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21 Genetically Engineered Microorganisms and Bioremediation FU-MIN MENN JAMES P. EASTER GARY S. SAYLER Knoxville, TN 37996-1605, USA 1 Introduction 443 2 Degradative (Catabolic) Genes 444 2.1 Branched Aromatic Hydrocarbons 445 2.1.1 Pseudomonas spp. 445 2.2 Chlorinated Compounds 445 2.2.1 Chlorobenzoates 445 2.2.1.1 Pseudomonas sp. B13 Hybrid Pathways 445 2.2.1.2 Pseudomonas aeruginosa AC869(pAC31) 446 2.2.1.3 Pseudomonas sp. US1 ex. 446 2.2.2 Polychlorinated Biphenyls (PCB) and Chlorobiphenyls 446 2.2.2.1 Ralstonia eutropha (formerly Alcaligenes eutrophus) AE707/AE1216 446 2.2.2.2 Pseudomonas sp. Hybrid Strains 447 2.2.2.3 Pseudomonas cepacia JHR22 447 2.2.2.4 Pseudomonas acidovorans M3GY 447 2.2.2.5 Pseudomonas putida (pDA261) 448 2.2.2.6 Comamonas testosteroni VP44(pE43)/VP44(pPC3) 448 2.2.2.7 Escherichia coli JM109 (pSHF1003)/(pSHF1007) Hybrid Biphenyl Dioxygenase 448 2.2.2.8 Pseudomonas putida IPL5 (Field Application Vectors) 449 2.2.3 Trichloroethylene (TCE) 449 2.2.3.1 Escherichia coli HB101/pMY402 and FM5/pKY287 449 2.2.3.2 Pseudomonas pseudoalcaligenes KF707-D2 and Pseudomonas putida KF715-D5 449 2.2.3.3 Pseudomonas sp. strain JR1A::ipb Hybrid Strains 449 2.2.3.4 Escherichia coli JM109(pDTG601) 450 2.2.3.5 Pseudomonas putida G786(pHG-2) 450 442 21 Genetically Engineered Microorganisms and Bioremediation 2.2.3.6 Pseudomonas putida F1/pSMMO20 450 2.2.3.7 Burkholderia cepacia G4 5223-PR1 450 2.2.3.8 Ralstonia eutropha AEK301/pYK3021 451 2.2.4 2,4-Dichlorophenoxyacetic acid (2,4-D) 451 2.2.4.1 Pseudomonas putida PPO300(pRO101) and PPO301(pRO103) 451 2.2.4.2 Pseudomonas cepacia RHJ1 451 2.3 Nitroaromatic Compounds 452 2.3.1 Pseudomonas 410PR(pWW0 pm) Hybrid Pathway 452 2.3.2 Pseudomonas sp. clone A (pWW0-Km) Hybrid Pathway 452 2.4 Sulfur Compounds 452 2.4.1 Pseudomonas aeruginosa EGSOX and Pseudomonas putida EGSOX 452 2.4.2 Pseudomonas sp. HL7bR(pC1) and Pseudomonas alcaligenes DM201(pC1) 452 3 Reporter Genes 453 3.1 Chlorinated Compounds 453 3.1.1 Ralstonia eutropha ENV307(pUTK60) 453 3.1.2 Burkholderia cepacia BRI6001L 453 3.1.3 Alcaligenes eutrophus H850Lr 453 3.1.4 Pseudomonas fluorescens 10586s/FAC510 and 10586/pUCD607 454 3.2 Aromatic Hydrocarbons 454 3.2.1 Pseudomonas putida TVA8 454 3.2.2 Pseudomonas fluorescens HK44 454 3.2.3 Escherichia coli HMS174(pOS25) 455 3.2.4 Pseudomonas sp. UG14Lr 455 3.3 Metals 455 3.3.1 Escherichia coli HMS174(pOS14/pOS15) 455 3.3.2 Staphylococcus aureus RN4220 (pC200)/(pC300) 455 3.4 Others 456 3.4.1 Alcaligenes eutrophus 2050 and Stenotrophomonas 3664 456 3.4.2 Pseudomonas FRD1(pUTK50) 456 3.4.3 Pseudomonas strain Shk1 456 3.4.4 Escherichia coli DPD2511 457 3.4.5 Escherichia coli WM1202(pRY002)/TV1061(pGrpElux.3 or pGrpElux.5) 457 4 Conclusion 457 5 References 457 1 Introduction 443 List of Abbreviations BBIC BTEX 2,4-D DBT FAV GEM IC IGP IPB IPTG PAH PCB PCP SDS-PAGE sMMO 2,4,5-T TCE TMO TNT TOL bioluminescent bioreporter integrated circuit benzene, toluene, ethylbenzene, and xylene 2,4-dichlorophenoxyacetic acid dibenzothiophene field application vector genetically engineered microorganisms integrated circuit Igepal CO-720 isopropylbenzene isopropyl thio- -galactoside phenanthrene, pyrene and benzo[a]pyrene polychlorinated biphenyls pentachlorophenol sodium dodecyl sulfate polyacrylamide gel electrophoresis soluble methane monooxygenase 2,4,5-trichloro phenoxy acetic acid trichloroethylene toluene monooxygenase 2,4,6-trinitrotoluene toluene plasmid pWWO 1 Introduction During the past 20 years, recombinant DNA techniques have been studied intensively to improve the degradation of hazardous wastes under laboratory conditions. Only one field test has been successfully implemented (SAYLER et al., 1999). Recombinant bacteria can be obtained by genetic engineering techniques or by natural genetic exchange between bacteria. Applications for genetically engineered microorganisms (GEM) in bioremediation have received a great deal of attention, but have largely been confined to the laboratory environment. This has been due to regulatory risk assessment concerns, and to a large extent the uncertainty of their practical impact and delivery under field conditions. There