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Applied Microbiology and Biotechnology (v.67, #3)
Strategies for efficient production of heterologous proteins in Escherichia coli by S. Jana; J. K. Deb (pp. 289-298).
In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost.
Influenza vaccines: recent advances in production technologies by N. Bardiya; J. H. Bae (pp. 299-305).
In spite of ongoing annual vaccination programs, the seasonal influenza epidemics remain a major cause of high morbidity and mortality. The currently used “inactivated” vaccines provide very short-term and highly specific humoral immunity due to the frequent antigenic variations in the influenza virion. These intra-muscularly administered vaccines also fail to induce protective mucosal immunity at the portal of viral entry and destruction of the virally infected cells by induction of cytotoxic T lymphocytes. Therefore, it is necessary to develop immunologically superior vaccines. This article highlights some of the recent developments in investigational influenza vaccines. The most notable recent developments of interest include the use of immunopotentiators, development of DNA vaccines, use of reverse genetics, and the feasibility of mammalian cell-based production processes. Presently, due to their safety and efficacy, the cold-adapted “live attenuated” vaccines are seen as viable alternatives to the “inactivated vaccines”. The DNA vaccines are gaining importance due to the induction of broad-spectrum immunity. In addition, recent advances in recombinant technologies have shown the possibility of constructing pre-made libraries of vaccine strains, so that adequately preparations can be made for epidemics and pandemics.
Applications of zeolite inorganic composites in biotechnology: current state and perspectives by Kengo Sakaguchi; Masayoshi Matsui; Fujio Mizukami (pp. 306-311).
The purpose of this short review is to introduce applications of inorganic composites, zeolites, in biotechnology. Although inorganic chemistry is generally considered distant from biotechnology, the two could be harmoniously integrated for biopolymer chromatography. New chromatographic carriers have been developed based on principles differing from those underlying conventional chromatography. Some can be used for the purification of proteins according to novel physicochemical principles, according to their isoelectric point (pI), molecular weight and shape. The amount of protein adsorbed is related to the pore size of the composites, which can recognize biomolecules with reference to these three parameters. Proteins adsorbed at their pI have been found to be desorbed at the pI by polyethylene glycol, but not by high ionic medium (NaCl), SDS, non-ionic detergents, ATP or urea. Therefore, inorganic composites synthesized in consideration of pore size and three-dimensional structure are suitable as new chromatographic carriers. Selective fractionation of biomaterials including proteins and nucleic acids should provide useful information regarding whether conjugated proteins in a precipitated state can be separated on net charge and whether cells can be directly fractionated in future.
Sucrose utilisation in bacteria: genetic organisation and regulation by Sharon J. Reid; Valerie R. Abratt (pp. 312-321).
Sucrose is the most abundant disaccharide in the environment because of its origin in higher plant tissues, and many Eubacteria possess catalytic enzymes, such as the sucrose-6-phosphate hydrolases and sucrose phosphorylases, that enable them to metabolise this carbohydrate in a regulated manner. This review describes the range of gene architecture, uptake systems, catabolic activity and regulation of the sucrose-utilisation regulons that have been reported in the Eubacteria to date. Evidence is presented that, although there are many common features to these gene clusters and high conservation of the proteins involved, there has been a certain degree of gene shuffling. Phylogenetic analyses of these proteins supports the hypothesis that these clusters have been acquired through horizontal gene transfer via mobile elements and transposons, and this may have enabled the recipient bacteria to colonise sucrose-rich environmental niches.
Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review by Sergi Maicas; José Juan Mateo (pp. 322-335).
The importance of monoterpenes on varietal flavour of must and other fruit juices has been reviewed. These compounds were mainly found linked to sugar moieties in grape juice and wines, showing no olfactory characteristics. In this way, analytical techniques developed to study these compounds, in both free or glycosidically forms, are discussed. Mechanisms to liberate terpenes were studied, making a comparative study between acidic and enzymic hydrolysis of terpene glycosides; as enzymic hydrolysis seems to be the most natural way to liberate terpenes, the ability to use glycosidases from grapes, yeasts, bacterial or exogenous, i.e. fungal commercial preparations, were reviewed. Re-arrangements of terpenes after acidic hydrolysis of glycoconjugated are discussed as well as potential adverse effects of enzyme preparations.
A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain by Roberto Parra; David Aldred; Naresh Magan (pp. 336-344).
A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl2, 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20–23 mg l−1 and 0.5–2 mg l−1, respectively). The maximum lysozyme yield achieved was about 23 mg l−1 by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl2). Response surface methodology was used to investigate the effect of pH and water activity (aw). The best medium pH was 4.5–5.0, and bead aw for optimum lysozyme yield was 0.94, regardless of polymer type.
Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process by Marie Svensson; Ingrid Svensson; Sven-Olof Enfors (pp. 345-350).
The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h−1. The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme β-lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of β-lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.
