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Applied Microbiology and Biotechnology (v.61, #3)
Polyene antibiotic biosynthesis gene clusters by J. F. Aparicio; P. Caffrey; J. A. Gil; S. B. Zotchev (pp. 179-188).
Over the past 15 years the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied and recently this work has been extended to the antifungal polyene macrolides. These compounds consist of large macrolactone rings that have a characteristic series of conjugated double bonds, as well as an exocyclic carboxyl group and an unusual mycosamine sugar. The biosynthetic gene clusters for nystatin, pimaricin, amphotericin and candicidin have been investigated in detail. These clusters contain the largest modular polyketide synthase genes reported to date. This body of work also provides insights into the enzymes catalysing the unusual post-polyketide modifications, and the genes regulating antibiotic biosynthesis. The sequences also provide clues about the evolutionary origins of polyene biosynthetic genes. Successful genetic manipulation of the producing organisms leading to production of polyene analogues indicates good prospects for generating improved antifungal compounds via genetic engineering.
Aspergillus niger citric acid accumulation: do we understand this well working black box? by Levente Karaffa; Christian P. Kubicek (pp. 189-196).
This Mini-Review summarizes the current knowledge on the biochemical and physiological events leading to massive citric acid accumulation by Aspergillus niger under industrially comparable conditions, thereby particularly emphasizing the roles of glycolytic flux and its control, excretion of citric acid from the mitochondria and the cytosol, and the critical fermentation variables. The potential of novel techniques for metabolic analysis and genomic approaches in understanding this fermentation is also discussed.
Yeast flocculation: what brewers should know by K. J. Verstrepen; G. Derdelinckx; H. Verachtert; F. R. Delvaux (pp. 197-205).
For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.
Effects of aeration and organic loading rates on degradation of trichloroethylene in a methanogenic-methanotrophic coupled reactor by D. Lyew; S. Guiot (pp. 206-213).
The effects of four aeration and four organic loading (OLR) rates on trichloroethylene (TCE) degradation in methanogenic-methanotrophic coupled reactors were studied using ethanol as the carbon source for the methanogens. Microcosm and PCR studies demonstrated that methanotrophs capable of mineralizing TCE and methanogens were present in the biomass throughout the study. The gene for the particulate form of methane monooxygenase (pMMO) was detected by PCR, but not that for the soluble form (sMMO). TCE mineralization by methanotrophs was therefore due primarily to pMMO activity. Low TCE concentrations were measured in effluent and off-gas samples in all cases. Volatilization losses were 0–5%. Dichloroethylene (DCE) was also observed, but vinyl chloride and ethylene were never detected. Changes in the aeration rate had no effect on TCE removal, but did influence DCE degradation. Reductive dechlorination of TCE to DCE was favored at low and no-aeration conditions, and DCE accumulation occurred due to slow DCE degradation. Low DCE levels were observed at the higher aeration rates, which indicated that conditions in these reactors were amenable to the aerobic co-metabolism of TCE and DCE. The OLR did have an effect on TCE removal. TCE and DCE removal were negatively affected when the OLR was increased. An OLR of 0.3 g COD lrx −1day−1 or lower with an aeration rate of 3 lO2 lrx −1day−1 and higher is the recommended operating condition of a coupled reactor for removal of TCE.
The trade-offs and effect of carrier size and oxygen-loading on gaseous toluene removal performance of a three-phase circulating-bed biofilm reactor by B.-I. Sang; E.-S. Yoo; B. J. Kim; B. E. Rittmann (pp. 214-219).
We conducted a series of steady-state and short-term experiments on a three-phase circulating-bed biofilm reactor (CBBR) for removing toluene from gas streams. The goal was to investigate the effect of macroporous-carrier size (1-mm cubes versus 4-mm cubes) on CBBR performance over a wide range of oxygen loading. We hypothesized that the smaller biomass accumulation with 1-mm carriers would minimize dissolved-oxygen (DO) limitation and improve toluene removal, particularly when the DO loading is constrained. The CBBR with 1-mm carriers overcame the performance limitation observed with the CBBR with 4-mm carriers: i.e., oxygen depletion inside the biofilm. The 1-mm carriers consistently gave superior removal of toluene and chemical oxygen-demand, and the advantage was greatest for the lowest oxygen loading and the greatest toluene loading. The 1-mm carriers achieved superior performance because they minimized the negative effects of oxygen depletion, while continuing to provide protection from excess biomass detachment and inhibition from toluene.
