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Applied Microbiology and Biotechnology (v.57, #4)
Deposition of biological material for patent protection in biotechnology
by D. Fritze; V. Weihs (pp. 443-450).
Biotechnological production of pyruvic acid by Y. Li; J. Chen; S.-Y. Lun (pp. 451-459).
Pyruvic acid is an important organic acid widely used in the chemical and drug, as well as agrochemical, industries. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost. An overview of biotechnological production of pyruvate, including direct fermentative production employing eukaryotic and prokaryotic microorganisms, production by a resting cell method and an enzymatic method as well as the recovery of pyruvate, is discussed. A multi-vitamin auxotrophic yeast strain, Torulopsis glabrata, has been used in the commercial production of pyruvate; emphasis is therefore placed on the mechanism and characteristics of pyruvate production by this strain.
Desulfurization and desulfonation: applications of sulfur-controlled gene expression in bacteria by M. Kertesz; C. Wietek (pp. 460-466).
Inorganic sulfate is the preferred sulfur source for the growth of most microorganisms but, in its absence, many organosulfur compounds can be degraded microbially to provide sulfur. Desulfurization of dibenzothiophene (DBT) by Rhodococcus sp. and of aromatic sulfonates by Pseudomonas sp. has considerable biotechnological potential. Both these pathways require non-flavin-containing FMNH2-dependent monoxygenases (DszC/DszA and SsuD, respectively). FMNH2 is provided from the freely diffusible FMNH2 pool in the cell, and is replenished by specific NAD(P)H:FMN oxidoreductases (DszD and SsuE). Overexpression of the DszD FMN reductase in a heterologous system increases the efficiency of DBT desulfurization but is detrimental to cell growth at high levels. Expression of the sulfonatase that cleaves aromatic sulfonates (surfactants, dyes) is accompanied by synthesis of a thiol-specific antioxidant protein, which may protect the cell from superoxide radicals generated by autoxidation of the reduced flavin. Effective application of DBT desulfurization in the biodesulfurization of crude oil, and of arylsulfonate desulfonation in bioremediation, may require optimization of both flavin reductase levels and antioxidant protection systems within the cell.
Molecular biology of peptide and polyketide biosynthesis in cyanobacteria by E. Dittmann; B. Neilan; T. Börner (pp. 467-473).
Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.
Biotechnological development of effective phytases for mineral nutrition and environmental protection by X. Lei; C. Stahl (pp. 474-481).
Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate (myo-inositol hexakisphosphate), the major phosphorus (P) form in animal feeds of plant origin. These enzymes can be supplemented in diets for food animals to improve P nutrition and to reduce P pollution of animal excreta. This mini-review provides a synopsis of the concept of "ideal phytase" and the biotechnological approaches for developing such an enzyme. Examples of Escherichia coli AppA and Aspergillus fumigatus PhyA are presented to illustrate how new phytases are identified from microorganisms and developed by genetic engineering based on the gene sequences and protein structures of these enzymes. We also discuss the characteristics of different heterologous phytase expression systems, including those of plants, bacteria, fungi, and yeast.
Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers by H. Aoyagi; Y. Kobayashi; K. Yamada; M. Yokoyama; K. Kusakari; H. Tanaka (pp. 482-488).
An efficient system to produce saikosaponins (saikosaponin-a and -d) in Bupleurum falcatum adventitious root fragments combined with signal transducers was developed. The roots are heterogeneous in terms of size and shape and sometimes form aggregates during cultivation. When the roots were cut to lengths of about 5 mm using a scalpel and cultivated, the root fragments did not form the aggregates, and root growth and saikosaponin production were not inhibited. After screening various signal transducers, it was clear that methyl jasmonate (MeJA) markedly promoted saikosaponin production. By comparing the effect of MeJA and related substances on saikosaponin production, we conclude that both the pentenyl and carboxylmethyl group of MeJA play an important role in the promotion of saikosaponin production. Addition of both 100 µM MeJA and 20 mM CaCl2 to the medium stimulated the content of saikosaponin in the root, with levels reaching 31.7 mg/g-dry root for 15 days of cultivation. A large amount of root fragments were prepared using a blender and cultivated (23 g-dry root/l) with 400 µM MeJA and 20 mM CaCl2, resulting in a high concentration of saikosaponins (747.3 mg/l).
Dual-substrate utilization by Bordetella pertussis by R. Neeleman; M. Joerink; C. Beuvery; T. Boxtel (pp. 489-493).
