Degradation of substituted benzoic acids by a Micrococcus species

Abstract a Micrococcus sp. isolated by isophthalate enrichment, utilized 8 of the 13 substituted benzoic acids tested as the sole source of carbon and energy. The organism degraded benzoic acid and anthranilic acid through the intermediate formation of catechol. While salicylate is metabolized through genetisic acid, p -hydroxybenzoic acid is degraded through protocatechuic acid. The organism grew well on isophthalate but failed to utilize phthalate and terphthalate. Catechol disoxygenase, gentisate dioxygenase and protocatechuate dioxygenase activities were shown in the cell-free extracts. Catechol and protocatechuate are further metabolized through an ortho -cleavage pathway.     

微生物降解苯甲酸的研究进展

苯甲酸在工业中的广泛应用使其成为环境中的常见污染物,对微生物好氧降解苯甲酸的邻位途径、间位途径、龙胆酸途径和原儿茶酸途径及厌氧降解途径等进行总结,并对苯甲酸降解过程中发挥重要作用的苯甲酸双加氧酶的种类、不同组分及苯甲酸降解基因和调控基因的基因簇进行介绍,同时展望微生物降解污染物的发展方向。     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2.

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Biodegradation of chlorinated biphenyls and benzoic acids by a Pseudomonas strain

Summary A Pseudomonas strain able to grown on biphenyl and 2- and 4-chlorobiphenyl has been isolated from soil. Benzoate-grown cultures of this strain were able to cometabolize other chlorobiphenyls to the corresponding chlorobenzoates. In contrast to most of the chlorobiphenyl-degrading strains described previously in the literature, which are reported to form chlorobenzoates as end metabolites from chlorobiphenyls, this strain is also able to further cometabolize chlorobenzoates to form ring-cleaved compounds.     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

Mineralization of mono- and dichlorobenzenes and simultaneous degradation of chloro- and methyl-substituted benzenes by the white rot fungus Phanerochaete chrysosporium.

Phanerochaete chrysosporium extensively degraded and mineralized chlorobenzene and o-, m-, and p-dichlorobenzenes. The rate of degradation was in the following order: monochlorobenzene > m-dichlorobenzene > o-dichlorobenzene > p-dichlorobenzene. Net level of degradation was generally higher than mineralization. Maximal degradation and mineralization of chlorobenzenes were observed in malt extract cultures in which the lignin peroxidases and manganese peroxidases are not known to be produced. The fungus degraded both chlorobenzene and toluene when presented as a mixture, indicating its ability to simultaneously degrade chloro-substituted and methyl-substituted benzenes.     

Containment of a genetically engineered microorganism during a field bioremediation application

A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10¹⁴ bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low. Received: 6 March 1998 /thinsp;Received revision: 16 September 1998 / Accepted: 16 October 1998     

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30°C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10⁵–10⁷ CFU ml⁻¹), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30°C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg⁻¹ MP and 25 mg kg⁻¹ carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.     

Degradation of phenol and benzoic acid in a three-phase fluidized-bed reactor

Degradation of phenol and benzoic acid was studied in a fluidized-bed reactor (liquid volume 2.17 L) under nonsterile conditions with special emphasis on maximizing the flow through the reactor and investigating reactor performance at fluctuating feeds. Reactor response to substrate pulses was investigated by applying substrate square-wave inputs at a liquid flow of 1.00 L h(-1). A twofold increase of the phenol and benzoic acid feed concentrations for 2.5 h did not lead to accumulation and breakthrough. The cells were able to survive four to fivefold increases of the feed concentration for 1 h without loss of viability, although the phenol pulse lead to phenol accumulation in the reactor. Reactor performance at constantly fluctuating loads was investigated by varying the feed concentrations using sine wave functions. No accumulation of phenol or benzoic acid was observed. Influence of induction was studied using shift experiments. After 35 days of operation (369 hydrodynamic residence times) with phenol as sole substrate (carbon source) the reactor was able to mineralize benzoic acid without any adaptation or lag phase. The capability of phenol degradation, on the other hand, was lost by most cells after only 3 days operation with benzoic acid as the sole substrate. The experiments underline the importance of induction. In order to maximize the flow through the reactor, the liquid flow was increased stepwise while the feed concentrations were reduced correspondingly, keeping the volumetric conversion rates of phenol (0.24 g L(-1) h(-1)) and benzoic acid (0.17 g L(-1) h(-1)) constant. By this means, liquid flow could be increased up to 13.32 L h(-1), which was more than 20-fold higher than the maximum liquid flow achievable in a chemostat using the same conditions.     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

