FEATURE
Genetic Engineering: The Frontier off Bioremediation New molecular tools and an improved understanding of biodegradative processes are slowly increasing prospects for successful technology deployment. P E T E R C. K. LAU A N D V I C T O R DE
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nterest is rapidly growing for using bioremediation methods to destroy contaminants at polluted sites. However, despite the enthusiasm of would-be practitioners and the newfound knowledge of the science underlying these apparently promising cleanup technologies, much remains to be learned before they can be more widely deployed. There are many things that we do not know about microbial processes involved in the natural attenuation of contaminant compounds such as petroleum hydrocarbons, chlorinated solvents, and explosives, said Perry McCarthy, a professor of civil engineering at Stanford University and the director of the Western Region Hazardous Substance Research Center in Stanford, Calif. For example, at the contaminated St. Joseph site in Michigan, studies indicated significant transformation of trichloroethylene (TCE) to ethene, but the disappearance of some process intermediates WHS difficult to understand, he explained. "This may be a result of not knowing the [site] hydrogeology well enough, or it may be [that] there are some microbiological processes ongoing that we have not yet discovered," he said. Regardless of what bioremediation method is used, the fundamental component of these technologies, which use living (micro)organisms, is the suite of detoxifying enzymes contained within them. The enzymes carry out the degradative processes that occur during contaminant remediation. For bioremediation to succeed, even if genes encoding the right enzymes are present in the microorganism used to perform the remediation process they may be useless unless they are expressed at the right time and under desired circumstances. To be effective for remediation expression conversion of a gene's coded 1 2 4 A • MARCH 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
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information into structures (such as enzymes) present and operating in cells—must occur when the contaminant to be degraded is present and (bio) available in amounts sufficient to justify the metabolic effort required for its degradation. *' Pseudomonas sutida mt-2, , n e ef the first toil microorganisms studied (isolated by professor K. Hosokawa of Japan in the early 1960s) for the catabolism (metabolic breakdown of substances into smaller products) of toluene, xylenes, and toluates, was unable to degrade 4-ethyl benzoate in spite of having all the genes required for the contaminant's metabolism. The [necessary] catabolic enzymes were simply not there," said J. L. Ramos, senior scientist of the National Research Council in Granada, Spain. The root of the problem was gene regulation. Regulatory genes control the transcription rate of other genes and cause expression to occur or stop at a given moment. Transcription is the process by which RNA is formed from DNA on a DNA template which is a molecular "model" for the synthesis of other macromolecules. The transcription process involves RNA polymerase which is the enzyme that catalyzes the synthesis of nucleic acids by linking nucleotides (DNA or RNA subunits of genes) to form polynucleotide chains The regulatory genes encode accessory proteins that switch on and off the expression of biodegradative enzvmes Lack of reSDonsiveness of the biodegradation svstem to the presence of the 4-ethyl benzoate substrate (the material that thp en7vmps were to react withl w a s solved hv m u t a t ine the rpenlatnrv ppne spmipnrp frv/Sl )n m a k e thp PY n PS ion nf t h p rfIs ad ti p pn v m p s rpsnnnsi p tn
4-ethyl benzoate, thereby unlocking its catabolism (i). The development of additional protocols aimed at monitoring and understanding existing processes © 1999 American Chemical Society
performance, bacterial catalysts used for bioremediation should express their catabolic enzymes in response to external, environmental signals present in locations where the bacteria are expected to perform. This behavioral requirement represents a major difference compared with other biotechnological processes in which working conditions can be fixed at the will of the operator (as in a bioreactor). Ideally, the physiological and genetic programming of bacteria expresses the desired phenotype (the observable physical character— genetically and environmentally determined of an organism) under physico-chemical circumstances in which there is little or no control. The main challenge of making Information bottlenecks Because of its presumed cost reduction potential, nat- well-characterized expression sysural attenuation is presently a preferred contami- tems that perform favorably outside nant management strategy. The method can be used the laboratory environment is deterto destroy contaminants and control contaminant mining which systems are functionplume spread, according to the National Research ing in the environment and whether Council (2). However, although extensive monitor- they can be exploited to express the ing and modeling are necessary to document bio- gene(s) of the remediator's choice. degradation, it is already evident mat treatment times Since bacteria cannot be forced to do something incompatible with their energetic balcan be very long—on the order of decades (2, 3). Cleanup times can be accelerated, but knowl- ance and their ecological fitness, engineering