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Applied Microbiology and Biotechnology (v.68, #5)
Global physiological understanding and metabolic engineering of microorganisms based on omics studies by S. J. Park; S. Y. Lee; J. Cho; T. Y. Kim; J. W. Lee; J. H. Park; M.-J. Han (pp. 567-579).
Through metabolic engineering, scientists seek to modify the metabolic pathways of living organisms to facilitate optimized, efficient production of target biomolecules. During the past decade, we have seen notable improvements in biotechnology, many of which have been based on metabolically engineered microorganisms. Recent developments in the fields of functional genomics, transcriptomics, proteomics, and metabolomics have changed metabolic engineering strategies from the local pathway level to the whole system level. This article focuses on recent advances in the field of metabolic engineering, which have been powered by the combined approaches of the various “omics” that allow us to understand the microbial metabolism at a global scale and to develop more effectively redesigned metabolic pathways for the enhanced production of target bioproducts.
Global physiological understanding and metabolic engineering of microorganisms based on omics studies by S. J. Park; S. Y. Lee; J. Cho; T. Y. Kim; J. W. Lee; J. H. Park; M.-J. Han (pp. 567-579).
Through metabolic engineering, scientists seek to modify the metabolic pathways of living organisms to facilitate optimized, efficient production of target biomolecules. During the past decade, we have seen notable improvements in biotechnology, many of which have been based on metabolically engineered microorganisms. Recent developments in the fields of functional genomics, transcriptomics, proteomics, and metabolomics have changed metabolic engineering strategies from the local pathway level to the whole system level. This article focuses on recent advances in the field of metabolic engineering, which have been powered by the combined approaches of the various “omics” that allow us to understand the microbial metabolism at a global scale and to develop more effectively redesigned metabolic pathways for the enhanced production of target bioproducts.
Functional genomics of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1 by Ralf Rabus (pp. 580-587).
Nitrate-reducing bacteria of the recently recognized Azoarcus/Thauera group within the Betaproteobacteria contribute significantly to the biodegradation of aromatic and other refractory compounds in anoxic waters and soils. Strain EbN1 belongs to a distinct cluster (new genus) and is the first member of this phylogenetic group, the genome of which has been determined (4.7 Mb; one chromosome, two plasmids) by [Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27–36]. Ten anaerobic and four aerobic aromatic-degradation pathways were recognized on the chromosome, with the coding genes mostly forming clusters. Presence of paralogous gene clusters (e.g. for anaerobic ethylbenzene degradation) suggests an even broader degradation spectrum than previously known. Metabolic versatility is also reflected by the presence of multiple respiratory complexes and is apparently controlled by an extensive regulatory network. Strain EbN1 is unique for its capacity to degrade toluene and ethylbenzene anaerobically via completely different pathways. Bioinformatical analysis of their genetic blueprints and global expression analysis (DNA-microarray and proteomics) of substrate-adapted cells [Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus R (2005) Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187:1493–1503] indicated coordinated vs sequential modes of regulation for the toluene and ethylbenzene pathways, respectively.
Functional genomics of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1 by Ralf Rabus (pp. 580-587).
Nitrate-reducing bacteria of the recently recognized Azoarcus/Thauera group within the Betaproteobacteria contribute significantly to the biodegradation of aromatic and other refractory compounds in anoxic waters and soils. Strain EbN1 belongs to a distinct cluster (new genus) and is the first member of this phylogenetic group, the genome of which has been determined (4.7 Mb; one chromosome, two plasmids) by [Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27–36]. Ten anaerobic and four aerobic aromatic-degradation pathways were recognized on the chromosome, with the coding genes mostly forming clusters. Presence of paralogous gene clusters (e.g. for anaerobic ethylbenzene degradation) suggests an even broader degradation spectrum than previously known. Metabolic versatility is also reflected by the presence of multiple respiratory complexes and is apparently controlled by an extensive regulatory network. Strain EbN1 is unique for its capacity to degrade toluene and ethylbenzene anaerobically via completely different pathways. Bioinformatical analysis of their genetic blueprints and global expression analysis (DNA-microarray and proteomics) of substrate-adapted cells [Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus R (2005) Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187:1493–1503] indicated coordinated vs sequential modes of regulation for the toluene and ethylbenzene pathways, respectively.