The cry3Aa gene of Bacillus thuringiensis Bt886 encodes a toxin against long-horned beetles by Jun Chen; Lian-Yun Dai; Xue-Pin Wang; Ying-Chuan Tian; Meng-Zhu Lu (pp. 351-356).
This report describes the identification of a new toxigenic strain of Bacillus thuringiensis specific for long-horned beetles. B. thuringiensis Bt866 encodes a cry3Aa-like gene (Bt886cry3Aa) that is 1,956 bp in length and is predicted to encode an 85.78-kDa protein. The gene is highly similar to cry3Aa1, differing in only six nucleotides and four amino acids. The four disparate amino acids occur within the conserved domains of the Cry3Aa toxin. The expression of Bt866cry3A in Escherichia coli cells resulted in a high level of toxicity toward Apriona germari Hope larvae. More than 75% of the larvae were killed; and the remaining survivors exhibited slower growth. These results indicate that the toxigenic strain Bt886cry3Aa encodes a protein that is specific against long-horned beetles. Genetic engineering of the Bt866cry3Aa gene into poplar plantations may provide resistance to long-horned beetles.
Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization by René Ullrich; Le Mai Huong; Nguyen Lan Dung; Martin Hofrichter (pp. 357-363).
The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l−1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l−1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the “blue copper oxidases”. The enzyme’s pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (Km, kcat): 63 μM, 21 s−1 for ABTS, 4 μM, 5 s−1 for syringaldazine, 1,026 μM, 15 s−1 for DMP and 4307 μM, 159 s−1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.
Secretory expression of heterologous protein in Kluyveromyces cicerisporus by X. P. Cai; J. Zhang; H.-Y. Yuan; Z.-A. Fang; Y.-Y. Li (pp. 364-369).
To explore the potential of heterologous protein expression in Kluyveromyces cicerisporus, three expression plasmids, pUK1-PIT, pUKD-PIT and pUKD-S-PIT, based on the vector pUK1 or pUKD were constructed and transformed, respectively, into yeast strain K. cicerisporus Y179U. Human interferon α-2a, used as an example protein, was successfully expressed and secreted by transformant Y179U/pUKD-PIT and Y179U/pUKD-S-PIT. In the flask culture, strain Y179U/pUKD-S-PIT could express interferon at 60 mg/l. The stability of plasmid pUKD-S-PIT in the host was higher than that of pUKD-PIT. This was consistent with their expression levels of interferon. There were two interferon-related bands found by Western blotting analysis. The possible reason for this is discussed.
Large plasmid pCAR2 and class II transposon Tn4676 are functional mobile genetic elements to distribute the carbazole/dioxin-degradative car gene cluster in different bacteria by Masaki Shintani; Takako Yoshida; Hiroshi Habe; Toshio Omori; Hideaki Nojiri (pp. 370-382).
The carbazole-catabolic plasmid pCAR1 isolated from Pseudomonas resinovorans strain CA10 was sequenced in its entirety; and it was found that pCAR1 carries the class II transposon Tn4676 containing carbazole-degradative genes. In this study, a new plasmid designated pCAR2 was isolated from P. putida strain HS01 that was a transconjugant from mating between the carbazole-degrader Pseudomonas sp. strain K23 and P. putida strain DS1. Southern hybridization and nucleotide sequence analysis of pCAR1 and pCAR2 revealed that the whole backbone structure was very similar in each. Plasmid pCAR2 was self-transmissible, because it was transferred from strain HS01 to P. fluorescens strain IAM12022 at the frequency of 2×10−7 per recipient cell. After the serial transfer of strain HS01 on rich medium, we detected the transposition of Tn4676 from pCAR2 to the HS01 chromosome. The chromosome-located copy of Tn4676 was flanked by a 6-bp target duplication, 5′-AACATC-3′. These results experimentally demonstrated the transferability of pCAR2 and the functionality of Tn4676 on pCAR2. It was clearly shown that plasmid pCAR2 and transposon Tn4676 are active mobile genetic elements that can mediate the horizontal transfer of genes for the catabolism of carbazole.
Adaptation of Rhodococcus erythropolis DCL14 to growth on n-alkanes, alcohols and terpenes by Carla C. C. R. de Carvalho; Beatriz Parreño-Marchante; Grit Neumann; M. Manuela R. da Fonseca; Hermann J. Heipieper (pp. 383-388).
Rhodococcus erythropolis DCL14 has the ability to convert the terpene (−)-carveol to the valuable flavour compound (−)-carvone when growing on a wide range of carbon sources. To study the effect of carbon and energy sources such as alkanes, alkanols and terpenes on the biotechnological process, the cellular adaptation at the level of fatty acid composition of the membrane phospholipids and the (−)-carvone production were examined. All tested carbon sources caused a dose-dependent increase in the degree of saturation of the fatty acids. The exception was observed with short-chain alcohols such as methanol and ethanol, to which the cells adapted with a concentration-dependent decrease in the saturation degree of the membrane phospholipids. This influence of the different carbon sources on the rigidity of the cell membrane also had an impact on the (−)-carvone productivity of the strain.
