. Codon optimization of both XIs did not improve the xylose fermentation ability of the strains. Adaption significantly increased XI activity resulting in improved growth and fermentation. The strains expressing codon-optimized XI showed a higher increase in xylose consumption and ethanol production compared to strains expressing wild XI. Among all strains, the adapted strain YCPA2E expressing XI from P. ruminicola showed the best performance in the fermentation of xylose to ethanol. After 48 h of fermentation, YCPA2E assimilated 16.95 g/L xylose and produced 6.98 g/L ethanol. These results indicate that YC-8 is a suitable host strain for XI expression, especially for the codon-optimized XI originating from P. ruminicola., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation., (Copyright © 2015 International Metabolic Engineering Society. All rights reserved.)

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L(-1) at t = 66 h and 32530 U L(-1) at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy.

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.

Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.

To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.

Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs) and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 °C, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.

The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.

Styrene is a large volume, commodity petrochemical with diverse commercial applications, including as a monomer building-block for the synthesis of many useful polymers. Here we demonstrate how, through the de novo design and development of a novel metabolic pathway, styrene can alternatively be synthesized from renewable substrates such as glucose. The conversion of endogenously synthesized l-phenylalanine to styrene was achieved by the co-expression of phenylalanine ammonia lyase and trans-cinnamate decarboxylase. Candidate isoenzymes for each step were screened from bacterial, yeast, and plant genetic sources. Finally, over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae (originally classified as ferulate decarboxylase) in an l-phenylalanine over-producing Escherichia coli host led to the accumulation of up to 260 mg/L in shake flask cultures. Achievable titers already approach the styrene toxicity threshold (determined as ~300 mg/L). To the best of our knowledge, this is the first report of microbial styrene production from sustainable feedstocks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H(2)O(2)) stress, on isoprene production were further examined. Heat, salt, and H(2)O(2) induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

Unlabelled: We determined the relative expression of ubiquitin (ub), glucosamine-6-phosphate-isomerase (gn6pi) and cyst wall protein (cwp) genes during encystment of the Portland-1 and Portland-1R strains of Giardia intestinalis. Encystment was induced with bile for different time periods. The presence of encystment-specific vesicles (ESVs) and the relative expression of genes (log(10)ΔRn) were determined by transmission electron microscopy and real-time PCR, respectively. Our results demonstrated the gene expression and the presence of ESVs after 6h of encystment. Values of cwp2 gene expression increased by 591-fold in strain Portland-1 and 78.2-fold in strain Portland-1R at this time point compared to values at 0h, after which values gradually decreased until reaching basal values between 8 and 18h after the encystment started. Expression of gn6pi was 43.5- and 46.3-fold higher than basal values, in Portland-1 and Portland-1R, respectively. Ub gene expression was 82.25-fold higher than its basal levels at 4h, after which expression decreased gradually until reaching basal values after 16h., Conclusions: This work showed the relationship between the presence of ESVs and encystment gene expression at 6h, and resistance to albendazole does not inhibit the encystment process. The results revealed important knowledge with implications in the control of parasite dissemination for preventing parasite transmission., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca(2+) concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L(-1), respectively. The interactions between Ca(2+) concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for L-ribulose production, the productivity was as high as 56.7 g L(-1) h(-1) at 400 g L(-1) substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of L-ribulose, particularly because of its high stability and low cost.

The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product ofxylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k(cat) of 14,504 min(-1) and a k(cat)/K(m) of 121 min(-1) mM(-1) for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.

Arthrobacter nicotinanae cells, producers of glucose isomerase, were immobilized in xerogel of silicium dioxide, and properties of the resulted heterogeneous biocatalysts were investigated in the process of isomerization of monosaccharide (glucose and fructose). The glucose isomerase activity of the resulted biocatalysts was shown to be 10 U/g, on average, taking into account the loss of the activity upon the immobilization, which amounted to 50% of the cell activity in suspension. The rate of the fructose isomerization increased linearly in the range of 55-80 degrees C with the temperature coefficient 1.3. The biocatalysts were stable in this range; they were rapidly inactivated, however, at increasing temperature. The half-inactivation time was six to seven h and five min or less at 80 degrees C and 85 degrees C, respectively. The half-inactivation time of heterogeneous biocatalysts was 50-90 h in the periodic process of isomerization of 2 M monosaccharides at 60 degrees C in the presence of the immobilized Arthrobacter nicotinanae cells.

