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Applied Microbiology and Biotechnology (v.65, #5)
Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase by Rubén Bottini; Fabricio Cassán; Patricia Piccoli (pp. 497-503).
This review focuses on studies with bacteria for which biosynthesis/production of the plant hormones gibberellins have been demonstrated. Actual data on gibberellin metabolism by bacteria are analyzed in comparison with the biosynthetic pathways known for vascular plants and fungi. The potential involvement of gibberellins produced by symbiotic and soil-endophytic microorganisms in plant growth promotion and yield increase is also discussed.
Biotechnological aspects of the production of the anticancer drug podophyllotoxin by Sunita Farkya; V. S. Bisaria; A. K. Srivastava (pp. 504-519).
The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l−1 in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l−1 day−1, when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.
Improvement of fermentative hydrogen production: various approaches by Kaushik Nath; Debabrata Das (pp. 520-529).
Fermentation of biomass or carbohydrate-based substrates presents a promising route of biological hydrogen production compared with photosynthetic or chemical routes. Pure substrates, including glucose, starch and cellulose, as well as different organic waste materials can be used for hydrogen fermentation. Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs, such as clostridia and enteric bacteria, are efficient producers of hydrogen. Despite having a higher evolution rate of hydrogen, the yield of hydrogen [mol H2 (mol substrate−1)] from fermentative processes is lower than that achieved using other methods; thus, the process is not economically viable in its present form. The pathways and experimental evidence cited in the literature reveal that a maximum of four mol of hydrogen can be obtained from substrates such as glucose. Modifications of the fermentation process, by redirection of metabolic pathways, gas sparging and maintaining a low partial pressure of hydrogen to make the reaction thermodynamically favorable, efficient product removal, optimum bioreactor design and integrating fermentative process with that of photosynthesis, are some of the ways that have been attempted to improve hydrogen productivity. This review briefly describes recent advances in these approaches towards improvement of hydrogen yield by fermentation.
Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products by P. T. Sangeetha; M. N. Ramesh; S. G. Prapulla (pp. 530-537).
Fructosyl transferase (FTase) production by Aspergillus oryzae CFR 202 was carried out by solid-state fermentation (SSF), using various agricultural by-products like cereal bran, corn products, sugarcane bagasse,cassava bagasse (tippi) and by-products of coffee and tea processing. The FTase produced was used for the production of fructo-oligosaccharides (FOS), using 60% sucrose as substrate. Among the cereal bran used, rice bran and wheat bran were good substrates for FTase production by A. oryzae CFR 202. Among the various corn products used, corn germ supported maximum FTase production, whereas among the by-products of coffee and tea processing used, spent coffee and spent tea were good substrates, with supplementation of yeast extract and complete synthetic media. FTase had maximum activity at 60°C and pH 6.0. FTase was stable up to 40°C and in the pH range 5.0–7.0. Maximum FOS production was obtained with FTase after 8 h of reaction with 60% sucrose. FTase produced by SSF using wheat bran was purified 107-fold by ammonium sulphate precipitation (30–80%), DEAE cellulose chromatography and Sephadex G-200 chromatography. The molecular mass of the purified FTase was 116.3 kDa by SDS-PAGE. This study indicates the potential for the use of agricultural by-products for the efficient production of FTase enzyme by A. oryzae CFR 202 in SSF, thereby resulting in value addition of those by-products.
Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli by Pyung Cheon Lee; Benjamin N. Mijts; Claudia Schmidt-Dannert (pp. 538-546).
Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and β-carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1–3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.
Cultivation of immobilized Dictyostelium discoideum for the production of soluble human Fas ligand by Yinghua Lu; Jaco C. Knol; Maarten H. K. Linskens; Karl Friehs; Peter J. M. van Haastert; Erwin Flaschel (pp. 547-552).