Biotechnological production and applications of phytases by Stefan Haefner; Anja Knietsch; Edzard Scholten; Joerg Braun; Markus Lohscheidt; Oskar Zelder (pp. 588-597).
Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.
Biotechnological production and applications of phytases by Stefan Haefner; Anja Knietsch; Edzard Scholten; Joerg Braun; Markus Lohscheidt; Oskar Zelder (pp. 588-597).
Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.
Complex media from processing of agricultural crops for microbial fermentation by Mette Hedegaard Thomsen (pp. 598-606).
This mini-review describes the concept of the green biorefinery and lists a number of suitable agricultural by-products, which can be used for production of bioenergy and/or biochemicals. A process, in which one possible agricultural by-product from the green crop drying industry, brown juice, is converted to a basic, universal fermentation medium by lactic acid fermentation, is outlined. The resulting all-round fermentation medium can be used for the production of many useful fermentation products when added a carbohydrate source, which could possibly be another agricultural by-product. Two examples of such products—polylactic acid and l-lysine—are given. A cost calculation shows that this fermentation medium can be produced at a very low cost ≈1.7 Euro cent/kg, when taking into account that the green crop industry has expenses amounting to 270,000 Euro/year for disposal of the brown juice. A newly built lysine factory in Esbjerg, Denmark, can benefit from this process by buying a low price medium for the fermentation process instead of more expensive traditional fermentation liquids such as corn steep liquor.
Complex media from processing of agricultural crops for microbial fermentation by Mette Hedegaard Thomsen (pp. 598-606).
This mini-review describes the concept of the green biorefinery and lists a number of suitable agricultural by-products, which can be used for production of bioenergy and/or biochemicals. A process, in which one possible agricultural by-product from the green crop drying industry, brown juice, is converted to a basic, universal fermentation medium by lactic acid fermentation, is outlined. The resulting all-round fermentation medium can be used for the production of many useful fermentation products when added a carbohydrate source, which could possibly be another agricultural by-product. Two examples of such products—polylactic acid and l-lysine—are given. A cost calculation shows that this fermentation medium can be produced at a very low cost ≈1.7 Euro cent/kg, when taking into account that the green crop industry has expenses amounting to 270,000 Euro/year for disposal of the brown juice. A newly built lysine factory in Esbjerg, Denmark, can benefit from this process by buying a low price medium for the fermentation process instead of more expensive traditional fermentation liquids such as corn steep liquor.
Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis by U. Rau; L. A. Nguyen; H. Roeper; H. Koch; S. Lang (pp. 607-613).
Two strains of Pseudozyma aphidis, DSM 70725 and DSM 14930, were used for the bioreactor production of mannosylerythritol lipids (MELs). Foam formation interfered substantially with the cultivation process. Soybean oil was simultaneously employed as both carbon source and anti-foam agent. Primary MEL formation occurred after nitrate limitation. After a first short time-period of nitrate limitation and further nitrate addition, MELs were secreted in spite of nitrate excess. The sedimentation of MEL-enriched beads indicated enhanced product formation. Maximum yield, productivity and yield coefficient of 165 g l−1, 13.9 g l−1 day−1 and 0.92 g g−1 were achieved using strain DSM 14930 with additional substrate-feeding (glucose, sodium nitrate, yeast extract) and a foam-controlled soybean oil supply.
Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis by U. Rau; L. A. Nguyen; H. Roeper; H. Koch; S. Lang (pp. 607-613).
