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Applied Microbiology and Biotechnology (v.62, #4)
Biotechnological applications of green fluorescent protein by J. C. March; G. Rao; W. E. Bentley (pp. 303-315).
Since its first use as a reporter gene in 1994, green fluorescent protein (GFP) has served as the researcher′s agent: slipping, virtually undetected, into unseen spaces, reporting back valuable information, and securing the delivery of precious cargo through hostile domains. GFP′s strength lies in its small size, formidable stability, and relative ease of use. It requires only oxygen and an energy source to do its work, which can be supplied at low cost and high precision, respectively. With such a low threshold for use, GFP is often the first line of inquiry into an unknown space. Here is provided a brief compendium of GFP′s contributions to biotechnology. They are linked by a need for a level of information that was previously inaccessible, both spatially and temporally. Protein fusions, transcriptional reporters, whole-organism visualization, and various other screening applications are reviewed with respect to biotechnological applications. Germane molecular improvements to GFP are also discussed.
Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophores in iron uptake and storage by H. Haas (pp. 316-330).
To acquire iron, all species have to overcome the problems of iron insolubility and toxicity. In response to low iron availability in the environment, most fungi excrete ferric iron-specific chelators—siderophores—to mobilize this metal. Siderophore-bound iron is subsequently utilized via the reductive iron assimilatory system or uptake of the siderophore-iron complex. Furthermore, most fungi possess intracellular siderophores as iron storage compounds. Molecular analysis of siderophore biosynthesis was initiated by pioneering studies on the basidiomycete Ustilago maydis, and has progressed recently by characterization of the relevant structural and regulatory genes in the ascomycetes Aspergillus nidulans and Neurospora crassa. In addition, significant advances in the understanding of utilization of siderophore-bound iron have been made recently in the yeasts Saccharomyces cerevisiae and Candida albicans as well as in the filamentous fungus A. nidulans. The present review summarizes molecular details of fungal siderophore biosynthesis and uptake, and the regulatory mechanisms involved in control of the corresponding genes.
Cleavage of β,β-carotene to flavor compounds by fungi by H. Zorn; S. Langhoff; M. Scheibner; R. G. Berger (pp. 331-336).
More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave β,β-carotene to flavor compounds. Ten strains discolored a β,β-carotene-containing growth agar, indicating efficient degradation of β,β-carotene. Dihydroactinidiolide was formed as the sole conversion product of β,β-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of β,β-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. β-Ionone proved to be the main metabolite in each case, whereas β-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the β,β-carotene cleaving enzyme activities of M. scorodonius were partially characterized.
Regioselective hydroxylation of quinolinic acid, lutidinic acid and isocinchomeronic acid by resting cells of pyridine dicarboxylic acid-degrading microorganisms by A. Uchida; T. Yoshida; M. Ogawa; T. Nagasawa (pp. 337-341).
Microorganisms aerobically degrading quinolinic acid, lutidinic acid or isocinchomeronic acid were isolated and the microbial regioselective hydroxylation of these pyridine dicarboxylic acids was studied. Alcaligenes sp. UK21 cells converted quinolinic acid into 6-hydroxypicolinic acid, suggesting the involvement of two enzyme reactions catalyzing hydroxylation at position C6 and decarboxylation at position C3 of quinolinic acid. Resting cells of Alcaligenes sp. UK21 accumulated 94.9 mM 6-hydroxypicolinic acid (13.2 g l−1), with a 96% molar conversion yield by 48 h incubation. Rhizobium sp. LA17 and Hydrogenophaga sp. IMA01 catalyzed the regioselective hydroxylation of lutidinic acid and isocinchomeronic acid into 6-hydroxylutidinic acid and 6-hydroxyisocinchomeronic acid, respectively. 6-Hydroxylutidinic acid accumulated up to 95.4 mM (17.5 g l−1) by 24 h incubation in the resting cells reaction, using Rhizobium sp. LA17, with a 99% molar conversion yield. Resting cells of Hydrogenophaga sp. IMA01 produced 88.7 mM 6-hydroxyisocinchomeronic acid (16.2 g l−1) by 24 h incubation, with a 81% molar conversion yield.
Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch by H.-K. Lim; S.-J. Choi; K.-Y. Kim; K.-H. Jung (pp. 342-348).
A DO-stat control strategy for two variables was introduced to the rGuamerin production process in Pichia pastoris and applied to repeated fed-batch culture. Two interrelated variables, namely the ratio of partial pressure of pure O2 in the inlet air-stream and the methanol feed rate, were controlled simultaneously. By using this control strategy, methanol feeding for induction could be controlled automatically while efficiently controlling the dissolved oxygen level. As a result, the cell concentration reached more than 140 g l−1 and rGuamerin expression level 450 iu l−1. rGuamerin was secreted into the culture medium and reached a level that was 40% higher than achieved in a fed-batch process using manual control of the methanol feeding rate. Repeated rGuamerin induction was achieved by repeating the methanol feeding and withdrawing the culture broth during extended production. During more than 250 h of culture, expression of rGuamerin was maintained at an average of about 430 iu l−1 (473 mg l−1), without causing the cell density to decrease. In addition to the rGuamerin production process, the proposed control system might be applied to cultivation of other methylotrophic yeasts in the production of therapeutic proteins.
Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application by E. Record; M. Asther; C. Sigoillot; S. Pagès; P. J. Punt; M. Delattre; M. Haon; C. A. M. J. J. van den Hondel; J.-C. Sigoillot; L. Lesage-Meessen; Marcel Asther (pp. 349-355).
A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l−1), improved FAEA activity 24.5-fold and a yield of 1 g l−1 of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification—close to 75%—and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector.
2-(2′-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2: purification and characterization by J. Konishi; K. Maruhashi (pp. 356-361).
2-(2′-Hydroxyphenyl)benzene sulfinate (HPBSi) desulfinase (TdsB), which catalyzes the final step of desulfurization of dibenzothiophene (DBT), was purified from a thermophilic DBT- and benzothiophene (BT)-desulfurizing bacterium: Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme was 31 kDa and 39 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting a monomeric structure. The optimal temperature and pH for the reaction involving TdsB was 55°C and the enzyme was more resistant to heat treatment than DszB, a counterpart purified from Rhodococcus erythropolis. The optimum pH for TdsB activity was pH 8. TdsB converted HPBSi to 2-hydroxybiphenyl (2-HBP) and sulfite stoichiometrically. The K m and k cat values for HPBSi were 0.33 mM and 0.32 s−1, respectively. TdsB was inactivated by SH reagents such as p-chloromercuribenzoic acid and 5,5′-dithio-bis-2-nitrobenzoic acid, but was not inhibited by chelating reagents such as EDTA and o-phenanthroline. TdsB was also inhibited by o-hydroxystyrene, the final desulfurized product of BT. However, 2-HBP and its derivatives showed only a weak inhibitory effect. TdsB desulfurized 2-(2′-hydroxyphenyl)ethen-1-sulfinate to yield o-hydroxystyrene, but DszB could not. A site-directed mutagenesis study revealed the cysteine residue at position 17 to be essential to the catalytic activity of TdsB.
Differential response to low temperature of two Δ6 fatty acid desaturases from Mucor circinelloides by Y. Michinaka; T. Aki; T. Shimauchi; T. Nakajima; S. Kawamoto; S. Shigeta; O. Suzuki; K. Ono (pp. 362-368).