are at least four principal approaches to GEM development for bioremediation application. These include: (1) modification of enzyme specificity and affinity, (2) pathway construction and regulation, (3) bioprocess development, monitoring, and control, and (4) bioaffinity bioreporter sensor applications for chemical sensing, toxicity reduction, and end point analysis. There are many reports on the degradation of environmental pollutants by different bacteria. However, only potential bioremediation related to GEM will be reviewed, and the construction and application of GEM will be the focus of this review. Some examples of the relevant use of genetic engineering technology to improve bioremediation are listed in Tab. 1. These genetically engineered microorganisms have higher degradative capacity and have been demonstrated successfully for the degradation of various pollutants under defined conditions. However, ecological and environmental concerns and regulatory constraints are major obstacles for testing GEM in the field. These problems must be overcome before GEM can provide an effective clean-up process at lower cost. The use of genetically engineered microorganisms has been applied to bioremediation 444 21 Genetically Engineered Microorganisms and Bioremediation Tab. 1. Genetic Engineering for Biodegradation of Contaminants Microorganism Modification Contaminants Reference Pseudomonas sp. B13 pathway mono/dichlorobenzoates P. putida P. putida KT2442 Pseudomonas sp. FR1 C. testosteroni VP44 pathway pathway pathway substrate specificity substrate specificity substrate specificity substrate specificity regulation 4-ethylbenzoate toluene/benzoate chloro-, methylbenzoates o-, p-monochlorobiphenyls REINEKE and KNACKMUSS, 1979, 1980 RAMOS et al., 1987 PANKE et al., 1998 ROJO et al., 1987 HRYWNA et al., 1999 Pseudomonas sp. LB400 E. coli JM109(pSHF1003) P. pseudoalcaligenes KF707-D2 E. coli FM5/pKY287 process monitoring, strain monitoring, stress response, end point analysis, and toxicity assessment. Examples of these applications are listed in Tab. 2. The range of tested contaminants includes chlorinated compounds, aromatic hydrocarbons, heavy metals, and nonpolar toxicants, etc. Development of effective and cost-efficient bioremediation processes is the goal for environmental biotechnology. The combination of microbiological and ecological knowledge, biochemical mechanisms, and field engineering designs are essential elements for successful in situ bioremediation using GEM. PCB, benzene, toluene ERICKSON and MONDELLO, 1993 KUMAMMRU et al., 1998 TCE, toluene, benzene SUYAMA et al., 1996 TCE, toluene WINTER et al., 1989 PCB 2 Degradative (Catabolic) Genes Extensive review information is available on the biochemical pathway analysis, operon structure, and molecular biology of biodegradative pathways important in bioremediation. Much of this information, critical to the development of appropriate GEM, is confined to aerobic catabolic and cometabolic pathways (SAYLER et al., 1998). Tab. 2. Genetic Engineering for Biodegradation Process Efficacy Microorganism Application Contaminants Reference A. eutrophus H850Lr P. putida TVA8 P. fluorescens HK44 process monitoring process monitoring process monitoring VAN DYKE et al., 1996 APPLEGATE et al., 1998 SAYLER et al., 1999 B. cepacia BRI6001L P. fluorescens 10586s/pUCD607 P. fluorescens 10586s/pUCD607 strain monitoring stress response PCB TCE, BTEX naphthalene, anthracene, phenanthrene 2,4-D BTEX toxicity assessment chlorobenzenes, chlorophenols, BTEX Pseudomonas strain Shk1 A. eutrophus 2050 toxicity assessment Cd, 2,4-dinitrophenol, hydroquinone nonpolar narcotics BOYD et al., 1998; SINCLAIR et al., 1999; GLOVER et al., 1999 KELLY et al., 1999 end point analysis MASSON et al., 1993 SOUSA et al., 1998 LAYTON et al., 1999 2 Degradative (Catabolic) Genes 2.1 Branched Aromatic Hydrocarbons 2.1.1 Pseudomonas spp. The TOL plasmid pathway of Pseudomonas putida has been manipulated extensively to expand its catabolic capability on different branched compounds. RAMOS et al. (1987) modified the TOL metabolic pathway to use 4-ethylbenzoate as a substrate. These restructured strains were mutants isolated by either altering a pathway regulator XylS (RAMOS et al., 1986) or by modifying substrate specificity of enzymes (catechol-2,3-dioxygenase). The complete upper TOL operon of plasmid pWW0 with its regulator gene, xylR, was reconstructed as a single gene cassette and yielded a hybrid mini-Tn5 [upp TOL] transposon. The transposon was further inserted into the chromosome of P. puida KT2442. The hybrid strain, KT2442::mini-Tn5 [upp TOL] [Kmr/xylE ], was stable and grew on toluene as the sole carbon source. Toluene was degraded to benzoate by the TOL upper pathway and benzoate was further metabolized through an o-cleavage pathway (PANKE et al., 1988). 