To improve the cultivation of Bordetella pertussis and take advantage of the newest techniques in monitoring and control, a quantitative description of substrate utilisation is necessary. Growth of the organism is limited by two main substrates. However neither interactive nor non-interactive modelling seem appropriate. A model that combines essential and enhanced kinetics was developed based on experimental observation. Instead of fitting all model parameters at once, a step-wise experimentation procedure was used. Finally two cultivations showed the accuracy of the model.
Effect of enzyme impurities on phenol removal by the method of polymerization and precipitation catalyzed by Coprinus cinereus peroxidase by M. Masuda; A. Sakurai; M. Sakakibara (pp. 494-499).
The removal of phenol by peroxidase-catalyzed polymerization was examined using the Coprinus cinereus peroxidases at different levels of impurity with respect to contamination. The phenol removal efficiency was improved by lowering the peroxidase purity. Acidic and high molecular weight proteins present as impurities in the peroxidase solution had some positive effect on the phenol-polymerizing reaction. The residual enzyme activity, either only in the solution or both in the solution and on the precipitate during the polymerizing reaction, was measured. The results indicate that the main effect of impurities in the peroxidase solution was the suppression of the adsorption of peroxidase molecules on the polymerized precipitate.
Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain by Y. Nakamura; S. Shibasaki; M. Ueda; A. Tanaka; H. Fukuda; A. Kondo (pp. 500-505).
The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of α-agglutinin under control of the 5′-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1–10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification.
Evaluation of an electrochemical bioreactor system in the biotransformation of 6-bromo-2-tetralone to 6-bromo-2-tetralol by H. Shin; M. Jain; M. Chartrain; J. Zeikus (pp. 506-510).
Biotransformation of 6-bromo-2-tetralone (Br-β-tetralone) to 6-bromo-2-tetralol (Br-β-tetralol) by yeast cells of Trichosporon capitatum (ATCC 74312) and its partially purified Br-β-tetralone reductase was evaluated in an electrochemical bioreactor. The biotransformation rates and final product formation were significantly affected by substrate concentration, biomass and electric potential. At 2 g/l of substrate, the initial reaction rate and final product were increased by 35% and 15%, respectively, with –1.5 V of electric potential compared to without electric potential. Additional substrate (2 g/l) provided by pulse feeding to the reaction mixture at different intervals resulted in 2.1 g/l Br-β-tetralol compared to a total of 1.2 g/l without feeding. However, the increased production was not proportionate to the amount of additionally fed substrate. Increased substrate availability by the addition of 5% (v/v) ethanol resulted in the highest reaction rate and product formation, but addition of ethanol at a concentration higher than 5% decreased the reaction rate. At low biomass, the initial reaction rates were enhanced significantly when electric potential was high, but a higher biomass was necessary to obtain a similar reaction rate when electric potential was reduced. The highest initial reaction rate (59.2 mg/l per min) was achieved with a two-fold biomass concentration of 15.6 g of dry cell weight/l, substrate at 4 g/l and electric potential at –6 V. The conversion of Br-β-tetralone to Br-β-tetralol with partially purified Br-β-tetralone reductase was slow in the presence of electric potential.
Improvement in the bioconversion of penicillin G to deacetoxycephalosporin G by elimination of agitation and addition of decane by Q. Gao; A. Demain (pp. 511-513).
The bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) using resting cells of Streptomyces clavuligerus could be a very valuable step in the economical production of semisynthetic cephalosporin antibiotics. The extent of the reaction, however, is very low due to inactivation of the ring expansion enzyme deacetoxycephalosporin C synthetase ("expandase") by reaction components. We show that elimination of agitation during the reaction lowers the rate but increases the amount of DAOG produced, presumably because the inactivation requires high levels of oxygen. Many additives to the medium were examined for their effect on the reaction. Clearly, the most effective compound was the organic solvent, decane.
Characteristics and N-terminal amino acid sequence of manganese peroxidase from solid substrate cultures of Agaricus bisporus
by Pauliina V. Lankinen; Alice M. Bonnen; Lori H. Anton; David A. Wood; Nisse Kalkkinen; Annele Hatakka; Christopher F. Thurston (pp. 514-514).
Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production by T. Matsumoto; S. Takahashi; M. Kaieda; M. Ueda; A. Tanaka; H. Fukuda; A. Kondo (pp. 515-520).
Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8–1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 °C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was71 wt% after a 165-h reaction at 37 °C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.
Differential expression of the Trichoderma reesei β-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis by R. Den Haan; W. Van Zyl (pp. 521-527).