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, or incubation period) and culture medium composition (mineral salts, carbon and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. On the other hand, the strain required for growth Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g. ammonium citrate). The bacterium had no requirement for sodium acetate for both growth and production of isomerases. The production rate of enzymes was increased when metal ions were added and mainly manganese (2.5 mM).

The effect of a specific substrate as well as other carbon sources on the biosynthesis of xylose isomerase in the actinobacterium Arthrobacter ureafaciens BIM B-6 has been studied. It was established that xylose and its structural analogue xylite induced the production of the enzyme by bacterial cells. The inducing effect peaked at a concentration of specific substrates of 0.025% (as carbon) and then remained unchanged irrespective of the substrate amount. It has been shown that the synthesis of xylose isomerase by A. ureafaciens is controlled by catabolite repression occurring at the transcription level and mediated by cyclic 3',5'-AMP.

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.

Plants have the ability to remediate environmental pollution. Especially, they have a high purification capability for airpollution. We have measured the purification characteristics of foliage plants for indoor airpollutants--for example, formaldehyde (HCHO), toluene, and xylene--using a tin oxide gas sensor. HCHO is an important intermediate for biological fixation of C1 compounds in methylotrophs. The ribulose monophosphate pathway of HCHO fixation is inherent in many methylotrophic bacteria, which can grow on Cl compounds. Two genes for the key enzymes, HPS and PHI, from the methylotrophic bacterium Mycobacterium gastri MB19 were introduced into tobacco. In this article, the HCHO-removal characteristic of the transformant was examined by using the gas sensor in order to evaluate quantitatively. The purification characteristics of the transformant for toluene, xylene, and styrene were also measured. The results confirmed an increase of 20% in the HCHO-removal capability. The differences of the purification capabilities for toluene, xylene, and styrene were not recognized.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

In Escherichia coli and most Gram-negative bacteria, KDO (3-deoxy-D-manno-octulosonate), a component of the lipopolysaccharide inner core, is essential for outer membrane biogenesis and cell viability. Two recently identified genes involved in KDO biosynthesis, kdsD and kdsC, belong to the yrbG-yhbG locus where four additional ORFs (yrbG, yrbK, yhbN and yhbG) with unknown function are located. We have constructed six conditional expression mutants in which the arabinose-inducible araBp promoter is respectively located upstream of each gene of the locus. Complementation analysis of these mutants indicates that the locus is organized in at least three operons and that the three distal genes (yrbK, yhbN and yhbG) are essential for E. coli viability. Surprisingly, kdsD and kdsC (encoding a D-arabinose 5-phosphate isomerase and a KDO 8-phosphate phosphatase, respectively) were shown to be non-essential, indicating genetic redundancy for these two functions. A preliminary characterization of the arabinose-dependent mutants under permissive conditions and upon depletion revealed increased sensitivity to hydrophobic toxic chemicals, suggesting that the mutants have a defective outer membrane. These genes may thus be implicated in cell envelope integrity.

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

A plate method was developed to screen for xylose isomerase-producing microorganisms based on the use of 2,3,5-triphenyltetrazolium as an indicator of D-xylulose, the D-xylose isomerization product. The use of this method allows microorganisms to be differentiated by the character of the enzyme synthesis (inducible or constitutive).

The enzyme, glucosamine-6-phosphate isomerase (GNPI) or deaminase (GNPDA) (EC 5.3.1.10), catalyzes the conversion of GNP to fructose-6-phosphate and ammonia, with an aldo/keto isomerization and an amination/deamination. A hamster sperm-derived protein (Oscillin) with high similarity to bacterial GNPI has been proved to be capable of inducing calcium oscillation in eggs at fertilization. GNPI/Oscillin was supposed to be an important factor in starting embryonic development. From the cDNA library of human dendritic cells (DC), we isolated a novel full-length cDNA encoding a 276-amino acid-residue protein that shares high homology with human GNPI/Oscillin. So, the novel molecule is named as GNPI2. The GNPI2 gene consists of seven exons and six introns. It is mapped to chromosome 4. Northern blot analysis indicated that the tissue distribution of GNPI2 mRNA is different from that of human GNPI or Oscillin mRNA. GNPI2 is ubiquitously expressed in most of human tissues with high expression in testis, ovary, placenta, and heart. Like GNPI, the recombinant GNPI2 has been proved to have the enzymatic activity to catalyze the conversion of GNP to fructose-6-phosphate. Our results indicated that GNPI2 is a novel protein with definite function as a GNPI., (Copyright 2003 Wiley-Liss, Inc.)