2.2 Chlorinated Compounds 2.2.1 Chlorobenzoates 2.2.1.1 Pseudomonas sp. B13 Hybrid Pathways The plasmid pWR1 encoding 3-chlorobenzoate and 4-chlorophenol degradation genes in strain B 13 have been modified extensively to generate hybrid pathways that extend vertical degradation of haloaromatic compounds. REINEKE and KNACKMUSS (1979, 1980) first reported the introduction of TOL plasmid pWW0, of P. putida mt-2 into strain B13 via conjugation. The transconjugants demonstrated the capability to utilize mono- and dichlorobenzoates as growth substrates. The inactivation of the TOL plasmid encoded m-pathway (by loss of catechol-2,3-dioxygenase activity) 445 was an obligatory requirement for the hybrid strain, Pseudomonas sp. WR211, to utilize 4-chlorobenzoate as the sole carbon source (REINEKE et al., 1982). SCHWIEN and SCHMIDT (1982) reported that a benzoate and phenol degrader, Alcaligenes sp. A7, acquired chlorocatechol degrading capacity from strain B13 through conjugation. The recombinant strain, Alcaligenes sp. A7-2, exhibited the ability to utilize all three isomeric chlorophenols as the sole source of carbon and energy. The xylXYZ (toluate-1,2-dioxygenase) and xylL (carboxylate dehydrogenase) genes from TOL plasmid (pWW0-161) of P. putida and nahG (salicylate hydroxylase) from NAH7 plasmid were cloned into strain B13 to extend its substrate range (LEHRBACH et al., 1984). A hybrid plasmid, including genes (xylXYZL), positive regulator (xylS ), and their native promoter (Pm), was introduced into strain B13 by conjugation. The resulting Pseudomonas sp. B13(TOL) was capable of utilizing 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate.The transconjugants containing the nahG gene were able to degrade salicylate, 3-, 4-, and 5-chlorosalicylate. ROJO et al. (1987) reported a modified ocleavage pathway, in strain B13, for simultaneous degradation of chloro- and methylaromatic compounds. This hybrid pathway included: (1) toluate 1,2-dioxygenase and carboxylate dehydrogenase from the TOL pathway to degrade 4-chlorobenzoate and transform methylbenzoates into methyl-2-enelactones (strain FR1), (2) 4-methyl-2-enelactone isomerase from Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 to transform 4-methyl-2-enelactones to 3-methyl-2enelactones to complete the o-pathway for 4-methylbenzoate [strain FR1(pRFC20P)], (3) phenol hydroxylase from B13 to mineralize chloro- and methylphenols (cresols) to corresponding catechols [strain FR1(pFRC20P)-1]. A hybrid plasmid, pFRC4P (Tn5::xylXYZLS ), containing Pm promoter was introduced into strain B13 through triparental mating using E. 446 21 Genetically Engineered Microorganisms and Bioremediation coli HB101(pFRC4P) as a donor, and E. coli HB101(pRK2013) as a helper. The hybrid Tn5 transposon that carried TOL genes was transposed into the chromosome of B13 and yielded strain FR1. Gene encoded 4-methyl-2-enelactone isomerase from R. eutropha JMP134 was cloned and transferred by conjugation into FR1. The resulting strain containing a hybrid plasmid, pFRC20P, was designated as B13FR1(pFRC20P) and was capable of growing on 4-methylbenzoate. Spontaneous mutants of FR1(pFRC20P), such as FR1(pFRC20P)-1 and -2, were able to use 4-methylphenol as a sole source of carbon and energy. The engineered bacterium Pseudomonas sp. B13FR1(pFRC20P)-1 was capable of growing on and mineralizing mixtures of 3-chloro-, 4-chloro-, and 4-methylbenzoate and 4-chloroand 4-methylphenol via the modified o-pathway. Studies on the survival rate of the engineered strain, the stability and expression of the recombinant pathways, and gene transfer in the environment were conducted in an activated sludge microcosm (N LEIN et al., 1992). Strain FR1(pFRC20P) was also examined in two different aquatic sediments and the results showed enhancement of the degradation rate of contaminants (PIPKE et al., 1993). 