Dictyostelium discoideum was immobilized by cultivation on inorganic porous matrices consisting of broken pumice or a ceramic catalyst carrier (CeramTec) to produce human soluble Fas ligand (hFasL). These supports were actively colonized by D. discoideum reaching cell (number) densities 10–20 times higher locally than those observed in suspension culture under similar conditions. In repeated batch or continuous operation, hFasL productivities of up to 15–25 μg h−1 l−1 pore volume were attained. The immobilized cell densities and hFasL productivities could be kept constant for a long period of time by repeated renewal or continuous feeding of complex or synthetic medium.
Influence of culture passages on growth kinetics and adenovirus vector production for gene therapy in monolayer and suspension cultures of HEK 293 cells by Min Tae Park; Myung Seop Lee; Sung Hyun Kim; Eui-Cheol Jo; Gyun Min Lee (pp. 553-558).
To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate (μ) of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The μ of 293M passage 43 (#43) was 0.29 day−1, while the average μ of 293M from #66 to #86 was 0.74±0.01 day−1 (average ± standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the μ, cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.
Differential regulation and xenobiotic induction of tandem P450 monooxygenase genes pc-1 (CYP63A1) and pc-2 (CYP63A2) in the white-rot fungus Phanerochaete chrysosporium by Harshavardhan Doddapaneni; Jagjit S. Yadav (pp. 559-565).
The two tandem P450 monooxygenase genes (pc-1 and pc-2) reported by us earlier in Phanerochaete chrysosporium were investigated for their regulation under nutrient-limited and nutrient-rich culture conditions. Transcript analysis based on real-time quantitative RT-PCR showed higher expression of pc-1 in defined low-nitrogen and pc-2 in defined high-nitrogen media, with maximum expression on day 4, indicating that the two genes, though tandemly linked, are regulated in a non-coordinate manner. Transcript levels of pc-1 and pc-2 were differentially influenced by the type of carbon source, incubation temperature, and oxygenation. Both genes were inducible by organic xenobiotic chemicals. Of the 42 xenobiotics tested in nutrient-rich cultures, pc-1 transcription was induced 2.12(±0.40)-fold to 6.27(±0.48)-fold in the presence of 19 compounds and pc-2 transcription was induced 2.00(±0.73)-fold to 29.39(±9.40)-fold in the presence of 22 compounds. Particularly, it is significant that both pc-1 and pc-2 are induced by polycyclic aromatic hydrocarbons (PAHs) of varying ring size, including naphthalene (4.35±0.09, 6.02±1.39), phenanthrene (2.82±0.12, 2.14±0.61), pyrene (3.93±0.01, 1.0±0.12), benzanthracene (1.67±0.03, 6.08±1.50), and benzo(a)pyrene (1.55±0.01, 5.54±2.75) respectively. This study constitutes the first report on the identification of P450 genes in a fungus that are responsive to environmentally significant pollutant chemicals (PAHs, DDT, long-chain alkyl phenols) and lignin derivatives.
Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA by N. Yoshida; Y. Saeki (pp. 566-575).
In the present study, Escherichia coli cells exhibited antibiotic resistance after transformation with exogenous plasmid DNA adsorbed onto chrysotile particles during agar-exposure. We previously demonstrated penetration of E. coli by chrysotile particles during agar-exposure. To further investigate the mechanism by which transformation of E. coli is achieved through the use of chrysotile fibers, the interaction between E. coli cells and chrysotile was examined during agar-exposure. Dispersion of chrysotile particles within the chrysotile solution was analyzed by flow cytometry. A suspension containing E. coli cells expressing blue fluorescence protein and chrysotile particles was exposed to agar using stirring apparatus, which allowed a constant vertical reaction force to be applied to the surface of the gel. Fluorescence microscopy was then used to illustrate the adsorption of fluorescein isothiocyanate-conjugated DNA oligomers to chrysotile. Larger aggregates were observed when increasing concentrations of chrysotile were added to the solution. With prolonged exposure, during which surface moisture diffused into the agar gel, greater concentrations of chrysotile were observed on the agar surface. In addition, chrysotile aggregates exceeding 50 μm developed on the agar surface. They were shaped like a chestnut bur. The chrysotile aggregates penetrated the cell membranes of adherent E. coli cells during agar-exposure due to sliding friction forces generated at the interface of the agar and the stirring stick. E. coli cells thus acquired plasmid DNA and antibiotic resistance, since the plasmid DNA had been adsorbed onto the chrysotile particles. The inoculation of plasmid DNA into E. coli cells demonstrates the usefulness of chrysotile for E. coli transformation.