Two strains of Pseudozyma aphidis, DSM 70725 and DSM 14930, were used for the bioreactor production of mannosylerythritol lipids (MELs). Foam formation interfered substantially with the cultivation process. Soybean oil was simultaneously employed as both carbon source and anti-foam agent. Primary MEL formation occurred after nitrate limitation. After a first short time-period of nitrate limitation and further nitrate addition, MELs were secreted in spite of nitrate excess. The sedimentation of MEL-enriched beads indicated enhanced product formation. Maximum yield, productivity and yield coefficient of 165 g l−1, 13.9 g l−1 day−1 and 0.92 g g−1 were achieved using strain DSM 14930 with additional substrate-feeding (glucose, sodium nitrate, yeast extract) and a foam-controlled soybean oil supply.
Phenol and 2-naphthol production by toluene 4-monooxygenases using an aqueous/dioctyl phthalate system by Ying Tao; William E. Bentley; Thomas K. Wood (pp. 614-621).
A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l−1 (39 mM), and wild-type T4MO, a 51±9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.
Phenol and 2-naphthol production by toluene 4-monooxygenases using an aqueous/dioctyl phthalate system by Ying Tao; William E. Bentley; Thomas K. Wood (pp. 614-621).
A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l−1 (39 mM), and wild-type T4MO, a 51±9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.
Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain by Rasmus Devantier; Sven Pedersen; Lisbeth Olsson (pp. 622-629).
Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved.
Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain by Rasmus Devantier; Sven Pedersen; Lisbeth Olsson (pp. 622-629).
Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved.
Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains by P. J. Slininger; M. A. Shea-Andersh (pp. 630-638).
The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as “take-all decline.” Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40–50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.
Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains by P. J. Slininger; M. A. Shea-Andersh (pp. 630-638).
The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as “take-all decline.” Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40–50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.
A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation by T. Ohta; A. Tani; K. Kimbara; F. Kawai (pp. 639-646).
A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36–52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca2+, but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C2–C14) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca2+-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.
A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation by T. Ohta; A. Tani; K. Kimbara; F. Kawai (pp. 639-646).
A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36–52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca2+, but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C2–C14) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca2+-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.
Test systems to study transcriptional regulation and promoter activity in Bacillus megaterium by Silke Schmidt; Nadine Wolf; Jan Strey; Hannes Nahrstedt; Friedhelm Meinhardt; Jens Waldeck (pp. 647-655).
Plasmid-located (multi-copy) and chromosomally located (single-copy) promoter test systems were developed for Bacillus megaterium by making use of the homologous β-galactosidase-encoding bgaM gene. The multi-copy system facilitates rapid promoter analyses and promoter trapping, whereas the single-copy system, integrated into the chromosome, allows investigation of tightly regulated promoters. As a prerequisite for both the multi- and the single-copy systems, a β-galactosidase-deficient B. megaterium strain was generated by deletion mutagenesis. Both test systems were verified using the promoter of the xylose operon (P xylA ) from B. megaterium along with its repressor (XylR). As expected, expression levels in the two systems differed significantly, although expression of the bgaM reporter gene was induced by xylose in both cases, thereby proving the functionality of both the multi- and the single-copy system.
Test systems to study transcriptional regulation and promoter activity in Bacillus megaterium by Silke Schmidt; Nadine Wolf; Jan Strey; Hannes Nahrstedt; Friedhelm Meinhardt; Jens Waldeck (pp. 647-655).
Plasmid-located (multi-copy) and chromosomally located (single-copy) promoter test systems were developed for Bacillus megaterium by making use of the homologous β-galactosidase-encoding bgaM gene. The multi-copy system facilitates rapid promoter analyses and promoter trapping, whereas the single-copy system, integrated into the chromosome, allows investigation of tightly regulated promoters. As a prerequisite for both the multi- and the single-copy systems, a β-galactosidase-deficient B. megaterium strain was generated by deletion mutagenesis. Both test systems were verified using the promoter of the xylose operon (P xylA ) from B. megaterium along with its repressor (XylR). As expected, expression levels in the two systems differed significantly, although expression of the bgaM reporter gene was induced by xylose in both cases, thereby proving the functionality of both the multi- and the single-copy system.
Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome by Rosa Alduina; Anna Giardina; Giuseppe Gallo; Giovanni Renzone; Clelia Ferraro; Alba Contino; Andrea Scaloni; Stefano Donadio; Anna Maria Puglia (pp. 656-662).