2.2.1.2 Pseudomonas aeruginosa AC869(pAC31) Strain P. putida, containing plasmid pAC25 that encodes genes for the degradation of 3chlorobenzoate, has been shown to be structurally homologous to pWR1 (CHATTERJEE and CHAKRABARTY, 1983). Genetic rearrangements occurred between plasmids pAC25 and TOL under chemostat maintenance and yielded pAC31. The new plasmid contained xylDGEF genes located on the chromosome. The resulting strain AC869(pAC31) showed the capability to degrade 3,5-dichlorobenzoate and 3- and 4-chlorobenzoate (CHATTERJEE and CHAKRABARTY, 1982). 2.2.1.3 Pseudomonas sp. US1 ex. A monochlorobenzoate and 2,4-D degrader, strain US1 ex. containing pJP4 was obtained through conjugation. The E. coli JMP397, harboring plasmid pJP4 (no expression), was used as a donor and the 4-chlorobenzoate degrader, US1, was the recipient.The new strain released stoichiometric amounts of chloride when grown on respective chloroaromatics as carbon source (SAHASRABUDHE and MODI, 1991). There are reports on the horizontal transfer of 3-chlorobenzoate catabolic plasmid (pBRC60) in a freshwater ecosystem (FULTHTHROPE and WYNDHAM, 1991) and in an activated sludge unit (pD10; MCCLURE et al., 1989). 2.2.2 Polychlorinated Biphenyls (PCB) and Chlorobiphenyls 2.2.2.1 Ralstonia eutropha (formerly Alcaligenes eutrophus) AE707/AE1216 Chromosomally located PCB catabolic genes of R. eutropha A5 (SHIELDS et al., 1985), Achromobacter sp. LBS1C1 (PETTIGREW et al., 1990), and Alcaligenes dentrificans sp. JB1 (PARSONS et al., 1988) were transferred into a heavy metal resistant strain R. eutropha CH34 through natural conjugation. All donor strains degraded biphenyl and monochlorobiphenyls to corresponding benzoate and chlorobenzoates. Benzoate was further metabolized via the o-cleavage pathway in strains A5 and LBS1C1, and via the m-cleavage pathway in strain JB1. Strain A5 harbored a catabolic transposon, Tn4317, which carried biphenyl and 4-chlorobiphenyl degradation genes (SPRINGEAL et al., 1993a). The dehalogenase activity was mediated by plasmid pSS50 in strain A5 (LAYTON et al., 1992). Transfer of PCB degradation genes from A5 to the heavy metal resistant strain CH34 was carried out by conjugation (SPRINGAEL et al., 1993b).The constructed strain,AE707, exhibited phenotype of BphcCbpc, and degraded 4-chlorobiphenyl to 4-chlorobenzoate in the presence of heavy metals. In resting cell assays (grown on biphenyl), strain AE707 cometabolized di- and trichlorinated congeners of Aroclor 1242 in the presence of heavy metals.The 2 Degradative (Catabolic) Genes PCB catabolic chromosomal genes of strain JB1 were transferred into CH34 through RP4::Mu3A mediated R-prime plasmid formation (SPRINGAEL et al., 1994). A transconjugant, strain AE1216, utilized 2-, 3- and 4-chlorobiphenyl and exhibited properties of metal resistance. 2.2.2.2 Pseudomonas sp. Hybrid Strains Pseudomonas putida BN10 grew on biphenyl and accumulated metabolites of 2-, 4-chlorobenzoate and 3-chlorocatechol from corresponding monochlorobiphenyls. 3-Chlorobiphenyl degraders were obtained from conjugation between Pseudomonas sp. B13 and strain BN10.The resulting Pseudomonas strain BN210 (gained chlorocatechol degradation genes) and B131 (acquired biphenyl degradation genes) were able to grow on 3-chlorobiphenyl. Both strains exhibited the capability to degrade monochlorobiphenyls and 2 of the dichlorobiphenyls found in Aroclor 1221 (MOKROSS et al., 1990). Another hybrid Pseudomonas sp. strain UCR2 was isolated from multi-chemostat mating between a chlorobenzoate degrader, P. aeruginosa JB2, and a 2-chlorobiphenyl utilizer, Arthrobacter sp. strain B1Barc (HICKEY et al., 1992). Strain UCR2 exhibited ability to mineralize 2-chloro- and 2,5-dichlorobiphenyl. The UCR2 showed higher phenotypic similarity and higher genomic DNA homology to strain JB2. No hybridization was observed when the parental strains were probed against each other. Recombinant Pseudomonas sp. strain CB15 was obtained by multi-chemostat mating by mixing Pseudomonas sp. strain HF1 (3-chlorobenzoate utilizer) and Acinetobacter sp. strain P6 (biphenyl utilizer) on ceramic beads (ADAMS et al., 1992). 