Identification and characterization of the main β-alanine uptake system in Escherichia coli by Frank Schneider; Reinhard Krämer; Andreas Burkovski (pp. 576-582).
In Escherichia coli, β-alanine is a direct precursor in the biosynthesis of pantothenic acid (vitamin B5). Although a sufficient β-alanine supply is crucial for biotechnological vitamin B5 production, nothing was known about β-alanine transport in E. coli until now. The aim of this work was the characterization of β-alanine transport by E. coli and the identification and overexpression of the corresponding carrier-encoding gene for the rational improvement of pantothenic acid-producing strains. β-Alanine uptake was found to be an active process catalyzed by the amino acid carrier CycA. The corresponding gene was cloned and overexpressed, resulting in an increase in the uptake rate, compared with the wild type. In all tested strains, this overexpression led to a strong sensitivity to β-alanine, but not to the other CycA substrates, such as l-alanine, d-alanine, and glycine. This prevented a direct application for the improvement of pantothenic acid-producing strains by an enhanced precursor supply.
Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme by Y. Hatada; Y. Hidaka; Y. Nogi; K. Uchimura; K. Katayama; Z. Li; M. Akita; Y. Ohta; S. Goda; H. Ito; H. Matsui; S. Ito; K. Horikoshi (pp. 583-592).
Arthrobacter globiformis T6 is unique in that it produces an enzyme yielding only isomaltose from dextran. In the present study, the organism was re-identified and its classification as a new species of the genus Arthrobacter, A. dextranlyticum, was proposed. The high G+C gene (66.8 mol%) for the isomalto-dextranase was sequenced. The deduced amino acid sequence, with a calculated molecular mass of 65,993 Da (603 amino acids), was confirmed by nanoscale capillary liquid chromatography coupled to tandem mass spectrometry, which covered 71.1% of the amino acid residues of the entire sequence. The enzyme was grouped into glycoside hydrolase family 27, and the C-terminal domain has homology to carbohydrate-binding module family 6. Hyper-exoproduction of the recombinant enzyme was achieved at a level corresponding to approximately 4.6 g l−1 of culture broth when proteases-deficient Bacillus subtilis cells were used as the host. The purified enzyme (65.5 kDa) had an optimal pH and temperature for activity of 3.5 and 60°C, respectively. It was crystallized using the sitting-drop vapor-diffusion method at 293 K.
Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes by Chun-Yi Kuo; Shu-Yu Chou; Ching-Tsan Huang (pp. 593-599).
The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5′ flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.
Cloning of l-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485 by S. G. Desai; M. L. Guerinot; L. R. Lynd (pp. 600-605).
The gene encoding l-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an l-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.
Growth and final product formation by Bifidobacterium infantis in aerated fermentations by R. González; A. Blancas; R. Santillana; A. Azaola; C. Wacher (pp. 606-610).