The metabolism of halogen-substituted benzoic acids by Pseudomonas fluorescens

1. Washed suspensions of Pseudomonas fluorescens, grown with benzoate as sole carbon source, oxidize monohalogenobenzoates in the following descending order of effectiveness: benzoate, fluorobenzoates, chlorobenzoates, bromobenzoates, iodobenzoates. 2. Cells grown on asparagine oxidize benzoate after an adaptive period of 90–120min. This adaptive period is increased by halogenobenzoates in the following approximate descending order of effectiveness: chlorobenzoates, fluorobenzoates (=bromobenzoates), iodobenzoates. This inhibition of adaptation by halogeno analogues depends on the concentration of benzoate and is thus apparently competitive. 3. Cells do not adapt to oxidize the halobenzoates when the halogeno analogues are inducers. However, the fluorobenzoates reduce the lag period taken to form the benzoate-oxidizing system. 4. The halogenobenzoates inhibit adaptation to citrate and nicotinate but not so effectively as benzoate itself. This is presumably a `diauxic' effect. The analogues do not inhibit adaptation to catechol. 5. The halogenobenzoates are not used as sole carbon source for growth nor do they increase growth when cells grow with asparagine as the main carbon source. 6. It is suggested that this inability to use the analogues for growth is due partly to inability of the cells to liberate the halogen and to carry the oxidation to a stage at which carbon may be assimilated and partly to the inhibition of the induction of the oxidizing enzymes.     

PCR—GeneScan法检测转基因产品

利用PCR－GeneScan技术检测转基因在豆和转基因玉米,该方法的特点是PCR扩增反应后,用Genescan扫描替代琼脂糖凝胶电泳检测PCR产物。用此方法共检测了35S启动子,NOS终止子和抗除草剂（草甘膦）和抗虫（Bt)4种转基因成分,结果显示,本方法比传统的PCR一琼脂糖凝胶电泳法灵敏度高,重现性好,结果易判断,为分析检测转基因产品提供了一个实用,灵敏的方法。     

Ethanol production by genetically modified strains of Saccharomyces

Summary Two polyploid yeast strains and two genetically manipulated yeast strains were subjected to anaerobic fermentations in whole corn mash and defined media. Carbohydrate utilization and ethanol production rates were investigated. Whilst the polyploid strains exhibited superior performance in the whole corn mash, the genetically manipulated strains were so in defined media with glucose as the substrate. The overall fermentation performance of the novel strains however was comparable to the polyploid strains with corn mash as the substrate when most of the solid material had been removed. The flocculating and dextrin utilizing properties of the yeast strains examined play an important role in such fermentations.     

Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil

One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68?g?l(-1). In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.     

Inhibitory effects of some synthetic monoethanolamine salts of para-substituted benzoic acids and corresponding benzoic acids on cucumber seed germination

Abstract

Benzoates and particularly, benzoic acids are known biologically for their effects in the regulation of seed germination. A series of monoethanolamine salts of para-substituted benzoic acids (MEASPBAs), the corresponding acids (BAs) and monoethanolamine (MEA) were tested at different concentrations, on Cucumis sativus L. germination in order to assess their biological activity. The correlation between the effects of different substituents of these salts and the corresponding acids with germination rate, root and shoot length, fresh and dry biomass, soluble protein content, isocitrate lyase (ICL, EC 4.1.3.1) and catalase (CAT, EC 1.11.1.6.) activity, was evaluated. Data showed that p-OH and p-CH₃ substituents had a lower inhibitory effect compared to the halogenated substituents. Moreover, the inhibition of root and shoot lengths and the dramatic decrease of fresh biomass for halogenated (p-Cl, p-Br, p-I) MEASPBAs and BAs followed the increase of the atomic size of the substituent.     

Scaffold-based bone engineering by using genetically modified cells

The first generation of clinically applied tissue engineering concepts in the area of skin, cartilage and bone marrow regeneration was based on the isolation, expansion and implantation of cells from the patient's own tissue. Although successful in selective treatments, tissue engineering needs to overcome major challenges to allow widespread clinical application with predictable outcomes. One challenge is to present the cells in a matrix to the implantation site to allow the cells to survive the wound healing contraction forces, tissue remodeling in certain tissues such as bone and biomechanical loading. Hence, several tissue engineering strategies focus on the development of load-bearing scaffold/cell constructs. From a cell source point of view, bone engineers face challenges to isolate and expand cells with the highest potential to form osseous tissue along with harvesting tissue without extensive donor site morbidity. A major hurdle to tissue engineering is de-differentiation and limited ability to control cell phenotype following in vitro expansion. Due to early successes with genetic engineering, bone tissue engineers have used different strategies to genetically alter various types of mesenchymal cells to enhance the mineralization capacity of tissue-engineered scaffold/cell constructs. Although the development of multi-component scaffold/osteogenic cell constructs requires a combination of interdisciplinary research strategies, the following review is limited to describe the general aspects of bone engineering and to present overall directions of technology platforms, which include a genetic engineering component. This paper reviews the most recent work in the field and discusses the concepts developed and executed by a collaborative effort of the multi-disciplinary teams of the two authors.     

Creating genetically modified pigs by using nuclear transfer

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture.     

Detection of genetically modified organisms by electrochemiluminescence PCR method

With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (CaMV35S) promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.