The transcriptional control of two native promoters and one heterologous promoter and the production of a heterologous protein from these promoters were evaluated in the xylose-fermenting yeast Pichia stipitis cultivated on xylose and glucose as carbon sources, using the β-xylanase II xyn2 gene of Trichoderma reesei. The xyn2 gene open reading frame was fused to the P. stipitis xylose reductase gene (XYL1) promoter, the P. stipitis transketolase gene (TKL) promoter and the Saccharomyces cerevisiae phosphoglycerate kinase gene (PGK1) promoter DNA sequences on episomal plasmids. The plasmids were transformed into Pichia stipitis and gene expression and β-xylanase production monitored. The XYL1 promoter was shown to be inducible in the presence of xylose, as xyn2 transcription and β-xylanase activity could be measured when the recombinant strain was cultivated on xylose but not when it was cultivated on glucose. TKL promoter expression was found to be constitutive when either glucose or xylose was used as sole carbon source. The PGK1 promoter did not promote xyn2 transcription in P. stipitis. The molecular size of the recombinant Xyn2 protein produced by P. stipitis was 20.7 kDa, which is similar to that of the native T. reesei Xyn2 protein. This indicates no or minimal glycosylation of the recombinant protein. The recombinant xyn2-expressing strain also yielded twice the amount of biomass yielded by the control strain when cultivated in medium containing 1% birchwood xylan as sole carbon source.
Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration by S. Shibasaki; M. Ueda; K. Ye; K. Shimizu; N. Kamasawa; M. Osumi; A. Tanaka (pp. 528-533).
We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.
H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate by H. Sekine; T. Shimada; C. Hayashi; A. Ishiguro; F. Tomita; A. Yokota (pp. 534-540).
A mutant of Corynebacterim glutamicum ('Brevibacterium flavum') ATCC14067 with a reduced H+-ATPase activity, F172–8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172–8 was reduced to about 25% of that of the parental strain. Strain F172–8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using a jar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated α-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172–8, because a revertant of strain F172–8, R2–1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172–8.
Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12 by H. Heipieper; P. Waard; P. Meer; J. Killian; S. Isken; J. Bont; G. Eggink; F. Wolf (pp. 541-547).
The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2Δ9-cis,Δ12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the Δ9-trans,Δ12-cis isomer, while the Δ9-cis,Δ12-trans and Δ9-trans,Δ12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27–36% of the chains) was obtained at 0.03–0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.
Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1 by J. Hage; F. Kiestra; S. Hartmans (pp. 548-554).
Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCA1 and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCA1 could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCA1. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2-dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.
Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost by Y. Ueno; S. Haruta; M. Ishii; Y. Igarashi (pp. 555-562).
Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticum was isolated in the enrichment culture with or without added peptone, and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.
Degradation of diphenyl ether herbicides by the lignin-degrading basidiomycete Coriolus versicolor by N. Hiratsuka; H. Wariishi; H. Tanaka (pp. 563-571).
Under ligninolytic conditions, the white-rot basidiomycete Coriolus versicolor metabolized chloronitrofen (2, 4, 6-trichloro-4'-nitrodiphenyl ether; CNP) and nitrofen (2, 4-dichloro-4'-nitrodiphenyl ether; NIP), which constitute the largest class of commercially produced diphenyl ether herbicides. The pathway of CNP degradation was elucidated by the identification of fungal metabolites upon addition of CNP and its metabolic intermediates. The metabolic pathway was initially branched to form four metabolites – 2, 4, 6-trichloro-3-hydroxy-4'-nitrodiphenyl ether, 2, 4-dichloro-6-hydroxy-4'-nitrodiphenyl ether, NIP, and 2, 4, 6-trichloro-4'-aminodiphenyl ether – indicating the involvement of hydroxylation, oxidative dechlorination, reductive dechlorination, and nitro-reduction. Of these reactions, hydroxylation was relatively major compared to the others. Extracellular ligninolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase did not catalyze the oxidation of either CNP or NIP. Piperonyl butoxide, an inhibitor of cytochrome P450, suppressed fungal oxidation of CNP and NIP to their hydroxylated products. The inhibition resulted in increasing the amount of reductively dechlorinated and nitro-reduced products. These observations strongly suggest that basidiomycetes may possess a mechanism for a strict substrate recognition system and a corresponding metabolic response system to effectively degrade environmentally persistent aromatic compounds.
Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments by Z. Zheng; G. Breedveld; P. Aagaard (pp. 572-578).
Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.