3-chlorobiphenyl, 3-chlorobenzoate, and biphenyl could be utilized as growth substrates by strain CB15. Results of DNA hybridization suggested strain CB15 was closely related to parent strain HF1. An in vivo recombinant plasmid, pDD530, containing the bphABCD genes from Burkholderia sp. strain LB400 was isolated from P. putida KT2442. The bph genes on pDD530 447 were further cloned into a pUT transposon vector to yield plasmid pDDPCB. Plasmid pDDPCB was mobilized into Pseudomonas sp. strain B13, and its genetically engineered derivative B13FR1 via conjugation. A transconjugant, Pseudomonas sp. strain B13FR1: :bph, with a chromosomally integrated bph gene removed approximately 90% of added 4chlorobiphenyl after 5 d in lake sediment microcosms (DOWLING et al., 1993). The plasmid, pDDPCB, was later transferred into a rhizosphere pseudomonad, Pseudomonas fluorescens F113, by conjugation to generate a genetically modified strain F113pcb (BRAZIL et al., 1995). The bph operon was chromosomally located and was stable in non-sterile soil microcosms for 25 d after inoculated onto sugar beet seeds. Strain F113pcb gained the ability to utilize biphenyl as a sole carbon source. 2.2.2.3 Pseudomonas cepacia JHR22 HAVEL and REINEKE (1991) reported a hybrid bacterium, Pseudomonas cepacia JHR2, was constructed by filter mating with a biphenyl-grown donor and a chlorobenzoate-grown recipient (P. cepacia JH230). Strain 230 is a hybrid strain that originated from the transfer of chlorocatechol degradative genes from strain B13 into Pseudomonas sp. WR401 (HARTMANN et al., 1989). Strain JHR2 was able to grow on 3- and 4-chlorobiphenyl. A 2-chlorobiphenyl degrader, JHR22, was obtained by growing JHR2 with 4-chlorobenzoate (1 mM) in the presence of 2-chlorobiphenyl (3 mM). The new hybrid strain, P. cepacia JHR22, showed capability to utilize 2-chloro-, 3-chloro-, 4-chloro-, 2,4-dichloro-, and 3,5-dichlorobiphenyl as sole source of carbon and energy. The strain JHR22 also exhibited ability to degrade all monochlorobiphenyls in Aroclor 1221 when tested with soils (HAVEL and REINEKE, 1993). 2.2.2.4 Pseudomonas acidovorans M3GY Recombinant bacterium, strain M3GY, was produced within a multi-chemostat culture by 448 21 Genetically Engineered Microorganisms and Bioremediation mixing P. acidovorans CC1 (a chloroacetate and biphenyl degrader) and Pseudomonas sp. strain CB15 (a hybrid strain) on ceramic beads that were coated with 3,3b-dichlorobiphenyl (MCCULLAR et al., 1994). Strain M3GY expressed catabolic ability to utilize 3,4b-dichlorobiphenyl as a sole carbon and energy source. The recipient strain was determined by phenotypic similarity and genetic homology between strains M3GY and CC1. In a recent study, 2,3b-dichloro- and 2,4bdichlorobiphenyl were mineralized by a twomember consortium, Burkholderia (formerly Pseudomonas) sp. strain LB400 and P. putida mt-2a (POTRAWFKE et al., 1998). The strain mt-2a was obtained by intergeneric mating with the chlorocatechol genes transferred from LB400 to mt-2 (TOL). Strain mt-2a exhibited the ability to grow on 3-chloro- and 4-chlorobenzoate. 2.2.2.5 Pseudomonas putida (pDA261) Different biphenyl degradative genes (bphABCD) from Comamonas testosteroni B-356 were subcloned into P. putida and E. coli separately, and their degradative capabilities were examined (AHMAD et al., 1992). The hphC and bphD genes were expressed well in both cells, however, bphA and bphB in E. coli were poorly expressed even though located downstream of the tac promoter. A review of the strain B-356 was published earlier (SYLVESTRE, 1995). 2.2.2.6 Comamonas testosteroni VP44(pE43)/VP44(pPC3) Introduction of dehalogenase genes into the biphenyl degrading strain C. testosteroni VP44 resulted in complete mineralization of o- and p-substituted monochlorobiphenyls (HRYWNA et al., 1999). Plasmid pE43, containing the ohbAB gene, encodes iron sulfur protein (ISPOHB) of the o-halobenzoate-1,2-dioxygenase in P. aerugrinosa 142. This plasmid, pE43, was transformed into strain VP44. The resulting recombinant strain VP44(pE43) mineralized 2-chlorobiphenyl and 2-chloroben- zoate. The other recombinant strain VP44(pPC3) contained the fcbABC genes (from Arthrobacter globiformis KZT1) that catalyze hydrolytic p-dechlorination of 4-chlorobenzoate. This strain demonstrated the capability to grow on 4-chlorobiphenyl and 4-chlorobenzoate. 