A recently identified Δ6 fatty acid desaturase in Mucor rouxii shows a low sequence homology (~24% at the amino acid level) to that isolated from Mortierella alpina, but is phylogenetically closer to a plant enzyme, suggesting the occurrence of Δ6 desaturase isozymes in Mucorales molds. In the present study, two types of Δ6 desaturases, mcD6-1 (Mo. alpina type) and mcD6-2 (M. rouxii type), were cloned from Mucor circinelloides. When the cloned genes were expressed in the yeast Saccharomyces cerevisiae in the presence of a linoleic acid substrate (C18:2Δ9, 12), a newly generated γ-linolenic acid (C18:3Δ6, 9, 12) was detected in the cells, which confirmed the suspected enzymatic function of the recombinant protein. This is the first report of Δ6 desaturase isozymes present in one organism. Northern analysis demonstrated that the amount of mcD6-2 mRNA was less than half of that of mcD6-1 mRNA in cells grown at 28 °C. However, upon cultivation of the cells at 15 °C for 0.5–1 h, mcD6-2 mRNA rapidly increased by up to 1.5-fold and then gradually decreased. By contrast, mcD6-1 transcripts levels did not fluctuate significantly for 1 h after the temperature shift, but declined by 75% over the next 2 h. The γ-linolenic acid content in total fatty acid from M. circinelloides decreased at 28 °C, but was maintained at approximately 30% at 15 °C. These data suggest that Δ6 desaturase isozymes play physiologically distinct roles in the maintenance of cellular lipids and adaptation to low temperature.
Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis by J. Olmos-Soto; R. Contreras-Flores (pp. 369-373).
The first amino acid residue from a proinsulin gene was fused in frame with the last amino acid residue of the aprE signal peptide sequence from Bacillus subtilis, using an overlapping PCR methodology. For expression of the fused DNA the aprE regulatory region (aprERR) was used. A six-protease-deficient strain of B. subtilis with the hpr2 and degU32 mutations was constructed for overproduction of the recombinant protein. The production of proinsulin was carried out in a mineral medium which facilitated the purification of proinsulin. Samples were taken during growth and analyzed by RIA and Western blot. Proinsulin was overproduced (1 mg ml−1) and 90% was secreted into the culture medium 1 h after stationary phase began.
Molecular breeding of transgenic rice expressing a xylanase domain of the xynA gene from Clostridium thermocellum by T. Kimura; T. Mizutani; T. Tanaka; T. Koyama; K. Sakka; K. Ohmiya (pp. 374-379).
The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60°C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.
Cloning, sequence analysis, and heterologous expression of the gene encoding a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297 by K. Abokitse; W. Hummel (pp. 380-386).
The gene encoding an (S)-specific NAD-dependent alcohol dehydrogenase (RE-ADH) was isolated from the genomic DNA of Rhodococcus erythropolis DSM 43297. The nucleotide sequence of 1,047 bp, coding for 348 amino acids, was cloned in Escherichia coli cells and successfully expressed. The subunit molecular mass as deduced from the amino acid sequence was determined to be 36.026 kDa. The recombinant enzyme exhibited high thermostability, which facilitated its purification by heat treatment, followed by two column-chromatography steps. RE-ADH shows high similarity to several zinc-containing medium-chain alcohol dehydrogenases. All zinc ligands seem to be conserved except one of the catalytic zinc ligands, where Cys is probably substituted by Asp. A similarity of 84% with a phenylacetaldehyde reductase from Corynebacterium sp. ST-10 was determined. Biochemical properties such as thermostability and substrate specificity of the two enzymes were compared.
Cloning of the KcURA3 gene and development of a transformation system for Kluyveromyces cicerisporus by J. Zhang; H. Yuan; T. Wen; F. Xu; Y. Di; K. Huo; Y.-Y. Li (pp. 387-391).
KcURA3 was cloned from Kluyveromyces cicerisporus CBS4857 by complementation of the ura3 mutation in Saccharomyces cerevisiae. KcURA3 encodes a 267-amino-acid protein with 80% sequence identity to that of S. cerevisiae. An ura3 mutant strain from K. cicerisporus CBS4857, named Y179U, was obtained by selection on 5-fluoroorotic acid plates. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +277. Two vectors, pUK1 and pUKD, bearing KcURA3 were constructed. Either the lithium acetate method or electroporation could be used to transform pUK1 and pUKD intoY179U. The transformation efficiency using electroporation was higher than that using the lithium acetate method.
