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Applied Microbiology and Biotechnology (v.61, #2)
Molecular genetic analysis and regulation of aflatoxin biosynthesis by D. Bhatnagar; K. C. Ehrlich; T. E. Cleveland (pp. 83-93).
Aflatoxins, produced by some Aspergillus species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular mechanism of AFB biosynthesis showed that the genes required for biosynthesis are in a 70 kb gene cluster. They encode a DNA-binding protein functioning in aflatoxin pathway gene regulation, and other enzymes such as cytochrome P450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid synthases. Information gained from these studies has led to a better understanding of aflatoxin biosynthesis by these fungi. The characterization of genes involved in aflatoxin formation affords the opportunity to examine the mechanism of molecular regulation of the aflatoxin biosynthetic pathway, particularly during the interaction between aflatoxin-producing fungi and plants.
The quest for microbial reductive dechlorination of C 2 to C 4 chloroalkanes is warranted by S. De Wildeman; W. Verstraete (pp. 94-102).
C 2 to C 4 chloroalkanes have been used for a wide range of industrial applications. Consequently, numerous leaks to the environment have occurred. It is generally observed that the lower chlorinated members of the group, containing 1–3 chlorine atoms, accumulate in environments where reductive conditions prevail. Their half-lives under these conditions often exceed several decades. To date, successes in rapid and complete in situ reductive dechlorination have only been obtained with tetrachloroethene (PCE) and trichloroethene (TCE), but not with chloroalkanes. Since the key-player PCE- and TCE-dechlorinating bacteria involved have been studied, these organisms could be used as very efficient tools for low-cost in situ bioremediation. Except for one 1,2-dichloroethane-dehalorespiring bacterium with limited application possibilities and a recent isolate which partly dechlorinates some polychloroethanes, all bacterial reductive conversions of C 2 to C 4 chloroalkanes are based on slow, mostly incomplete and poorly controllable cometabolic dechlorinations. Furthermore, metals such as Fe(0) cannot dechlorinate most lower-chlorinated C 2 to C 4 alkanes. Hence, pump and treat, or aerobic degradation are the applied technologies, although they are expensive and time-intensive. However, energetic consideration of chloroalkane dechlorination suggests that metabolizing anaerobes may exist. Isolation and characterization of these organisms is warranted in order to develop cost-efficient, controlled, fast and complete in situ remediation technologies.
Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment by A. Hiraishi; S. T. Khan (pp. 103-109).
Application of polyhydroxyalkanoates (PHAs) and related biodegradable polymers has gained momentum in various areas of biotechnology. A promising application that started appearing in the past decade is the use of PHAs as the solid substrate for denitrification of water and wastewater. This type of denitrification, termed here "solid-phase denitrification", has several advantages over the conventional system supplemented with liquid organic substrate. PHAs serve not only as constant sources of reducing power for denitrification but also as solid matrices favorable for development of microbial films. In addition, in contrast to conventional processes, the use of PHAs has no potential risk of release of dissolved organic carbon with the resultant deterioration of effluent water quality. If the production cost of PHAs can be brought down, its application to the denitrification process will become economically more promising. A number of PHA-degrading denitrifying bacteria have been isolated and characterized from activated sludge and continuous flow-bed reactors for denitrification with PHAs. Most of these isolates have been assigned phylogenetically to members of β-Proteobacteria, especially those of the family Comamonadaceae. The metabolic and regulatory relationships between PHA degradation and denitrification, and the interactive relationship between PHA-degrading cells and the solid surface structure are important subjects awaiting future studies, which would provide a new insight into our comprehensive understanding of the solid-phase denitrification process.
Continuous gluconic acid production by isolated yeast-like mould strains of Aureobasidium pullulans by S. Anastassiadis; A. Aivasidis; C. Wandrey (pp. 110-117).
By extensive microbial screening, about 50 strains with the ability to secrete gluconic acid were isolated from wild flowers. The strains belong to the yeast-like mould Aureobasidium pullulans (de Bary) Arnaud. In shake flask experiments, gluconic acid concentrations between 23 and 140 g/l were produced within 2 days using a mineral medium. In batch experiments, various important fermentation parameters influencing gluconic acid production by A. pullulans isolate 70 (DSM 7085) were identified. Continuous production of gluconic acid with free-growing cells of the isolated yeast-like microorganisms was studied. About 260 g/l gluconic acid at total glucose conversion could be achieved using continuous stirred tank reactors in defined media with residence times (RT) of about 26 h. The highest space-time-yield of 19.3 g l−1 h−1) with a gluconic acid concentration of 207.5 g/l was achieved with a RT of 10.8 h. The possibility of gluconic acid production with biomass retention by immobilised cells on porous sinter glass is discussed. The new continuous gluconate fermentation process provides significant advantages over traditional discontinuous operation employing Aspergillus niger. The aim of this work was the development of a continuous fermentation process for the production of gluconic acid. Process control becomes easier, offering constant product quality and quantity.
Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W by S. Chauhan; S. Wu; S. Blumerman; R. D. Fallon; J. E. Gavagan; R. DiCosimo; M. S. Payne (pp. 118-122).
