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Applied Microbiology and Biotechnology (v.60, #5)
Sterols in microorganisms by J. Volkman (pp. 495-506).
Sterols are vital components of all eukaryotic cells. This review describes the variety of sterol structures found in microalgae, yeasts, fungi, protozoans and microheterotrophs. Reports of the occurrence of sterols in prokaryotic cells are critically assessed. Methylotrophic bacteria contain unusual 4-methylsterols, but reports of 4-desmethyl sterols in cyanobacteria and other bacteria are limited and many of these seem dubious. Possible application areas for sterols derived from mass culture of microalgae and other microorganisms are highlighted.
Biotechnological applications of penicillin acylases: state-of-the-art by M. Arroyo; I. de la Mata; C. Acebal; M. Pilar Castillón (pp. 507-514).
This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic β-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.
Regulation of gene expression in industrial fungi: Trichoderma by R. Mach; S. Zeilinger (pp. 515-522).
The genus Trichoderma comprises a group of filamentous ascomycetes that are now widely used in industrial applications because of their ability to produce extracellular hydrolases in large amounts. In addition, strong inducible promoters together with high secretory capacity have made Trichoderma an attractive host for heterologous protein production. Several promoters of genes encoding hydrolytic enzymes have been investigated in detail regarding their cis-acting elements and trans-acting factors. Potent inducer molecules, for both xylanolytic and cellulolytic enzyme systems, have been identified and characterized. Furthermore, models for the recognition of the insoluble substrates cellulose and xylan have been developed based on a large set of experiments. This mini-review summarises the considerable amount of data accumulated over the past three decades.
Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems by K. Terpe (pp. 523-533).
In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.
Optimization of isonovalal production from α-pinene oxide using permeabilized cells of Pseudomonas rhodesiae CIP 107491 by P. Fontanille; C. Larroche (pp. 534-540).
Optimization studies on the synthesis of isonovalal from α-pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented. Three key parameters are identified. The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether. This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates. The second is that the enzyme α-pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l–1 g–1 biomass. The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion. It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l–1 (400 g l–1) from 25 g biomass l–1 in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min–1 g–1 cells (160 g l–1 h–1).
Effect of major nutrients on podophyllotoxin production in Podophyllum hexandrum suspension cultures by S. Chattopadhyay; R. Mehra; A. Srivastava; S. Bhojwani; V. Bisaria (pp. 541-546).
The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.
Metabolic flux redistribution in Corynebacterium glutamicum in response to osmotic stress by C. Varela; E. Agosin; M. Baez; M. Klapa; G. Stephanopoulos (pp. 547-555).
Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes – trehalose first and proline at a later stage of the gradient – increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways – glycolysis and the tricarboxylic acid cycle – while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and α-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.
Development of a low-cost technology for mass production of the free-living nematode Panagrellus redivivus as an alternative live food for first feeding fish larvae by M. Ricci; A. Fifi; A. Ragni; C. Schlechtriem; U. Focken (pp. 556-559).
The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm3 sponge cubes) in autoclavable plastic bags (size range: 50×30 cm to 75×67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11–13 days) at 25 °C, the average yield (number of nematodes/g medium) was 241×103 for OM and 333×103 for PIM in 12-l bags (50×30 cm). The production scale has currently reached a bag volume of 50 l (75×67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3×109 nematodes/bag (291×103 nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259×103 and 290×103 nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.
Increasing ethanol productivity during xylose fermentation by cell recycling of recombinant Saccharomyces cerevisiae by C. Roca; L. Olsson (pp. 560-563).
The influence of cell recycling of xylose-fermenting Saccharomyces cerevisiae TMB3001 was investigated during continuous cultivation on a xylose-glucose mixture. By using cell recycling at the dilution rate (D) of 0.05 h–1, the cell-mass concentration could be increased from 2.2 g l–1 to 22 g l–1. Consequently, the volumetric ethanol productivity increased ten-fold, from 0.5 g l–1 h–1 to 5.35 g l–1 h–1. By increasing the biomass concentration, the xylose consumption rate increased from 0.75 g xylose l–1 h–1 without recycling to 1.9 g l–1 h–1 with recycling. The specific ethanol productivity was in the range of 0.23–0.26 g g–1 h–1 with or without cell recycling, showing that an increased cell-mass concentration did not influence the efficiency of the yeast.
Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-β-galactosidase gene lacG by T. Takala; P. Saris; S. Tynkkynen (pp. 564-570).
A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-β-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-β-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.
Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.) by G. Menzel; H.-J. Harloff; C. Jung (pp. 571-576).
Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.
Construction of a sodA::luxCDABE fusion Escherichia coli: comparison with a katG fusion strain through their responses to oxidative stresses by H. Lee; M. Gu (pp. 577-580).