2.2.2.7 Escherichia coli JM109 (pSHF1003)/(pSHF1007) Hybrid Biphenyl Dioxygenase The bphA1 genes of biphenyl dioxygenase in Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 were recombined randomly by DNA shuffling (KUMAMARU et al., 1998). The shuffled bphA1 genes were cloned into pJHF18 (MulI), upstream of bphA2A3A4BC, to replace the disrupted bphA1 genes in the pJHF18 (HIROSE et al., 1994). The resulting plasmids containing bphA1 (shuffled) and bphA2A3A4BC (KF707) were transformed into E. coli JM109 by electroporation. Some chimeric biphenyl dioxygenases in E. coli cells exhibited enhanced degradation of different biphenyl compounds. Biphenyl dioxygenase (pSHF 1003) showed 3 amino acid substitutions in KF707 bphA1 (H255Q, V258I, and D303E), derived from strain LB400, and acquired degradation capabilities not only for PCB, but also for benzene and toluene. Plasmid pSHF1007 showed 4 amino acid substitutions (H255Q, V258I, G268A, and T376N) in KF707 bphA1 gene and increased substrate affinities for some PCB congeners. The above results suggested the substitutions of His with Gln at 255 and of Val with Ile at 258 leading to the differences in substrate specificity and mode of oxygenation between the two enzymes. Site-directed mutagenesis on the LB400 bphA gene was reported previously (ERICKSON and MONDELLO, 1993). 4 amino acids in LB400 were converted to the corresponding KF707 sequence. The modified biphenyl dioxygenase exhibited broader substrate specificity with PCB. The bphB gene, encoding the cis-biphenyl dihydrodiol dehydrogenase from P. putida OU83 was cloned into the pQE31 vector and 2 Degradative (Catabolic) Genes expressed in IPTG-induced E. coli recombinant cells (KHAN et al., 1997). 2.2.2.8 Pseudomonas putida IPL5 (Field Application Vectors) Field application vectors (FAV) are a combination of a selective substrate that can be used easily by host (not indigenous) microorganisms, and a cloning vector to provide a temporary niche for the host bacterium in harsh environments (LAJOIE et al., 1992). FAV can stabilize and enhance the expression of foreign genes in contaminated sites. The chromosomally encoded PCB catabolic genes (bphABC ) from Pseudomonas sp. strain ENV307 were cloned into broad host range plasmid pRK293. The resulting plasmid was transferred to the host Sphingomonas paucimobilis 1IGP that utilizes non-ionic surfactant Igepal CO-720 (IGP) as selective substrate. The recombinant strain 1IGP4(pCL3) exhibited ability to degrade individual PCB congeners in Aroclor 1242 without biphenyl as an inducer (LAJOIE et al., 1993). The transposon encoded PCB degradative genes (bphABC ) were more stable than plasmid encoded after insertion into the surfactant utilizing strain, Pseudomonas putida IPL5 (LAJOIE et al., 1994). 2.2.3 Trichloroethylene (TCE) 2.2.3.1 Escherichia coli HB101/pMY402 and FM5/pKY287 A XhoI fragment (4.7 kb), containing toluene monooxygenase (TMO) genes from Pseudomonas mendocina KR-1, was subcloned into a broad host range vector, pMMB66EH, which contains an E. coli tac promoter to yield plasmid pMY402. The same fragment was also inserted into another E. coli expression vector, pCFM1146, containing the temperature inducible E. coli phage PL promoter, to yield plasmid pKY287. Recombinant E. coli strains, E. coli HB101/pMY402 and FM5/pKY287, were able to oxidize both toluene and TCE (WINTER et al., 1989). 449 Recently, TMO genes in pMMB503EH, a broad host range vector, were introduced into P. putida KT2440 by electroporation (WARD et al., 1998). 