Keywords: Kluyveromyces cicerisporus ; KcURA3 ; Transformation system
Profiling local gene expression changes associated with Eimeria maxima and Eimeria acervulina using cDNA microarray by W. Min; H. S. Lillehoj; S. Kim; J. J. Zhu; H. Beard; N. Alkharouf; B. F. Matthews (pp. 392-399).
Eimeria parasites show preferential sites of invasion in the avian intestine and produce a species-specific host immune response. Two economically important species, Eimeria acervulina and Eimeria maxima, preferentially invade and develop in the avian duodenum and jejunum/ileum, respectively. To investigate local host immune responses induced by parasite infection, global transcriptional changes in intestinal intraepithelial lymphocytes (IELs) induced by oral inoculation of chickens with E. acervulina or E. maxima were monitored using cDNA microarrays containing 400 unique chicken genes. Multiple gene transcripts were significantly up- or down-regulated following primary or secondary infection with E. acervulina or E. maxima. In general, infection by either parasite resulted in the expression changes of more genes following primary infection than following secondary infection, and E. acervulina caused more changes than did E. maxima. Although different regions of the small intestine were infected, similar changes in the levels of several cytokine mRNAs were observed in both Eimeria species following primary infection. Also identified was a set of transcripts whose expression was commonly enhanced or repressed in intestinal IELs of chickens infected with either parasite. Microarray analysis of chicken genes induced or repressed following Eimeria infection offers a powerful tool to enhance our understanding of host–parasite interactions leading to protective immunity.
Phylogenetic analysis of an anaerobic microbial consortium deiodinating 5-amino-2,4,6-triiodoisophthalic acid by D. Lecouturier; J.-J. Godon; J.-M. Lebeault (pp. 400-406).
The dehalogenating performance of an anaerobic 5-amino-2,4,6-triiodoisophthalic acid (ATIA) fixed-bed reactor was evaluated. The reactor operating conditions were set for ATIA deiodination. A phylogenetic survey for a stable anaerobic ATIA-deiodinating microbial consortium was carried out using 16S rDNA restriction fragment length polymorphism, and unique clones were sequenced. Four phylotypes were identified. Two sequences were related to those of Desulfitobacterium frappieri species and another was closest to that of Desulfitobacterium hafniense, but may have represented a new Desulfitobacterium species. Desulfitobacteria were previously described as aryl-dechlorinating and debrominating bacteria. The new strains identified in this study were probably responsible for the ATIA deiodination. The fourth clone was related to the Clostridium-Flavobacterium-Bacteroides group.
Antifungal mechanism of an anti-Pythium protein (SAP) from the marine bacterium Streptomyces sp. strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp. by J.-H. Woo; Y. Kamei (pp. 407-413).
Previously we reported an antifungal protein specific to Pythium porphyrae, a causative agent of red rot disease afflicting seaweed Porphyra spp. This study was carried out to identify the antifungal mechanism of the antifungal protein to P. porphyrae. When we first examined the effect of an anti-Pythium protein (SAP) on the P. porphyrae cell walls, SAP did not decompose the six structural polysaccharides in Pythium cell walls. However, hyphal growth was significantly inhibited in Pythium cells treated with 50 μg/ml of SAP by MTT assay. Protoplasmic leakage was observed in P. porphyrae hyphae treated with SAP for 1 h, followed by hyphal swelling and disintegration, using SYTOX Green, and SAP permeabilized the membrane of P. porphyrae in a dose-dependent manner. Treating P. porphyrae cells with SAP in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a membrane-depolarizing agent, significantly reduced the membrane permeability to SYTOX Green. Moreover, a similar effect was observed when the P. porphyrae cells were treated with SAP in the presence of MgCl2. In contrast, identical treatment in the presence of KCl significantly increased the membrane permeability to SYTOX Green. These results suggested that anti-Pythium mechanism of SAP was related to alteration of the membrane permeability in P. porphyrae.
SEM and AFM images of pyrite surfaces after bioleaching by the indigenous Thiobacillus thiooxidans by H.-L. Liu; B.-Y. Chen; Y.-W. Lan; Y.-C. Cheng (pp. 414-420).