Fermentation conditions were developed to allow Bifidobacterium infantis to grow in the presence of air. Batch fermentations in TPYG medium, starting from anoxic conditions followed by the application of low airflow rates [0.02–0.1 air volume, per liquid media volume, per minute (vvm)], were analyzed for growth, oxygen uptake, and product formation by the bacterium. Under all aerated fermentations, B. infantis showed high aerotolerance, with a maximum oxygen-specific consumption rate of 0.34 mmol oxygen per gram dry cell weight per hour in the presence of 0.06 vvm. Similar growth yields were obtained under oxic and anoxic conditions (0.11–0.13 and 0.11 g dry cell weight per mmol glucose, respectively). Oxygen also influenced metabolite formation since lactate production and its molar relation to acetate increased and formate decreased with aeration rate. Under anoxic conditions, a maximum concentration of 8.1 mM lactate and an acetate/lactate ratio of 3.5:1 were obtained, while under oxic conditions the lactate concentration increased more than two-fold and the acetate/lactate molar ratio decreased to 1.5:1. The possibility of balancing acetate/lactate molar ratios for organoleptic purposes as well as for obtaining good growth under microaerated conditions was demonstrated.
Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems by Y.-J. An; Y.-H. Joo; I.-Y. Hong; H.-W. Ryu; K.-S. Cho (pp. 611-619).
The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.
Mesophilic aerobic degradation of a metal lubricant by a biological consortium by Sachiyo Iwashita; Timothy P. Callahan; Juan Haydu; Thomas K. Wood (pp. 620-626).
The metal-forming industries require the use of greases to lubricate metal surfaces during manufacturing operations, and the residues of these lubricants must be removed prior to finishing processes to protect and improve the appearance of the final product. An aqueous, biological metal-cleaning process operating under mild conditions (pH 9, 42°C) eliminates the use of environmentally unfriendly cleaning materials such as chlorinated solvents by employing microorganisms to degrade greases and oils naturally. This process was characterized in terms of initial degradation rates of a representative metal lubricant and by phylogenetic identification of the active bacteria. The metal lubricant in a surfactant solution was degraded by a bacterial consortium, and its concentration was determined by a novel gas chromatography assay. The maximum degradation rate Vmax and the apparent Km were obtained as 45 mg/(day mg protein) and 24 g/l on cellular basis, and 4.6 g/(day l) and 33 g/l on a volumetric basis, respectively. Mineralization of the metal lubricant was shown by analyzing the evolved CO2 and Cl−, and the bacterial consortium utilized the metal lubricant as a sole carbon and energy source (μ=0.05±0.01 h−1 at 0.5 vol% lubricant concentration). The active bacteria in the biological metal-cleaning process were identified as Bacillus licheniformis for the higher lubricant concentrations (3, 5, and 7.5 vol%), Bacillus cereus at 1 vol%, and Pseudomonas aeruginosa, Rhizobiaceae strain M100, and Achromobacter sp. LMG 5431 at 0.3 vol%.
Evaluation of chemical pretreatment of contaminated soil for improved PAH bioremediation by Reetta Piskonen; Merja Itävaara (pp. 627-634).
The efficiency of several chemical treatments as potential enhancers of the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil was evaluated by analyzing the mineralization of 14C-labeled phenanthrene, pyrene, and benzo(a)pyrene. The effect of nonionic surfactants with Fenton oxidation and combinations of surfactants with the Fenton oxidation was evaluated in a microtiter plate assay. The surfactants selected for the study were Tween 80, Brij 35, Tergitol NP-10, and Triton X-100. The addition of Fenton’s reagent significantly enhanced the mineralization of pyrene at the two concentrations studied: 2.8 M H2O2 with 0.1 M FeSO4 and 0.7 M H2O2 with 0.025 M FeSO4. Phenanthrene mineralization was also positively induced by the Fenton treatments. However, none of the treatments had a significant effect on benzo(a)pyrene mineralization. Surfactant additions at concentrations of 20% and 80% of the aqueous critical micelle concentration did not significantly affect the mineralization rates. When surfactant addition was combined with the Fenton oxidation, reduced mineralization rates were obtained when compared with mineralization after Fenton’s treatment alone. The results indicate that the addition of Fenton’s reagent may enhance the mineralization of PAHs in contaminated soil, whereas the addition of surfactants has no significant beneficial effect. The efficiency of the Fenton oxidation may decrease when surfactants are added simultaneously with Fenton’s reagent to contaminated soil.