Transcriptome analysis of the secretion stress response of Bacillus subtilis by Hanne-Leena Hyyryläinen; Matti Sarvas; Vesa P. Kontinen (pp. 389-396).
Transcription profiling of all protein-encoding genes of Bacillus subtilis was carried out under several secretion stress conditions in the exponential growth phase. Cells that secreted AmyQ α-amylase at a high level were stressed only moderately: seven genes were induced, most significantly htrA and htrB, encoding quality control proteases, and yqxL, encoding a putative CorA-type Mg2+ transporter. These three genes were induced more strongly by severe secretion stress (prsA3 mutant secreting AmyQ), suggesting that their expression responds to protein misfolding. In addition, 17 other genes were induced, including the liaIHGFSR (yvqIHGFEC) operon, csaA and ffh, encoding chaperones involved in the pretranslocational phase of secretion, and genes involved in cell wall synthesis/modification. Severe secretion stress caused downregulation of 23 genes, including the prsA paralogue yacD. Analysis of a cssS knockout mutant indicated that the absence of the CssRS two-component system, and consequently the absence of the HtrA and HtrB proteases, caused secretion stress. The results also suggest that the htrA and htrB genes comprise the CssRS regulon. B. subtilis cells respond to secretion/folding stress by various changes in gene expression, which can be seen as an attempt to combat the stress condition.
Reductive transformation of TNT by Escherichia coli: pathway description by Hong Yin; Thomas K. Wood; Barth F. Smets (pp. 397-404).
The reductive transformation of 2,4,6-trinitrotoluene (TNT) was studied using aerobically grown Escherichia coli cultures. In the absence of an external carbon or energy source, E. coli resting cells transformed TNT to hydroxylaminodinitrotoluenes (2HADNT, 4HADNT, with 4HADNT as the dominant isomer), aminodinitrotoluenes (4ADNT, with sporadic detection of 2ADNT), 2,4-di(hydroxylamino)-6-nitrotoluene (24D(HA)6NT), 2,4-diamino-6-nitrotoluene (24DA6NT), and an additional compound which was tentatively identified as a (hydroxylamino)aminonitrotoluene isomer via gas chromatography/mass spectroscopy and spectral analysis. The resting cell assay, performed in an oxygen-free atmosphere, avoided formation of azoxy dimers and provided good mass balances. Significant preference for reduction in the para versus ortho position was detected. The formation of 24D(HA)6NT, but not ADNT, appeared inhibited by the presence of TNT. The rate and extent of TNT reduction were significantly enhanced at higher cell densities, or by supplying an exogenous reducing power source, revealing the importance of enzyme concentration and reducing power. Whether the oxygen-insensitive E. coli nitroreductases, encoded by nfsA and nfsB, directly catalyze the TNT reduction or account for the complete TNT transformation pathway, remains to be determined.
Fungal biotransformation of benzo[f]quinoline, benzo[h]quinoline, and phenanthridine by John B. Sutherland; E. Lynn Cross; Thomas M. Heinze; James P. Freeman; Joanna D. Moody (pp. 405-411).
Cultures of Umbelopsis ramanniana (=Mucor ramannianus) were grown in fluid Sabouraud medium for 3 days, dosed with 0.23 mM benzo[f]quinoline, benzo[h]quinoline, or phenanthridine (benzo[c]quinoline), and incubated for another 18 days. Cultures were extracted and metabolites (66–75% of the UV absorbance) were separated by high-performance liquid chromatography. They were identified by mass spectrometry and nuclear magnetic resonance spectroscopy. Benzo[f]quinoline was metabolized to benzo[f]quinoline trans-7,8-dihydrodiol, benzo[f]quinoline N-oxide, and 7-hydroxybenzo[f]quinoline, benzo[h]quinoline was metabolized to benzo[h]quinoline trans-5,6-dihydrodiol, benzo[h]quinoline trans-7,8-dihydrodiol, and 7-hydroxybenzo[h]quinoline, and phenanthridine was metabolized to phenanthridine N-oxide and phenanthridin-6(5H)-one. At least one of the metabolites produced from each compound was mutagenic and could not be considered detoxified.
Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes by S. M. Tiquia; J. M. Ichida; H. M. Keener; D. L. Elwell; E. H. Burtt Jr.; F. C. Michel Jr. (pp. 412-419).
Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13×108 cfu g−1 feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO2 and NH3 evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts.
Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria by Pia Arentsen Willumsen; Jens Efsen Johansen; Ulrich Karlson; Bjarne Munk Hansen (pp. 420-428).
The azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment. Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention. This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils. Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated. Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth. Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species. Only a few strains were able to mineralize the homocyclic analogue, phenanthrene. Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds. These strains can provide further information on the fate of azaarenes in the environment.