The bioleaching mechanism of pyrite by the indigenous Thiobacillus thiooxidans was examined with the aid of scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of the pyrite surface. The presence of pyrite eliminated the lag phase during growth of this microorganism. This was due to the stimulatory effect on cell growth of the slight amount of Cu2+ that had leached from the pyrite. Zn2+ was found to be much more readily solubilized than Cu2+. The efficiency of bioleaching was four times higher than that of chemical leaching. SEM images provided evidence of direct cell attachment onto the pyrite surface, thereby enhancing the bioleaching rate. Furthermore, extracellular polymeric substances (EPSs) were found on the pyrite surface after 4 days of oxidation. AFM images showed that the pyrite surface area positively correlated with the oxidation rate. A combination of direct and indirect mechanism is probably responsible for the oxidation of pyrite by T. thiooxidans.
Production of verbenol, a high valued food flavourant from a fusant strain of Aspergillus niger by C. M. Vidya; R. Agrawal (pp. 421-422).
A hyperperformer for the production of verbenol was produced from the fusion of two improved strains of Aspergillus niger. A 2-deoxy glucose de-repressed mutant [high sporulation (50%), viability (80%) showing a conversion of 15.6% of initial α-pinene to verbenol in 6 h under the conditions used] was fused with another strain enriched with α-pinene (26.4% of α pinene converted to verbenol) to obtain a final verbenol conversion yield of 48.6% of initial α pinene.
Bacterial community structure and physiological state in a biofilm reactor degrading 4-chloroaniline. by H. Radianingtyas; G. K. Robinson; A. T. Bull (pp. 423-429).
Degradation of 4-chloroaniline in the presence of aniline by a microbial community in a laboratory-scale biofilm reactor was evaluated. The starter inoculum was isolated and reconstructed from a percolating column enrichment of Indonesian agricultural soil. The capacity to mineralise and detoxify 4-chloroaniline in the presence of aniline was demonstrated by the biofilm reactor when operated at high hydraulic retention time (HRT; 0.87 h). At low HRT (0.23 h and 0.39 h) 4-chlorocatechol accumulated in the effluent, accompanied by a decrease in dechlorination and detoxification. When returned to high HRT (2.14 h), the accumulation of 4-chlorocatechol stopped and the extent of dechlorination and detoxification increased. Bacteria other than the original inoculum appeared in the reactor when the operating mode was switched from closed cycle to open cycle. One of these bacteria, identified as Pseudomonas putida R1 by partial 16S rDNA sequencing, subsequently dominated the reactor at every HRT imposed. PCR-based single-strand conformational polymorphism of 16 s rDNA and traditional cultivation procedures indicated that the bacterial composition in the reactor shifted in response to applied HRT. The relationship between the bacterial abundance and the degradation capacity of the reactor is discussed.
Development and characteristics of phosphorus-accumulating microbial granules in sequencing batch reactors by Y.-M. Lin; Y. Liu; J.-H. Tay (pp. 430-435).
Phosphorus (P)-accumulating microbial granules were developed at different substrate P/chemical oxygen demand (COD) ratios in the range of 1/100 to 10/100 by weight in sequencing batch reactors. The soluble COD and PO4-P profiles showed that the granules had typical P-accumulating characteristics, with concomitant uptake of soluble organic carbon and the release of phosphate in the anaerobic stage, followed by rapid phosphate uptake in the aerobic stage. The size of P-accumulating granules exhibited a decreasing trend with the increase in substrate P/COD ratio, while the structure of the granules became more compact and denser as the substrate P/COD ratio increased. The P uptake by granules fell within the range of 1.9% to 9.3% by weight, which is comparable with uptake obtained in conventional enhanced biological phosphorus removal (EBPR) processes. It was further found that low aerobic respirometric activity of granules in terms of specific oxygen utilization rate favors P uptake by granules. The results presented would be useful for the further development of a novel granule-based EBPR technology.