A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.
Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome by Rosa Alduina; Anna Giardina; Giuseppe Gallo; Giovanni Renzone; Clelia Ferraro; Alba Contino; Andrea Scaloni; Stefano Donadio; Anna Maria Puglia (pp. 656-662).
A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.
Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation by Adam G. Lawrence; Joerg Schoenheit; Aimin He; Jiamin Tian; Pinghua Liu; JoAnne Stubbe; Anthony J. Sinskey (pp. 663-672).
Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.
Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation by Adam G. Lawrence; Joerg Schoenheit; Aimin He; Jiamin Tian; Pinghua Liu; JoAnne Stubbe; Anthony J. Sinskey (pp. 663-672).
Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.
Transportation mechanism for vanillin uptake through fungal plasma membrane by Motoyuki Shimizu; Yoshinori Kobayashi; Hiroo Tanaka; Hiroyuki Wariishi (pp. 673-679).
Protoplasts of the basidiomycete, Fomitopsis palustris (formerly Tyromyces palustris), were utilized to study a function of the fungal plasma membrane. Fungal protoplasts exhibited metabolic activities as seen with intact mycelial cells. Furthermore, the uptake of certain compounds into the protoplast cells was quantitatively observed by using non-radioactive compounds. Vanillin was converted to vanillyl alcohol and vanillic acid as major products and to protocatechuic acid and 1,2,4-trihydroxybenzene as trace products by protoplasts prepared from F. palustris. Extracellular culture medium showed no activity responsible for the redox reactions of vanillin. Only vanillic acid was detected in the intracellular fraction of protoplasts. However, the addition of disulfiram, an aldehyde dehydrogenase inhibitor, caused an intracellular accumulation of vanillin, strongly suggesting that vanillin is taken up by the cell, followed by oxidation to vanillic acid. The addition of carbonylcyanide m-chlorophenylhydrazone, which dissipates the pH gradient across the plasma membrane, inhibited the uptake of either vanillin or vanillic acid into the cell. Thus, the fungus seems to possess transporter devices for both vanillin and vanillic acid for their uptake. Since vanillyl alcohol was only observed extracellularly, the reduction of vanillin was thought to be catalyzed by a membrane system.
Transportation mechanism for vanillin uptake through fungal plasma membrane by Motoyuki Shimizu; Yoshinori Kobayashi; Hiroo Tanaka; Hiroyuki Wariishi (pp. 673-679).
Protoplasts of the basidiomycete, Fomitopsis palustris (formerly Tyromyces palustris), were utilized to study a function of the fungal plasma membrane. Fungal protoplasts exhibited metabolic activities as seen with intact mycelial cells. Furthermore, the uptake of certain compounds into the protoplast cells was quantitatively observed by using non-radioactive compounds. Vanillin was converted to vanillyl alcohol and vanillic acid as major products and to protocatechuic acid and 1,2,4-trihydroxybenzene as trace products by protoplasts prepared from F. palustris. Extracellular culture medium showed no activity responsible for the redox reactions of vanillin. Only vanillic acid was detected in the intracellular fraction of protoplasts. However, the addition of disulfiram, an aldehyde dehydrogenase inhibitor, caused an intracellular accumulation of vanillin, strongly suggesting that vanillin is taken up by the cell, followed by oxidation to vanillic acid. The addition of carbonylcyanide m-chlorophenylhydrazone, which dissipates the pH gradient across the plasma membrane, inhibited the uptake of either vanillin or vanillic acid into the cell. Thus, the fungus seems to possess transporter devices for both vanillin and vanillic acid for their uptake. Since vanillyl alcohol was only observed extracellularly, the reduction of vanillin was thought to be catalyzed by a membrane system.
Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor by Miguel Angel Gómez; Belén Rodelas; Florentina Sáez; Clementina Pozo; María Victoria Martínez-Toledo; Ernesto Hontoria; Jesús González-López (pp. 680-685).