Isolation and cDNA cloning of novel hydrogen peroxide-dependent phenol oxidase from the basidiomycete Termitomyces albuminosus by T. Johjima; M. Ohkuma; T. Kudo (pp. 220-225).
A novel hydrogen peroxide-dependent phenol oxidase (TAP) was isolated from the basidiomycete Termitomyces albuminosus. TAP is an extracellular monomeric enzyme with an estimated molecular weight of 67 kDa. The purified enzyme can oxidize various phenolic compounds in the presence of hydrogen peroxide, but cannot oxidize 3,4-dimethoxybenzyl (veratryl) alcohol. MnII was not required for catalysis by TAP. The optimum pH for TAP activity was 2.3, which is the lowest known optimum pH for a fungal phenol oxidase. The cDNA encoding TAP was cloned with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers based on the N-terminal amino acid sequence of TAP and 5′ rapid amplification of cDNA ends (RACE)-PCR. The cDNA encodes a mature protein of 449 amino acids with a 55-amino-acid signal peptide. The deduced amino acid sequence of TAP showed 56% identity with dye-decolorizing heme peroxidase (DYP) from the ascomycete Geotrichum candidum Dec 1, but no homology with other known peroxidases from fungi.
Characterization of a Paenibacillus cell-associated xylanase with high activity on aryl-xylosides: a new subclass of family 10 xylanases by O. Gallardo; P. Diaz; F. I. J. Pastor (pp. 226-233).
The sequence of gene xynB encoding xylanase B from Paenibacillus sp. BP-23 was determined. It revealed an open reading frame of 999 nucleotides encoding a protein of 38,561 Da. The deduced amino acid sequence of xylanase B shows that the N-terminal region of the enzyme lacks the features of a signal peptide. When the xylan-degrading system of Paenibacillus sp. BP-23 was analysed in zymograms, it revealed that xylanase B was not secreted to the extracellular medium but instead remained cell-associated, even in late stationary-phase cultures. When xynB was expressed in a Bacillus subtilis secreting host, it also remained associated with the cells. Sequence homology analysis showed that xylanase B from Paenibacillus sp. BP-23 belongs to family 10 glycosyl hydrolases, exhibiting a distinctive high homology to six xylanases of this family. The homologous enzymes were also found to be devoid of a signal peptide and seem to constitute, together with xylanase B, a separate group of enzymes. They all have two conserved amino acid regions not found in the other family 10 xylanases, and cluster in a separate group after dendrogram analysis. We propose that these enzymes constitute a new subclass of family 10 xylanases, that are cell-associated, and that hydrolyse the xylooligosaccharides resulting from extracellular xylan hydrolysis. Xylanase B shows similar specific activity on aryl-xylosides and xylans. This can be correlated to some, not yet identified, trait of catalytic activity of the enzyme on plant xylan.
High-level expression of a lacZ gene from a bacterial artificial chromosome in Escherichia coli by T.-S. Chang; W.-J. Wu; H.-M. Wan; T.-R. Shiu; W.-T. Wu (pp. 234-239).
The GlnAP2 element has been proved to be an effective and inducible—by exogenous acetate—promoter in Escherichia coli with glnL/pta double mutations. Based on this feature, a single-copy expression vector was constructed via coupling of the glnAP2 promoter-regulated T7 RNA polymerase gene and the T7-promoter-controlled lacZ gene on a bacterial artificial chromosome. After induction with 20 mM potassium acetate, the glnL/pta double mutant E. coli harboring the single-copy plasmid produced 47,500 Miller units of β-galactosidase activity. This high level expression, corresponding to 27% of total cell protein, was comparable to that determined with the commercial multi-copy expression vector, pET-14b, in strain E. coli Tuner (DE3) (64,300 Miller units, 41% of total cell protein). Moreover, this single-copy expression vector could be maintained for at least 150 generations even in the presence of inducers. In contrast, the multi-copy expression vector was extensively lost after induction. The results indicate that the single-copy expression system has the potential for high-level heterologous protein production for industrial applications.
Characterization of pNI10 plasmid in Pseudomonas, and the construction of an improved Escherichia and Pseudomonas shuttle vector, pNUK73 by N. Itoh; T. Kawanami; C. Nitta; N. Iwata; S. Usami; Y. Abe; Y. Koide (pp. 240-246).