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance ( tsr(R)) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr(R) transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI ( GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI.

A homolog of the glucosamine-6-phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site-directed mutant indicated that the Myb-binding sequence (5'-AACTG-3') localized in the upstream region is important for gene expression. The results of gel-shift assays showed the presence of an Myb-related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages.

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.

The occurrence of promoter-generating mutations allowing the transcription of heterologous genes has been studied in a system based on the plasmid-mediated conjugal transfer of histidine biosynthetic genes from a donor bacterium (Azospirillum brasilense) into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability and initially unable to recognize the transcriptional signal of the introgressed gene(s). Under selective stressful conditions, His+ revertants accumulated in the E. coli His- culture. The number of His+ colonies was dependent on the time of incubation under selective conditions, the strength of selective pressure, and on the crowding of cells plated; moreover, it was independent of the physiological status of the cell (i.e. the growth phase). Sequence analysis of plasmid DNA extracted from E. coli His+ revertants revealed that single base substitutions in the region upstream of the A. brasilense his operon resulted in an adjustment of the pre-existing sequence that was rendered similar to the E. coli -10 promoter sequence and transcriptable by the host RNA-polymerase. One particular transition (C --> T) was predominant in the His+ revertants. Data presented here indicated that the barriers to the expression of horizontally transferred heterologous genes or operons may be overcome in a short time scale and at high frequency, and supported the selfish operon model on the origin and evolution of gene clusters.

A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (ThR) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions.

The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria. It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway. For this purpose, a segment of dxr was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with the PCR segment led to the identification of a 26. 5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing. The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate=DMADP) was significantly (P

It was reported that a hamster protein, called "oscillin," with a sequence related to that of an Escherichia coli GNPDA triggered Ca(2+) oscillations in mammalian oocytes when introduced into their cytoplasm upon fertilization. Recently, it was shown that GNPDA/oscillin is ubiquitously expressed in rat tissues and that a recombinant hamster GNPDA/oscillin protein does not exhibit oscillin activity when injected into oocytes. In the mouse, the nature and role of such a GNPDA/oscillin is not known, but another candidate protein, tr-kit, has been proposed as a sperm factor causing oocyte activation. In order to clarify this issue, we have characterized the mouse homolog of hamster and human GNPDA/oscillin, and examined its expression along with that of tr-kit, in parallel. We report here the molecular cloning and sequencing of mouse GNPDA/oscillin, which shows over 96% identity with the hamster and human homologs. Using specific primers, we performed an RT-PCR analysis to determine the tissue distribution of mouse GNPDA/oscillin mRNA. Unlike tr-kit mRNA which is expressed solely in mouse testis, GNPDA/oscillin mRNA is detected in unfertilized oocytes and in all tissues examined including testis, heart, thymus, liver, ovary, uterus, kidney, spleen, and lung. The protein itself is also detected in all tissues examined by Western blots. Indirect immunofluorescence studies, using an antibody raised against hamster GNPDA, demonstrate that GNPDA is lost with the acrosome reaction of mouse spermatozoa, is localized in the equatorial and neck regions of the human spermatozoa and the post-acrosomal region of the hamster spermatozoa. Our results thus indicate that mouse GNPDA/oscillin, the homolog of hamster oscillin, unlike tr-kit, does not exhibit some of the required characteristics expected from a putative sperm-derived oocyte-activating factor., (Copyright 2000 Wiley-Liss, Inc.)

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells. As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized. Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated. To reduce the negative effects of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expression. In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium. However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium. GPI was induced to a factor of two under ammonium-containing medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium., (Copyright 1999 John Wiley & Sons, Inc.)

Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.