them edge of baseline cleanup performance—an under- requires considerable understanding of how regustanding of what naturally occurring bacterial strains latory elements like promoters (DNA sites on which can do—must first be obtained before considering RNA polymerase binds prior to transcription) are natwhat improvements can be realized through ge- urally assembled in front of genes and operons to renetic engineering. Civil engineers currently tend to spond adequately to novel chemical compounds (Operons are DNA segments that contain regulaoverlook this consideration. Their efforts are intory stead chiefly aimed at geophysical and chemical charregions jjrifi pj acterizations of polluted sites. Professor Peter Adrilinear sequence of genes They aens of the University of Michigan-Ann Arbor, found in prokarvotes such as bacteria) Environmental and Water Resources Engineering DeThe comparative study of biodegradative-pathway partment, believes attention to bacterial behavior is (sequences of degradative reactions) regulation of typmarginal. At best, some MPN (most probcible numbers) or fatty acid ciricilyses 3xe included in the nat- ical biodegraders, such as Pseudomonas, ,aises puzural attenuation protocol. Beyond this circum- zling questions about how regulatory circuits evolve. stance microbiology is "hesitantly controlled or Analysis of DNA and the protein- sequences of the enzymes that form catabolic pathways reveals that, as a assayed for" he said. This lack of knowledge prevails despite the cur- rule, enzymes that catalyze similar steps within catarent availability of useful biological tools and instru- bolic pathways tend to be highly homologous, even in mentation for molecular analysis and genetic ma- instances where the substrates of the pathway, as a nipulations. The behavior and properties of regulatory whole, are very different. Regulation of expression of catabolic operons, on genes, which impose major limitations on the perthe contrary, may be extremely diverse. The organism formance of any bacterial catalyst used in bioremediating toxic pollutants, are not well understood. Most Pseudomonas putida Fl (PpFl) is capable of deof the factors and mechanisms that control expres- grading toluene (andTCE by cometabolism, which ocsion of catabolic enzymes in bacteria such as Pseudo- curs when, without using process energy to support mimonas remain largely unknown and have not been crobial growth, an organism oxidizes substances). The characterized. Similarly, remediation opportunities organism can also degrade p-cymene (p-isopropyloffered by constructions of genetically modified or- toluene) through a second catabolic pathway that has ganisms (GMOs also known as GEMs, or geneti- its own specificity and regulation. According to oncally engineered microorganisms) have not been suf- going research in Lau's laboratory, this strain also carries genes that encode 3. potential solvent efflux ficiently investigated and cire underexploited Moreover the importance of purified enzymes as bio- pump, a property that may explain the organism's surcatalysts in special applications has not been exten- vival in a potentially toxic solvent environment. Contaminant destruction can be a complex prosively investigated cess. The degradation of toluene by the "tod" (tolThe environment in which remediation measures uene degradation) pathway requires at least 10 are carried out is also important. The adaptation of nat- genes, 7 enzymatic steps, and 2 regulatory proural bacteria to degrade contaminant chemicals par- teins. The p-cymene {cym) degradation pathway reallels their ability to regulate the expression of the gene quires at least 11 enzymatic steps, 15 genes, and 1 and the enzymes suitable to that end (5)) For optimal regulatory protein. The cym pathway is normally "turned off" by a repressor molecule (CymR) by is under way (2, 3), concurrent with efforts aimed at the eventual deployment of improved treatment methods. Classical wait-and-see strategies of how pollutants naturally disappear are beginning to give way to efforts aimed at the rational improvement of underlying microbial degradation processes. A significant motivation is the long amount of time it takes for natural degradation processes to completely remediate a particular site. At a JP-4 jet-fuel site, it was estimated that it would require more than 20 years to completely deplete BTEX (benzene, toluene, ethylbenzene, and xylene isomers) and other contaminants by natural attenuation (4).
Contaminant destruction can be a complex process.
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The mechanics of degradative processes Regulation of gene expression of a degradative pathway for environmental pollutants in bacteria takes several forms. One of the most sophisticated, the two-component signal transduction system, is like a telephone circuit that requires special characteristics to ensure privacy (specificity) and reliability—a misconnection means nonaction.
(a: Single line circuit) A simple two-component system involves two proteins. A "sensor," often located in the cytoplasmic membrane monitors environmetal cues, such as oxygen tension, pH change, or nutrient deprivation. A "response regulator" mediates a cellular response, usually turning "on" a gene. The signalling process is carried out by phosphorylation reactions (addition or removal of a phosphate (P) at specific sites of the designated protein modules. The sensor protein (TodS) that regulates toluene degradation in Pseudomonas putida Fl (6) is a prototype and multifaceted hybrid structure.