The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication (ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene (bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.
Telomeric repeat sequence of Aspergillus oryzae consists of dodeca-nucleotides by K.-I. Kusumoto; S. Suzuki; Y. Kashiwagi (pp. 247-251).
Four telomeres in the chromosomes of Aspergillus oryzae NFRI1599 were cloned and sequenced. The telomeric repeat sequence of A. oryzae consisted of dodeca-nucleotides: TTAGGGTCAACA. The length of the telomeric repeat tract was 114–136 bp, which corresponds to 9–11 repeats of the dodeca-nucleotide sequence. Compared to a chromosome internal control (18S rDNA), the telomeric sequences were found to be sensitive to BAL31 exonuclease digestion, thus proving that the identified telomeric repeat sequences were located at the most terminal tract of the chromosomes. The length of the telomeric repeat tract of A. oryzae is similar to that of Aspergillus nidulans, whose repeat unit is TTAGGG, indicating that the regulatory mechanism of telomere length might be conserved among Aspergillus species.
Hydrolysis of fenamiphos and its oxidation products by a soil bacterium in pure culture, soil and water by M. Megharaj; N. Singh; R. S. Kookana; R. Naidu; N. Sethunathan (pp. 252-256).
A bacterium, identified as Brevibacterium sp. MM1, readily hydrolysed fenamiphos, a widely used organophosphorus insecticide and its toxic oxides (fenamiphos sulfoxide, fenamiphos sulfone), which all contain a common P–O–C bond, in a mineral salts medium. The bacterium also hydrolysed fenamiphos and its oxides in soil and groundwater. Interestingly, fenamiphos phenol, fenamiphos sulfoxide phenol and fenamiphos sulfone phenol, formed during bacterial hydrolysis of fenamiphos and its oxides, persisted in the mineral salts medium, but were transitory in soil and groundwater due to their further metabolism by indigenous micro-organisms. The cell-free preparation (crude enzyme) of this bacterium was very effective in hydrolysing fenamiphos. This is the first report on exceptionally rapid hydrolysis of fenamiphos by a bacterium in pure cultures, soil and groundwater.
Phosphate feeding strategy during production phase improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) storage by Ralstonia eutropha by C. R. Squio; C. Marangoni; C. S. De Vecchi; G. M. F. Aragão (pp. 257-260).
The effect of a phosphate feeding strategy and the optimal rate of biomass production (r x) during the production phase of P(3HB-co-3HV) in a 6-l fermentor were determined in cultures of Ralstonia eutropha with the goal of enhancing polymer productivity. Rates of biomass production (r x) between 0.00 and 0.20 g×r l−1 h−1 were monitored during the production phase. When a low rate of cell growth was maintained (r x of 0.02 g×r l−1 h−1), polymer production improved, resulting in a final cell mass, P(3HB-co-3HV) mass, and P(3HB-co-3HV) content of 98.2 g, 62.0 g and 63.1 wt%, respectively, after 27.3 h. The maximum polymer productivity obtained during the production phase was 1.36 g l−1 h−1.
Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors by R. Muñoz; B. Guieysse; B. Mattiasson (pp. 261-267).
An algal-bacterial consortium formed by Chlorella sorokiniana and a phenanthrene-degrading Pseudomonas migulae strain was able to biodegrade 200–500 mg/l of phenanthrene dissolved in silicone oil or tetradecane under photosynthetic conditions and without any external supply of oxygen. Phenanthrene was only removed when provided in organic solvent, which confirms the potential of two-phase systems for toxicity reduction. Phenanthrene was degraded at highest rates when provided in silicone oil rather than in tetradecane since this solvent probably sequestered the PAH, reducing its mass transfer to the aqueous phase. The influence of phenanthrene concentration, amount of inoculum and light intensity on pollutant removal was also investigated and, under the best conditions, phenanthrene was degraded at 24.2 g m−3·h−1. In addition to being cost-effective and mitigating the release of greenhouse gases into the atmosphere, photosynthetic oxygenation was especially beneficial to the use of two-phase partitioning bioreactors since it prevented solvent emulsification and/or volatilization and evidence was found that the microalgae release biosurfactants that could further enhance phenanthrene degradation.