A recombinant bioluminescent Escherichia coli strain, EBHJ, (sodA::luxCDABE), containing the promoter for the manganese superoxide dismutase (sodA) gene fused to the Vibrio fischeri luxCDABE operon, was successfully constructed and characterized. Redox-cycling agents, such as paraquat and chromium, strongly induced a sodA- regulated response in dose-dependent manners, resulting in an increase of the bioluminescence. In a comparison with an existing oxidative stress responsive strain, DPD2511 (katG::luxCDABE), which is sensitive to H2O2, the mechanism of chemicals that cause oxidative damage was elucidated via the key transcriptional factors involved in induction of the sodA and katG promoters, i.e. SoxRS and OxyR, respectively. It was found that responses from the katG- and sodA-based strains were significantly different dependent upon the chemicals being tested. Therefore, EBHJ, alone or in parallel with DPD2511, can be used to characterize and monitor chemicals that cause oxidative damage.
Isolation of a novel thermophilic fungus Chaetomium sp. nov. MS-017 and description of its palm-oil mill fiber-decomposing properties by Suyanto; T. Ohtsuki; S. Yazaki; S. Ui; A. Mimura (pp. 581-587).
Palm-oil mill fiber (POMF) is a fibrous, natural hard material discharged in enormous amounts from palm-oil mills in tropical plantations; therefore, research to find microorganisms that decompose POMF was conducted. As the result of screening, a new thermophilic fungus, Chaetomium sp. nov. MS-017, exhibiting rapid growth on POMF was isolated from rotted wood. Based on partial characterization of the decomposition of POMF, it was shown that MS-017 preferentially assimilates polysaccharides, especially hemicelluloses such as xylan. A preliminary composting study indicated that MS-017 produced 855 g of decomposed product from 1,000 g of intact POMF in 12 days under optimized solid-culture conditions. The decomposition rate of POMF was 23% (w/w), and the cell yield calculated from consumed POMF was as high as 36% (w/w). These results indicate that MS-017 has a very high potential to decompose POMF and that it is suitable for economical production of compost to recycle by-product biomass from oil-palm plantations.
Characteristics of a bioflocculant produced by Bacillus mucilaginosus and its use in starch wastewater treatment by S. Deng; R. Bai; X. Hu; Q. Luo (pp. 588-593).
A bioflocculant, MBFA9, was produced from a strain of bioflocculant-producing bacteria isolated from a soil sample and identified as Bacillus mucilaginosus. MBFA9 had a good flocculating capability and could achieve a flocculating rate of 99.6% for kaolin suspension at a dosage of only 0.1 ml/l. The major component of MBFA9 was found to be polysaccharide composed mainly of uronic acid (19.1%), neutral sugar (47.4%) and amino sugar (2.7%). Infrared spectrum analysis showed the presence of carboxyl and hydroxyl groups in the bioflocculant. MBFA9 is nontoxic and can be used in food industries for suspended solids (SS) recovery. When applied to starch wastewater treatment, MBFA9 greatly accelerated the formation of flocs and the settling of organic particles in the presence of Ca2+ salt. After 5 min of settling, the removal rate of SS and chemical oxygen demand were up to 85.5% and 68.5%, respectively, which is better than traditional chemical flocculants.
Functional and structural analyses of trichloroethylene-degrading bacterial communities under different phenol-feeding conditions: laboratory experiments by H. Futamata; S. Harayama; A. Hiraishi; K. Watanabe (pp. 594-600).
The effects of different phenol-feeding conditions on trichloroethylene (TCE) biodegradation and bacterial population structure in an aquifer soil community were studied. The soil sample, minerals, phenol, and TCE were mixed in glass bottles, which were then incubated under three different phenol-feeding conditions. First, phenol was supplied only once at 0.2 mM (condition 0.2P); second, it was added at 2.0 mM (condition 2.0P); and third, it was periodically supplied ten times at 0.2 mM (condition 0.2PS). TCE concentrations remained stable under conditions 0.2P and 2.0P. In contrast, TCE was completely degraded under condition 0.2PS. TCE/phenol-degrading bacteria were enumerated indirectly and functionally by quantitative PCR. The low-K s (half saturation constant) group of phenol-degrading bacteria, exhibiting high TCE-degrading activity, yielded a 50-fold higher population under condition 0.2PS than under condition 2.0P. The bacterial community structure under condition 0.2PS was studied by denaturing gradient gel electrophoresis targeting the genes encoding 16S rRNA and the largest subunit of multicomponent phenol hydroxylase. Sequence analysis of the major bands detected indicated the predominance of the low-K s group of TCE/phenol-degrading bacteria belonging to β-Proteobacteria. These results suggest that continuous supplementation with phenol at a low concentration increases the population of the low-K s group of TCE/phenol-degrading bacteria.