(b: Party line circuit) The indicated sequential flow of information is largely hypothetical. Although the precise signaling mechanism is still under investigation, it can be envisaged that any misrelay or undue interference of message within the protein modules will deactivate the degradative pathway. The target DNA sequence to which the response regulator (TodT) binds in order to illicit its action has been located {6).
binding to operators; repressor molecules prevent the operator-controlled transcription of regulatory genes. Operators, which are found in operons, are DNA sequences to which a repressor protein can bind. The protein binding to these DNA sequences can regulate gene functioning. In the presence of the pollutant (the inducing substrate— inducers cause transcription to occur by binding to a repressor), the machinery, which drives contaminant degradation, is "turned on." On the other hand, the tod pathway is regulated by a sophisticated "twocomponent signal transduction" system having a complexity not found in any other existing counterpart two-component regulators (6) (see figure above). Other toluene degraders cire regulcited by dissimilar mccricinisms thcvt in cill esses ensure an or)timal yield of biomass versus the amount of biodegraded pollutant (5) Process complexities can seriously limit remediation success. For bioremediation purposes, coupling degradation with growth of remediating microorganisms is undesirable. The best, fastgrowing, natural degraders may not be efficient in situ biocatalysts of contaminants. Wells and tight spots in aquifers can become clogged with microbial growtii. Under tiiese circumstances, it would be 1 2 6 A •
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undesirable to add phosphate or oxygen to stimulate die growdi of a consortium of bacteria in die site to be remediated. Phosphates and odier added nutrients can be food for one organism but poison for others. Improving process options Most genes can now be cloned easily. Once die genes are known, amplified (bulk replication of a gene widi the result that the number of copies of a DNA fragment is greatly increased), and isolated, they can be recombined with other genes to make hybrids that can either evolve new specificities or result in a broader substrate specificity man die original strains. A notable engineering feat of this nature was accomplished in the laboratory of K. Furukawa (Japan). Furukawa used a DNA-shuffling technique (a novel process that uses recombination to accelerate the rate of gene evolution) to generate a new biphenyl dioxygenase (7). It has an enriched capability to degrade polychlorinated biphenyls (PCBs) and some aromatic hydrocarbons, which are normally poor substrates for die dioxygenase, a key initial biocatalyst for the patiiway. In a related system, amino acid replacements (ranging from one to a few) substantially affected die range of substrates the modified enzyme could tackle (8).
Another promising recent development is the ability to engineer a recombinant Deinococcus sadiodurans, based on the recruitment of the toluene dioxygenase genesfromPpFl, so that the organism can degrade organic solvents in a radioactive, mixed-waste environment (9). Other engineered systems involving toluene dioxygenase genes include the recruitment of a specific cytochrome P450 to process pentachloroethane and the degradation of phenylmercury by combination of specific mercuric operon genes and those of toluene dioxygenase. By using genetic design methods, it is also possible to address a general ecotoxicological problem that arises from the formation of protoanemonin (a broad-spectrum antibiotic) during the degradation of 4-chlorobenzoate by bacterial strains that can grow on benzoate. Studies carried out at the GBF (National Research Centre for Biotechnology) laboratory in Braunschweig, Germany, showed that the poor survival of PCB-metabolizing organisms at contaminated sites could be traced to toxicity resulting from the specific misrouting of the 4-chlorobenzoate. This resulted from partial degradation of pollutants such as 4-chlorobiphenyl and led to the formation of the antibiotic. Similarly, problematic dead-end products are known to form in numerous other degradcition systems These Ccin inhibit the performance of the entire degradation Identifying and removing these molecular bottlenecks is one of the most exciting tasks now being addressed by biotechnologists. A paradigmatic case is the inhibition caused by 3-chlorocatechol (an intermediate in the degradation of many chloroaromatic pollutants) on the enzyme catechol 2,3dioxygenase (C230), which is the key for the biodegradation of a range of aromatic compounds. The generation and expression in vivo of C230 variants that are able to resist inhibition by 3-chlorocatechol allows construction of recombinant strains with a considerably broadened substrate range. Tremendous progress has also been made in the development of bioreporters—live bacteria that can sense pollutants for the purpose of site assessment and characterization studies. This technology is made possible by the availability of cloned genes and some knowledge of regulatory signals (10). An in-depth risk assessment is obligatory before releasing one of these living sensors (naphthalene biosensor) into the environment {11). In some instances, genetic engineering has also solved the critical issue of biomass production versus degradation of the pollutant, by uncoupling expression of degradative enzymes from growth. At Stanford University in California, Matin pioneered the notion of using starvation signals for the expression of biodegradative enzymes. Starvation signals are special regulatory elements (promoters) that are selectively switched on in slowly growing cells under conditions of stress. The stress can be in the form of cold shock, heat shock, starvation for carbon, nitrogen, phosphate, or another nutrient. In this way, it is possible to ensure a minimum of biomass with a maximum of catalytic activity. This approach has been instrumental in the construction of bacterial