Xanthobacter autotrophicus strains with the ability to reduce nitrate and nitrite to either nitrous oxide or molecular nitrogen were isolated from submerged fixed-film reactors. Isolated strains were Gram-negative rods able to grow on methanol, ethanol and sucrose. The yellow cellular pigmentation, pleomorphic appearance, and the presence of poly-β-hydroxybutyrate granules suggest that the organisms might belong to the genus Xanthobacter. Comparison of 16S rDNA gene sequences demonstrated the affiliation of the strains to X. autotrophicus species. The results show that X. autotrophicus may play a role in inorganic nitrogen removal from a denitrifying submerged filter used for the treatment of contaminated groundwater. To our knowledge, no data on denitrifying activity in X. autotrophicus strains have been reported previously.
Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor by Miguel Angel Gómez; Belén Rodelas; Florentina Sáez; Clementina Pozo; María Victoria Martínez-Toledo; Ernesto Hontoria; Jesús González-López (pp. 680-685).
Xanthobacter autotrophicus strains with the ability to reduce nitrate and nitrite to either nitrous oxide or molecular nitrogen were isolated from submerged fixed-film reactors. Isolated strains were Gram-negative rods able to grow on methanol, ethanol and sucrose. The yellow cellular pigmentation, pleomorphic appearance, and the presence of poly-β-hydroxybutyrate granules suggest that the organisms might belong to the genus Xanthobacter. Comparison of 16S rDNA gene sequences demonstrated the affiliation of the strains to X. autotrophicus species. The results show that X. autotrophicus may play a role in inorganic nitrogen removal from a denitrifying submerged filter used for the treatment of contaminated groundwater. To our knowledge, no data on denitrifying activity in X. autotrophicus strains have been reported previously.
Extracellular enzyme activities of Bjerkandera adusta R59 soil strain, capable of daunomycin and humic acids degradation by A. Belcarz; G. Ginalska; T. Kornillowicz-Kowalska (pp. 686-694).
Geotrichum-like strain R59, the anamorphic form of the white-rot fungus, Bjerkandera adusta, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post-production effluent during 10 days of incubation at 26°C. Strain R59 produced only low levels of ligninolytic enzymes when grown on wheat straw- or beech sawdust-containing media, but in the presence of humic acids derived from brown coal it synthesized significant amounts of laccase and lipase. This phenomenon was coupled with the fungus entering the idiophase and the appearance of aerial mycelium. B. adusta strain R59 was found to completely decolorize 0.03% humic acids from brown coal and lessive soil and to partially decolorize humic acids isolated from a chernozem during 14 days of growth. This ability of strain R59 could be useful in constructing a new generation of biologically active filters for the purification of humic acids-contaminated drinking waters.
Extracellular enzyme activities of Bjerkandera adusta R59 soil strain, capable of daunomycin and humic acids degradation by A. Belcarz; G. Ginalska; T. Kornillowicz-Kowalska (pp. 686-694).
Geotrichum-like strain R59, the anamorphic form of the white-rot fungus, Bjerkandera adusta, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post-production effluent during 10 days of incubation at 26°C. Strain R59 produced only low levels of ligninolytic enzymes when grown on wheat straw- or beech sawdust-containing media, but in the presence of humic acids derived from brown coal it synthesized significant amounts of laccase and lipase. This phenomenon was coupled with the fungus entering the idiophase and the appearance of aerial mycelium. B. adusta strain R59 was found to completely decolorize 0.03% humic acids from brown coal and lessive soil and to partially decolorize humic acids isolated from a chernozem during 14 days of growth. This ability of strain R59 could be useful in constructing a new generation of biologically active filters for the purification of humic acids-contaminated drinking waters.
Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR by Han Vervaeren; Kurt De Wilde; Jorg Matthys; Nico Boon; Lutgarde Raskin; Willy Verstraete (pp. 695-704).
Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r 2=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.
Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR by Han Vervaeren; Kurt De Wilde; Jorg Matthys; Nico Boon; Lutgarde Raskin; Willy Verstraete (pp. 695-704).
Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r 2=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.