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Applied Microbiology and Biotechnology (v.69, #6)
Autodisplay: efficient bacterial surface display of recombinant proteins by Joachim Jose (pp. 607-614).
To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 105 recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.
Autodisplay: efficient bacterial surface display of recombinant proteins by Joachim Jose (pp. 607-614).
To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 105 recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.
Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering by Masato Ikeda (pp. 615-626).
The aromatic amino acids, l-tryptophan, l-phenylalanine, and l-tyrosine, can be manufactured by bacterial fermentation. Until recently, production efficiency of classical aromatic amino-acid-producing mutants had not yet reached a high level enough to make the fermentation method the most economic. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. Many recent activities in this metabolic engineering have led to several effective approaches, which include modification of terminal pathways leading to removal of bottleneck or metabolic conversion, engineering of central carbon metabolism leading to increased supply of precursors, and transport engineering leading to reduced intracellular pool of the aromatic amino acids. In this review, advances in metabolic engineering for the production of the aromatic amino acids and useful aromatic intermediates are described with particular emphasis on two representative producer organisms, Corynebacterium glutamicum and Escherichia coli.
Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering by Masato Ikeda (pp. 615-626).
The aromatic amino acids, l-tryptophan, l-phenylalanine, and l-tyrosine, can be manufactured by bacterial fermentation. Until recently, production efficiency of classical aromatic amino-acid-producing mutants had not yet reached a high level enough to make the fermentation method the most economic. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. Many recent activities in this metabolic engineering have led to several effective approaches, which include modification of terminal pathways leading to removal of bottleneck or metabolic conversion, engineering of central carbon metabolism leading to increased supply of precursors, and transport engineering leading to reduced intracellular pool of the aromatic amino acids. In this review, advances in metabolic engineering for the production of the aromatic amino acids and useful aromatic intermediates are described with particular emphasis on two representative producer organisms, Corynebacterium glutamicum and Escherichia coli.
Ethanol fermentation from biomass resources: current state and prospects by Yan Lin; Shuzo Tanaka (pp. 627-642).
In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the technology of ethanol fermentation. This review provides practical examples and gives a broad overview of the current status of ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and fermentation, and sugar conversion into ethanol.
Ethanol fermentation from biomass resources: current state and prospects by Yan Lin; Shuzo Tanaka (pp. 627-642).
In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the technology of ethanol fermentation. This review provides practical examples and gives a broad overview of the current status of ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and fermentation, and sugar conversion into ethanol.
A membrane bioreactor for the biotransformation of α-pinene oxide to isonovalal by Pseudomonas fluorescens NCIMB 11671 by A. Boontawan; D. C. Stuckey (pp. 643-649).
In this work the biotransformation of α-pinene oxide to isonovalal using resting cells of Pseudomonas fluorescens NCIMB 11671 was evaluated in a membrane bioreactor for biotransformations (MBB). Since the membrane area required to obtain optimum productivities was calculated to be very large (1,000 m2 m−3), and not possible to fit into the laboratory reactor used, we initially evaluated performance with lower membrane areas (71 m2 m−3) in a batch system with both the substrate and product in the organic phase. This resulted in low productivities due to mass transfer limitations, so an optimum feeding rate of 0.1 g α-pinene oxide h−1 gcells −1 added directly to the reactor contents was determined in batch culture to minimise inhibition. The MBB was then operated continuously for the production of isonovalal, and a final concentration of 108 g l−1 was obtained in the organic reservoir after nearly 400 h of operation (0.32 g-isonovalal l−1 h−1), and the reaction was found not to be mass transfer limited. Finally, the relative viability of the cells was measured using fluorescent probes, and their half-life was found to be almost 2 months, confirming the ability of the MBB to facilitate biotransformations with inhibitory substrates and products.
A membrane bioreactor for the biotransformation of α-pinene oxide to isonovalal by Pseudomonas fluorescens NCIMB 11671 by A. Boontawan; D. C. Stuckey (pp. 643-649).
In this work the biotransformation of α-pinene oxide to isonovalal using resting cells of Pseudomonas fluorescens NCIMB 11671 was evaluated in a membrane bioreactor for biotransformations (MBB). Since the membrane area required to obtain optimum productivities was calculated to be very large (1,000 m2 m−3), and not possible to fit into the laboratory reactor used, we initially evaluated performance with lower membrane areas (71 m2 m−3) in a batch system with both the substrate and product in the organic phase. This resulted in low productivities due to mass transfer limitations, so an optimum feeding rate of 0.1 g α-pinene oxide h−1 gcells −1 added directly to the reactor contents was determined in batch culture to minimise inhibition. The MBB was then operated continuously for the production of isonovalal, and a final concentration of 108 g l−1 was obtained in the organic reservoir after nearly 400 h of operation (0.32 g-isonovalal l−1 h−1), and the reaction was found not to be mass transfer limited. Finally, the relative viability of the cells was measured using fluorescent probes, and their half-life was found to be almost 2 months, confirming the ability of the MBB to facilitate biotransformations with inhibitory substrates and products.
Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture by Yogender Pal; Amardeep Khushoo; K. J. Mukherjee (pp. 650-657).
Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h−1 for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l−1 h−1.
Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture by Yogender Pal; Amardeep Khushoo; K. J. Mukherjee (pp. 650-657).
Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h−1 for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l−1 h−1.
Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture by Daosheng Zhang; Robert W. Lovitt (pp. 658-664).
The growth performance of malolactic fermenting bacteria Oenococcus oeni NCIMB 11648 and Lactobacillus brevis X2 was assessed in continuous culture. O. oeni grew at a dilution rate range of 0.007 to 0.052 h−1 in a mixture of 5:6 (g l−1) of glucose/fructose at an optimal pH of 4.5, and L. brevis X2 grew at 0.010 to 0.089 h−1 in 10 g l−1 glucose at an optimal pH of 5.5 in a simple and safe medium. The cell dry weight, substrate uptake and product formation were monitored, as well as growth kinetics, yield parameters and fermentation balances were also evaluated under pH control conditions. A comparison of growth characteristics of two strains was made, and this showed significantly different performance. O. oeni has lower maximum specific growth rate (μmax=0.073 h−1), lower maximum cell productivity (Q x max=17.6 mg cell l−1 h−1), lower maximum biomass yield (Y x/s max=7.93 g cell mol−1 sugar) and higher maintenance coefficient (m s=0.45 mmol−1 sugar g−1 cell h−1) as compared with L. brevis X2 (μmax=0.110 h−1; Q x max=93.2 g−1 cell mol−1 glucose; Y x/s max=22.3 g cell mol−1 glucose; m s=0.21 mmol−1 glucose g−1 cell h−1). These data suggest a possible more productive strategy for their combined use in maturation of cider and wine.
Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture by Daosheng Zhang; Robert W. Lovitt (pp. 658-664).
The growth performance of malolactic fermenting bacteria Oenococcus oeni NCIMB 11648 and Lactobacillus brevis X2 was assessed in continuous culture. O. oeni grew at a dilution rate range of 0.007 to 0.052 h−1 in a mixture of 5:6 (g l−1) of glucose/fructose at an optimal pH of 4.5, and L. brevis X2 grew at 0.010 to 0.089 h−1 in 10 g l−1 glucose at an optimal pH of 5.5 in a simple and safe medium. The cell dry weight, substrate uptake and product formation were monitored, as well as growth kinetics, yield parameters and fermentation balances were also evaluated under pH control conditions. A comparison of growth characteristics of two strains was made, and this showed significantly different performance. O. oeni has lower maximum specific growth rate (μmax=0.073 h−1), lower maximum cell productivity (Q x max=17.6 mg cell l−1 h−1), lower maximum biomass yield (Y x/s max=7.93 g cell mol−1 sugar) and higher maintenance coefficient (m s=0.45 mmol−1 sugar g−1 cell h−1) as compared with L. brevis X2 (μmax=0.110 h−1; Q x max=93.2 g−1 cell mol−1 glucose; Y x/s max=22.3 g cell mol−1 glucose; m s=0.21 mmol−1 glucose g−1 cell h−1). These data suggest a possible more productive strategy for their combined use in maturation of cider and wine.
Isolation and properties of Aspergillus niger IBT-90 xylanase for bakery by Irena Romanowska; Jacek Polak; Stanisław Bielecki (pp. 665-671).
Xylanase of low molecular weight (K II) was isolated from the fungus Aspergillus niger IBT-90 cultivated in medium with wheat bran. K II was purified by precipitation with ammonium sulphate (20–80% saturation) and gel filtration on Biogel P-10. This enzyme is most active in hydrolysis of birchwood xylan at 50°C and pH 5.5. Xylanase K II has an ability to degrade 1,4-β-bonds and to debranch substrates. It degrades not only xylans but also cellulose, an important factor for its application in bakery. Ag+, Fe3+ and NBS are strong inhibitors of the enzyme. DTT and Na+ activate xylanase K II by 24 and 13%, respectively. Enzyme K II used as additive to flour improves dough properties, increases the volume of wheat–rye and whole meal bread, and increases the porosity of crumb and the moisture of the final product, consequently extending the shelf life of bread.