2.2.3.2 Pseudomonas pseudoalcaligenes KF707-D2 and Pseudomonas putida KF715-D5 HIROSE et al. (1994) reported a hybrid dioxygenase gene cluster between the tod and the bph operons in E. coli JM109. Plasmid pJHF101, containing todC1::bphA2orf3A3A4, was constructed by deleting a 1.3 kb PpuMI fragment from bphB and bphC genes from pJHF10 (FURUKAWA et al., 1993). The vector pUC118 was used for the construction of pJHF10. This recombinant strain, E. coli (pJHF101), degraded TCE at an initial rate of 1.8 g mLP1 hP1 which was much faster than E. coli cells carrying the toluene dioxygenase genes (todC1C2BA) or the biphenyl dioxygenase genes (bphA1A2A3A4, FURUKAWA et al., 1994). The hybrid gene cluster, todC1: :phA2orf3A3A4, was further inserted into the chromosomal bph operons by SUYAMA et al. (1996). The plasmid pJHF101 was subcloned into suicide vector, pSUPB30, to yield plasmid pASF101. The pASF101 was inserted into E. coli S17-1 (chromosomally integrated RP4-2Tc::Mu-Km::Tn7) cells via transformation, and the resulting strain was used as a donor in the mating with the recipient biphenyl utilizing P. pseudoalcaligenes KF707 and P. putida KF715. The resulting double crossover strains, KF707D2 and KF715-D5, maintained todC1 in LB broth under no selective pressure. Both single and double crossover strains carrying todC1 on chromosomes of KF707 and KF715 degraded TCE efficiently, and grew on toluene and benzene. 2.2.3.3 Pseudomonas sp. strain JR1A::ipb Hybrid Strains A multi-component isopropylbenzene (IPB) dioxygenase from strain Pseudomonas sp. JR1, growing on IPB exhibited the capability to co-oxidize TCE (PFLUGMACHER et al., 450 21 Genetically Engineered Microorganisms and Bioremediation 1996). Organization of ipb genes encodes IPB dioxygenase (ipbA1A2A3A4), 2,3-dihydro2,3-dihyroxy-IPB dehydrogenase (ipbB), and 3-IPB-2,3-dioxygenase (ipbC ). Recently, a recombinant, Pseudomonas sp. JR1A, exhibited constitutive TCE oxidation activity was reported (BERENDES et al., 1998). A transposon vector pC8 (pUT/miniTn5Km::ipbABC ) was constructed, then transformed into E. coli S17.1( pir).The pC8 vector in E. coli was conjugatively transferred into different recipients. The ipb genes were detected in following transconjugants: strain JR1A (spontaneous IPB negative mutants of strain JR1), P. putida 548, and Pseudomonas sp. strain CBS-3. Two of these hybrid strains, JR1A::ipb and CBS3::ipb, were stable for more than 120 generations in antibiotic free medium and degraded TCE and IPB constitutively. Further studies showed that strain JRlA::ipb can oxidize TCE without inducer. 2.2.3.4 Escherichia coli JM109(pDTG601) Toluene dioxygenase from P. putida F1 has been reported as an enzyme responsible for degradation of TCE (WACKETT and GIBSON, 1988). The structural genes (todC1C2BA) of toluene dioxygenase were cloned from plasmid pKK223-3 then transformed into strain JM109. The resulting strain E. coli JM109 (pDTG601) degraded TCE at a slower initial rate when compared to P. putida F39/D, a mutant strain of strain F1 that does not contain cis-toluene dihydrodiol dehydrogenase (ZYLSTRA et al., 1989). 2.2.3.5 Pseudomonas putida G786(pHG-2) A recombinant P. putida G786(pHG-2), containing two multicomponent enzyme systems, cytochrome P450cam, and toluene dioxygenase, capable of degrading pentachloroethane and TCE was reported by WACKETT et al. (1994, 1995). The toluene dioxygenase genes (todC1C2BA) and tac promoter (Ptac) were cloned into plasmid pKT230 to form plasmid pHG-1. The lacIQ gene cassette from plasmid pMMB24 was cloned into plasmid pHG-1 to yield pHG-2, then transformed into E. coli DH5. Plasmid pRK2073 was used as a helper to transconjugate plasmid pHG-2 into P. putida G786 containing cytochrome P450cam genes on the CAM plasmid. The new hybrid strain, G786(pHG-2), transformed pentachloroethane to TCE by the cytochrome P450cam, then TCE was further mineralized by the toluene dioxygenase. 2.2.3.6 Pseudomonas putida F1/pSMMO20 Slow growth rate, copper repression of the smmo locus, and strong competition between TCE and methane for soluble methane monooxygenase (sMMO) are restrictions to the use of strain OB3b to degrade TCE. To overcome these impediments, the complete smmo cluster of Methylosinus trichosporium OB3b was cloned into a wide host range vector, pMMB277, to form pSMMO20 (JAHNG and WOOD, 1994). pSMMO20 is a 14.7 kb plasmid and contains the IPTG-inducible tac promoter upstream of the smmo cluster. Plasmid pSMMO20 was transformed into P. putida F1, P. putida KT2440, P. mendocina KR1, B. cepacia G4, and B. cepacia G4 PR1 through electroporation. P. putida F1/pSMMO20 was the only bacterium that was able to degrade TCE. However, inconsistent sMMO activity was a major drawback in the recombinant strain. Furthermore, the sMMO protein bands were detected on SDS-PAGE gels only when TPTG was present to induce the tac promoter. The constructed strain, P. putida F1/pSMMO20, showed a lower TCE degradation rate and a much higher growth rate than strain OB3b. This recombinant strain also demonstrated the capability to mineralize chloroform. 