strains with a superior capacity to degrade TCE. Since the growth of bacteria in the environment occurs mostly under nutrient conditions unable to support exponential growth, starvation might be considered a universal signal that is potentially useful to express heterologous genes when no other manageable signal is available. Promoters responsive to carbon, nitrogen, iron, and phosphate starvation have been characterized in many gram-negative bacteria. In principle, they are excellent building blocks for expression systems of biodegradative pathways of Pseudomonas, which 3X6 particularly attractive for use in designing heterologous expression systems in the field. Another approach involves using known regulatory proteins (and their cognate promoters) that are responsive to the pollutants to be degraded or their structural analogs. The most extensively studied regulatory proteins are those of the catabolic plasmids TOL and NAH of Pseudomonas putida. These systems become activated when host bacteria encounter various alkyl- and halo-benzoates, alkyl- and halotoluenes, or alkyl- and halo-salicylates in the medium. These expression systems can be induced with very low concentrations of cheap inducers, thus making extensive induction operations feasible, unrealistic for applications in other expression systems it only takes 1 part per million of benzoate to activate the benzoate-responsive regulatory protein XylS for the expression of heterologous genes at reasonable levels These expression systems also have a broad host range and are functional in a variety of genera GEM research initiatives "The growing knowledge [of] catabolic pathways, critical enzymes, and expression systems is providing the basis for the rational genetic design of new and improved proteins and pathways for the development of more performant processes," said Ken Timmis of GBF, Braunschweig, Germany, in reference to GEMs used in bioremediation applications. Timmis recently organized the International Workshop on Innovative Potential and Advanced Biological Systems for Remediation, which was held last March in Hamburg, Germany. Two important topics addressed at this meeting were "enhanced degradation through biostimulation and bioaugmentation" and "circumvention of ecological problems." In his keynote address, Ron Unterman of Envirogen, Lawrenceville, NJ., acknowledged that intrinsic bioremediation is the first choice of remediation technology and said "Genetic engineering represents the future of biological treatment technologies by providing a mechanism for improving the degradative efficiency of microorganisms selected for treating specific contaminants." Practical concerns surround the use of this novel technology, including regulations, public acceptance, and economic issues. One recurrent concern is that engineered organisms not only must perform well in the laboratory, but also in the field under complex environmental conditions, including a heterogeneous habitat, differential adhesion or affinity of a compound, and the ability of organisms to colonize a particular niche. MARCH 1, 1999/ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 1 2 7 A
A recent study illustrating that the use of indigenous bacteria for bioremediation and as hosts for constructing GEMs does not provide any advantage in dynamic, highly competitive environments {12} is relevant to these considerations. The study recommended engineering a site to provide a temporary niche for the introduced organism rather than the reverse. For safety reasons, the precautionary use was recommended of a limited number of wellcharacterized strains that lack pathogenic potential and for which ecological information can be obtained. In an earlier study, GBF researchers dismissed the dogma that laboratory bacteria may be too adapted to the monoculture lifestyle of the lab and are therefore ill-adapted to survive and thus function in natural environments; the Pseudotnoficis sp B13 strain which has been in the laboratory for 20 found to survive for long periods of time in a targetted aquifer
carried out. Environmental gene expression and regulation in such settings represents a research pathway presently being pursued by only a few investigators, among whom, notably, is Gary Sayler at the University of Knoxville, Tenn. Sayler a n d coworkers pioneered the extraction and measurement of messenger RNA (mRNA) from the environment; this allows in situ measurement of biological activities and provides a direct method of monitoring the induction of enzymatic pathways involved in bioremeidiation {14). The group also introduced the concept of "field application vectors," a development of organisms designed to respond to selective agents under field conditions. Besides environmental gene expression and regulation other demanding challenges include intensive research and development of novel inoculation techniques and largescale bioprocess engineering technologies Practical results in these fields forthcoming in the near
Bioprotection, an essential element of genetic engineering approaches, has been investigated {13). The study used a Pseudomonss that was designed to degrade mixtures of chloro- and methylphenols. It was found that the organism also protected the microfauna and fauna of waste treatm e n t plants. This application has relevance to solving ecotoxicological problems associated with certain environmental pollutants. At the University of Umea in Sweden, professor Victoria Shingler is trying to test "if improved degradation via a regulatory mutation results in improved degradation and survival." Her research team is differentially tagging wild-type as well as regulatorymutant pVI150 plasmids and host strains to perform competition and survival experiments in microcosms. The work is in the preliminary stages. "We do not even know if the experimental setup will work since we have such high rates of degradation by native microbes and other technical p r o b lems," said Shingler.