Isolation and properties of Aspergillus niger IBT-90 xylanase for bakery by Irena Romanowska; Jacek Polak; Stanisław Bielecki (pp. 665-671).
Xylanase of low molecular weight (K II) was isolated from the fungus Aspergillus niger IBT-90 cultivated in medium with wheat bran. K II was purified by precipitation with ammonium sulphate (20–80% saturation) and gel filtration on Biogel P-10. This enzyme is most active in hydrolysis of birchwood xylan at 50°C and pH 5.5. Xylanase K II has an ability to degrade 1,4-β-bonds and to debranch substrates. It degrades not only xylans but also cellulose, an important factor for its application in bakery. Ag+, Fe3+ and NBS are strong inhibitors of the enzyme. DTT and Na+ activate xylanase K II by 24 and 13%, respectively. Enzyme K II used as additive to flour improves dough properties, increases the volume of wheat–rye and whole meal bread, and increases the porosity of crumb and the moisture of the final product, consequently extending the shelf life of bread.
Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans by Shin-ichi Hirano; Mitsuru Haruki; Kazufumi Takano; Tadayuki Imanaka; Masaaki Morikawa; Shigenori Kanaya (pp. 672-681).
Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.
Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans by Shin-ichi Hirano; Mitsuru Haruki; Kazufumi Takano; Tadayuki Imanaka; Masaaki Morikawa; Shigenori Kanaya (pp. 672-681).
Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.
The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties by Anna Michniewicz; René Ullrich; Stanisław Ledakowicz; Martin Hofrichter (pp. 682-688).
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C, and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4°C. Kinetic constants (K m, k cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.
The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties by Anna Michniewicz; René Ullrich; Stanisław Ledakowicz; Martin Hofrichter (pp. 682-688).
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C, and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4°C. Kinetic constants (K m, k cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.
Direct mating between diploid sake strains of Saccharomyces cerevisiae by Shinji Hashimoto; Kazuo Aritomi; Takafumi Minohara; Yoshinori Nishizawa; Hisashi Hoshida; Susumu Kashiwagi; Rinji Akada (pp. 689-696).
Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.
Direct mating between diploid sake strains of Saccharomyces cerevisiae by Shinji Hashimoto; Kazuo Aritomi; Takafumi Minohara; Yoshinori Nishizawa; Hisashi Hoshida; Susumu Kashiwagi; Rinji Akada (pp. 689-696).
Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.
Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage by Phatchaneeya Thammawong; Watchara Kasinrerk; Raymond J. Turner; Chatchai Tayapiwatana (pp. 697-703).
A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand–receptor tracing).
Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage by Phatchaneeya Thammawong; Watchara Kasinrerk; Raymond J. Turner; Chatchai Tayapiwatana (pp. 697-703).
A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand–receptor tracing).
A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast by Megumi Shobayashi; Nobuhiko Mukai; Kazuhiro Iwashita; Yoshikazu Hiraga; Haruyuki Iefuji (pp. 704-710).
S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.
A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast by Megumi Shobayashi; Nobuhiko Mukai; Kazuhiro Iwashita; Yoshikazu Hiraga; Haruyuki Iefuji (pp. 704-710).
S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.
A new method for screening and isolation of hypersecretion mutants in Aspergillus niger by Xavier O. Weenink; Peter J. Punt; Cees A. M. J. J. van den Hondel; Arthur F. J. Ram (pp. 711-717).
Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein.
A new method for screening and isolation of hypersecretion mutants in Aspergillus niger by Xavier O. Weenink; Peter J. Punt; Cees A. M. J. J. van den Hondel; Arthur F. J. Ram (pp. 711-717).
Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein.
Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol by Gemma Assante; Adriana Bava; Gianluca Nasini (pp. 718-721).
The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l−1 to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l−1: it rather increased at higher ratios between 150 and 200 mg l−1. The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l−1) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.
Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol by Gemma Assante; Adriana Bava; Gianluca Nasini (pp. 718-721).
The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l−1 to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l−1: it rather increased at higher ratios between 150 and 200 mg l−1. The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l−1) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.
Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor by Niu Qiuhong; Huang Xiaowei; Tian Baoyu; Yang Jinkui; Liu Jiang; Zhang Lin; Zhang Keqin (pp. 722-730).
An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography, and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi. Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50°C. The purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge, this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship between bacterial pathogen and host and in improving the nematocidal activity in biological control.
Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor by Niu Qiuhong; Huang Xiaowei; Tian Baoyu; Yang Jinkui; Liu Jiang; Zhang Lin; Zhang Keqin (pp. 722-730).
An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography, and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi. Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50°C. The purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge, this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship between bacterial pathogen and host and in improving the nematocidal activity in biological control.