2.2.3.7 Burkholderia cepacia G4 5223-PR1 An aerobic bacterium, Burkholderia cepacia G4, cometabolically degrades TCE to CO2 and non-volatile products by toluene o-monooxygenase (SHIELDS et al., 1989). Tn5 mutants of strain G4 were constructed via triparental 2 Degradative (Catabolic) Genes mating among G4, E. coli C600(pRZ102), and E. coli HB101(pRK2013). The resulting Tn5 mutants could not express toluene o-monooxygenase activity, and were unable to degrade TCE, toluene, and phenol. A mutant spontaneously reverted to express toluene omonooxygenase constitutively and was designated as G4 5223-PR1. This strain metabolized TCE and m-trifluoromethyl phenol without induction (SHIELDS and REAGIN, 1992). 2.2.3.8 Ralstonia eutropha AEK301/pYK3021 Strain AEK301, a Tn5 mutant that lost phenol hydroxylase activity, was derived from strain JMP134. Plasmid pYK3021 encoding phenol hydroxylase was subcloned into pMMB67EH (vector from pJP4) and exhibited TCE degradation capability without phenol induction. Triparental mating was used to transfer plasmid pYK3021 from E. coli to R. eutropha AEK301 with the helper plasmid pRK2013. The recombinant strain AEK301/ pYK3021 expressed phenol hydroxylase activity constitutively and degraded TCE efficiently (KIM et al., 1996). The removal rate of TCE by the strain could be influenced by growth substrates (AYOUBI and HARKER, 1998). 2.2.4 2,4-Dichlorophenoxyacetic Acid (2,4-D) 2.2.4.1 Pseudomonas putida PPO300(pRO101) and PPO301(pRO103) Plasmid pJP4 encodes the degradation genes of 2,4-D and 3-chlorobenzoate in R. eutropha JMP134 (DON and PEMBERTON, 1985). GEM containing catabolic genes for the degradation of 2,4-D to 2-chloromaleylacetate were reported by HARKER et al. (1989). Plasmid pRO101 (pJP4::Tn1721) was constructed by insertion of transposon Tn1721 into pJP4, then further transferred by conjugation to different Pseudomonas strains. One of the resulting strains, P. putida PPO300(pRO101), 451 with chromosomally encoded phenol hydroxylase, also degrades phenoxyacetate in the presence of an inducer (2,4-D or 3-chlorobenzoate) of the 2,4-D pathway. A mutant plasmid, pRO103, derived from pRO101 by the spontaneous deletion of negative regulatory gene (tfdR) was isolated from strain PPO300(pRO101). The deletion of tfdR resulted in the constitutive expression of the tfdA gene (encodes 2,4-D monooxygenase), and enabled mutant strain PPO300(pRO103) to grow on phenoxyacetate as the sole carbon source. Strain PPO301, derived from PPO300 (ATCC 17514) and resistant to nalidixic acid, was used to harbor plasmid pRO103 to yield a constitutive 2,4-D degrader, PPO301(pRO103), that was used in agricultural soil studies (SHORT et al., 1990). A dual substrate (2,4-D and succinate) chemostat study with P. cepacia DBO1(pRO101) indicated that succinate can act as repressor of the 2,4-D catabolic pathway. But, this repression can be relieved with appropriate adjustments, such as lower 2,4-dichlorophenol accumulation or reduced succinate concentration (~2,4-D) in the media (DAUGHERTY and KAREL, 1994). 2.2.4.2 Pseudomonas cepacia RHJ1 Burkholderia cepacia (formerly P. cepacia) RH1 is a recombinant strain created by performing conjugation between R. eutropha JMP134 (2,4-D degrader) and B. cepacia AC1100 (2,4,5-trichlorophenoxyacetate degrader). The self-transmissible 2,4-D degradative plasmid, pJP4, in A. eutrophus JMP134 was transferred into a 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) degrader, B. cepacia AC1100. This new strain, designated RHJ1, was capable of degrading mixtures of 2,4-D and 2,4,5-T simultaneously (HAUGLAND et al., 1990).

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