term but microbial biotechnologists are currently making substantial progress Enzyme crystallography is another technological development capability now on the horizon and promises long-term research and development benefits. The most recent accomplishment has been the structure determination of the oxygenase component of naphthalene dioxygenase at David Gibson's laboratory at the University of Iowa in Iowa City. The first result of its kind, this work lays the foundation for others and provides an opportunity to discover how this key group of enzymes really works and allow us to consider the prospect of designing new biocatalysts.
Outreach and outlook Molecular techniques allow construction of recombinant gram-negative and some gram-positive strains with novel phenotypes for various aerobic biotransformations of environmental interest. For bioremediating sites polluted with recalcitrant chemicals likely mixed with heavy metals, recombinant bacteria can be deployed that replace preferential use of naturally occurring microorganisms. Strain characterization, contrary to a "black box" approach in natural attenuation scenarios, and gene elucidation (including structural and regulatory aspects) remain important parameters upon which genetic improvements and developments can be built. An immediate benefit of current efforts is a diversified gene probe database for biomonitoring purposes. Gene probe technology, however, will not address remediation problems entirely it is not a "be-all and end-all" technology. The study of the molecular basis for the regulation and the enzymology of catabolic pathways should be accompanied by an improved understanding of the environments in which applications are 1 2 8 A • MARCH 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
References (1) Ramos, J. L.; Stolz, A.; Reineke, W.; Timmis, K. N. Proc. Natl. Acad. Sci. USA 1986, 83, 8467-8471. (2) National Research Council. Innovations in Groundwater and Soil Cleanup-From Concepts to Commercialization; National Academy Press: Washington, DC, 1997. (3) Renner, R. Environ. Sci. Technol. 1998, 32, 180A-182A. (4) Cho, J. S.; Wilson, J. X; DiGiulio, D. C; Vardy, J. A.. Choi, W Biodegradation 1997, 8, 265-273. (5) de Lorenzo, V; Perez-Martin, J. Mol. Microbiol. 1996,19, 1177-1184. (6) Lau, P C. K.; Wang, Y.; Patel, A.; Labbe, D.; Bergeron, H.; Brousseau, R.; Konishi, Y.; Rawlings, M. Proc. Natl. Acad. Sci. USA 1997, 94, 1453-1458. (7) Kumamaru, T.; Suenaga, H.; Mitsuoka, M.; Watanabe, X; Furukawa, K. Nat. Biotech. 1998, 16, 663-666. (8) Trombly, J. Environ. Sci. Technol. 1995, 29, 560A-564A. (9) Lange, C. C; Wackett, L. R; Minton, K. W.; Daly, M. J. Natt Biotech. 1998, 16, 929-933. (10) Sayler, G. S.; Matrubutham, U.; Menn, F-M.; Johnston, W. H.; Stapleton, Jr. R. D. Bioremediation: Principles and Practice; Sikdar, S. K.; Irvine, R. L., Eds.. Teclhnomic Publlshing Co., Inc.: Lancaster, PA, 1997; Vol. 1, pp. 385-434. (11) Sayre, P; In Biotechnology in the Sustainable Environment, Sayler, G. S.; Sanseverino, J.; Davis, K. L., Eds., Plenum Press: New York, 1997; pp. 269-279. (12) Blumenroth, R; Wagner-Dobler, I. Microbial Ecol. 1998, 35, 279-288. (13) Erb, R. W.; Eichner, C. A.; Wagner-Dobler, I.; Timmis, K. N. Nat. Biotech. 1997, 15, 378-382. (14) Hart, S. Environ. Scii Technol. 1996, 30, 398A-401A. Peter C. K. Lau is a senior research officer at the National Research Council of Canada at the Biotechnology Research Institute in Montreal. Victor de Lorenzo is a senior scientist at the Centro National de Biotecnologia in Madrid, Spain.