A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.
Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression by P. Oelschlaeger; S. Lange; J. Schmitt; M. Siemann; M. Reuss; R. D. Schmid (pp. 123-132).
The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.
Synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate using fabG-homologues by H. Yamamoto; A. Matsuyama; Y. Kobayashi (pp. 133-139).
This paper is a report on the successful application of bioinformatics to enzyme screening. The synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate (ECHB) by asymmetric reduction of ethyl 4-chloroacetoacetate (ECAA) using fabG-homologues was studied. β-Ketoacyl-acyl carrier protein reductases from both Escherichia coli and Bacillus subtilis, which are components of type II fatty acid synthase, could reduce ECAA to (S)-ECHB with 94–98% ee. Furthermore, acetoacetyl-CoA reductases (ARs) from both Ralstonia eutropha and Zoogloea ramigera, whose genes are significantly similar to fabG genes and play a physiological role in the biosynthesis of poly-β-3-hydroxybutyrate, could also catalyze the asymmetric reduction of ECAA to (S)-ECHB with >99% ee. (S)-ECHB was synthesized to 48.7 g/l with an optical purity of 99.8% ee, using recombinant E. coli cells coexpressing AR from R. eutropha and glucose dehydrogenase from B. subtilis for the regeneration of NADPH.
Molecular cloning, sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp. PE2, and its domain structure by E. Kitamura; Y. Kamei (pp. 140-149).
The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBDPchA was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBMPchA showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBDPchA might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBMPchA also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.
Transformation and mineralization of 2,4,6-trinitrotoluene by the white rot fungus Irpex lacteus by H.-Y. Kim; H.-G. Song (pp. 150-156).
Unlike other 2,4,6-trinitrotoluene (TNT)-degrading white rot fungi, including Phanerochaete chrysoporium, initial metabolism of TNT by Irpex lacteus was found to occur through two different transformation pathways. Metabolites of the nitro group reduction pathway were confirmed with the standard compounds, and the formation of hydride-Meisenheimer complex of TNT (H−-TNT) formed in the denitration pathway was identified with LC/MS and by LC/photodiode array (PDA) detection. The molecular weight of the H−-TNT complex was identified as 228 m/z, and the UV-visible absorption spectrum, recorded with a PDA detector, proved the identity of this metabolite (RT, 18.7 min; λ max 254, 474, 557 nm) by comparison with the authentic synthetic H−-TNT (RT 18.7 min; λ max 261, 474, 563 nm). Mineralization of [U-14C]TNT by I. lacteus was also measured in static and shaken cultures. The mineralization rate of TNT in the static culture was higher than that in the shaken culture, and addition of Tween 80 (final concentration 1%) enhanced the mineralization of TNT in the static culture, resulting in 30.57% of CO2 evolution from the radiolabeled TNT added. The high TNT mineralization capability of I. lacteus seemed to be the result of simultaneous utilization of the denitration pathway, which is more favorable for the ring cleavage and mineralization of TNT, together with the nitro group reduction pathway.
Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production by M. G. Aguilar Uscanga; M.-L. Délia; P. Strehaiano (pp. 157-162).
The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0–300 l h–1 (0–0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O2 l–1h–1. In this case, there was an air flow rate of 60 l h–1 (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h–1 (0.3 vvm, OTR =105 mg O2 l–1 h–1), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g l–1) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.
Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement by L. Peng; K. Shimizu (pp. 163-178).
Regulation of the main metabolic pathways of Escherichia coli K12 was investigated based on 2-dimensional electrophoresis (2DE) and the measurement of enzyme activities. The cells were grown aerobically in different carbon sources, such as glucose, acetate, gluconate or glycerol. Microaerobic cultivation was also conducted with glucose as a carbon source. Fifty-two proteins could be identified based on 2DE, and 26 enzyme activities from the main metabolic pathways—including glycolysis, pentose phosphate pathway, TCA cycle, Entner-Doudoroff pathway and fermentative pathway—were assayed. These enzyme activities, together with global and quantitative protein expression, gave us a clear picture of metabolic regulation. The results show that, compared with the control experiment with glucose as a carbon source under aerobic conditions, glycolytic enzymes were slightly up-regulated (<2-fold), TCA cycle enzymes were significantly down-regulated (2- to 10-fold), and fermentative enzymes such as pfl and adhE were highly up-regulated (>10-fold) under microaerobic conditions in glucose medium. When acetate was used as a carbon source, pfkA, pykF, ppc and zwf were down-regulated, while fbp, pckA, ppsA and mez were significantly up-regulated. Glyoxylate enzymes such as aceA and aceB were strongly up-regulated (>10-fold) and TCA-cycle-related enzymes were also up-regulated to some extent. With gluconate as a carbon source, edd, eda, fbp and TCA cycle enzymes were up-regulated. With glycerol as a carbon source, fbp and TCA cycle enzymes were up-regulated, while ackA was significantly down-regulated. Protein abundance obtained by 2DE correlated well with enzyme activity, with a few exceptions (e.g., isocitrate dehydrogenase), during aerobic growth on acetate.