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Environmental biotechnology


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  • 1. FEATURESReferences cells from embryonal and neonatal rat livers. Dig. Dis. Sci. 44, 1 Langer, R. and Vacanti, J.P. (1993) Tissue engineering. Science 260, 364–371 920–926 13 Overturf, K. et al. (1997) Serial transplantation reveals the stem-cell- 2 Platt, J.L. (1996) The immunological barriers to xenotransplantation. like regenerative potential of adult mouse hepatocytes. Am. J. Pathol. Crit. Rev. Immunol. 16, 331–358 151, 1273–1280 3 Thomson, J.A. et al. (1998) Embryonic stem cell lines derived from 14 Alison, M. et al. (1998) Wound healing in the liver with particular human blastocysts. Science 282, 1145–1147 reference to stem cells. Philos. Trans. R. Soc. London Ser. B Biol. Sci. 4 Solter, D. and Gearhart, J. (1999) Putting stem cells to work. Science 353, 677–684 283, 1468–1470 15 Cornelius, J.G. et al. (1997) In vitro-generation of islets in long-term 5 Langer, R. and Vacanti, J.P. (1999) Tissue engineering: the challenges cultures of pluripotent stem cells from adult mouse pancreas. Horm. ahead. Sci. Am. 280, 86–89 Metab. Res. 29, 271–277 6 McKay, R. (1997) Stem cells in the central nervous system. Science 16 Bruder, S.P. et al. (1997) Growth kinetics, self-renewal, and the 276, 66–71 osteogenic potential of purified human mesenchymal stem cells 7 Bruder, S.P. et al. (1998) Bone regeneration by implantation of during extensive subcultivation and following cryopreservation. purified, culture-expanded human mesenchymal stem cells. J. Orthop. J. Cell Biochem. 64, 278–294 Res. 16, 155–162 17 Kallos, M.S. and Behie, L.A. (1999) Inoculation and growth condi- 8 Reddi, A.H. (1998) Role of morphogenic proteins in skeletal tissue tions for high-cell-density expansion of mammalian neural stem cells engineering and regeneration. Nat. Biotechnol. 16, 247–252 in suspension bioreactors. Biotechnol. Bioeng. 63, 473–483 9 Williams, J.T. et al. (1999) Cells isolated from adult human skeletal 18 Awad, H.A. et al. (1999) Autologous mesenchymal stem cell- muscle capable of differentiating into multiple mesodermal phenotypes. mediated repair of tendon. Tissue Eng. 5, 267–277 Am. Surg. 65, 22–26 19 Grande, D.A. et al. (1995) Repair of articular cartilage defects using10 Nevo, Z. et al. (1998) The manipulated mesenchymal stem cells in mesenchymal stem cells. Tissue Eng. 1, 345–353 regenerated skeletal tissues. Cell Transplant. 7, 63–70 20 Klug, M.G. et al. (1996) Genetically selected cardiomyocytes from11 Flax, J.D. et al. (1998) Engraftable human neural stem cells respond differentiating embryonic stem cells form stable intercardiac grafts. to developmental cues, replace neurons, and express foreign genes. J. Clin. Invest. 98, 1–9 Nat. Biotechnol. 16, 1033–1039 21 Pedersen, R.A. (1999) Embryonic stem cells for medicine. Sci. Am.12 Brill, S. et al. (1999) Expansion conditions for early hepatic progenitor 280, 68–73Environmental biotechnologyLawrence P. WackettThere is an increasing interest in environmental biotechnology owing to a worldwide need to feed the world’s growing popu-lation and to maintain clean soil, air and water. The major technological developments are in plant and microbial biology. Plantscan be more readily engineered for resistances that enhance yield or produce new products whereas microorganisms areexploited for their catalytic diversity and ease of genetic engineering. harmaceutical biotechnology has matured; several dealing with environmental protection, biocatalysisP companies manufacturing largely biomedical products are today the pillars of biotechnology.This industry is based on products that are expensive and biomaterials for green chemistry, and agricultural biotechnology.and slow to develop, but which bring in a high profit Environmental protectionmargin. Fossil fuels Against this backdrop comes the next wave of Biotechnology can have an impact on the environ-biotechnology, with the focus on products of lesser unit ment by providing cleaner large-scale processes; per-value, but of massive volume. These biotechnology haps no greater benefit could accrue than to makeproducts and services deal with the agricultural, spe- energy generation cleaner. The burning of fossil fuelsciality chemical and bioremediation markets. Although (Fig. 1) causes pollution in several ways; for example,a genetically engineered vegetable can sell for pennies, the most problematic being the liberation of sulfurthe overall value of the market for food products is dioxide. This can be prevented by removing the sulfurtrillions of US dollars. This provides more than enough from fossil fuels. However, it is particularly difficult toincentive to develop new biotechnological products. remove the sulfur ‘tied up’ in organic compounds whileThis review will focus on these new developments, preserving a high thermal value of the fuel. In this con- text, there have been investigations aimed at removingL.P. Wackett ( is at the Department sulfur from coal and oil by microorganisms containingof Biochemistry, Molecular Biology and Biophysics and BioProcess enzymes that selectively cleave the carbon-to-sulfurTechnology Institute, University of Minnesota, MN 55108, USA. bonds in the fuel. Recently, the use of thermophilicTIBTECH JANUARY 2000 (Vol. 18) 0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01399-2 19
  • 2. FEATURES Figure 1 The burning of fossil fuels is a major source of pollution (illustration courtesy of Photodisc). bacteria that are capable of desulfurizing model com- the black root rot disease of tomato, which is caused by pounds, such as those found in fossil fuels1, were inves- the fungus Thielaviopsis basicola4. Hydrogen cyanide was tigated. It is thought that any large-scale process must shown to be biosynthesized from glycine by P fluorescens . ideally be carried out at temperatures approaching the expressing the hcnABC gene cluster. Mutants that failed upper range tolerated by microorganisms. to fully express the hcnABC genes were partially impaired in their ability to suppress the growth of Alternative energy T. basicola. This could potentially form the basis of a Of even greater potential application would be a new biological control mechanism for hard-to-manage biotechnological process that would eliminate the need plant fungal pathogens. for current high-volume fossil-fuel usage by providing an alternative energy source. For example, cars that use Hazardous wastes hydrogen fuel are currently under development. One long-standing environmental use of microor- Hydrogen is an ideal non-polluting energy source as it ganisms is for the remediation of hazardous wastes, and burns to produce water as the only product. However, new approaches are emerging to treat the most drastic for widespread acceptance of this new technology, it mixed waste problem. One of the legacies of the Cold will be necessary to generate hydrogen cheaply, for War remains: the nuclear-weapons production facilities example, using the energy freely available from sun- and their decades of spent wastes. These contain a light. One recent study sought to link the photosystem mixture of organic wastes, heavy metals and high- of bacteria directly to a reversible hydrogenase to trans- energy radionuclides. In the USA alone, the Department form the energy from sunlight into biogenic hydro- of Energy has estimated the cost of cleaning up these gen2. The critical issue here is the level of efficiency in sites to be as high as US$250 billion. Innovative tech- capturing photosynthetic-electron flux; it must approach niques are sought, but it is appreciated that the vast the chemical-energy efficiency of natural photosyn- majority of microorganisms would not survive the radi- thetic organisms to make the process economically ation fluxes present at these sites. In this context, an feasible. This has not yet been attained, but the goal is extremely radiation-resistant bacterium, Deinococcus well worth greater effort. radiodurans, is being genetically engineered with biodegradation genes to render it suitable for the Chemical pesticides treatment of mixed wastes5. The broad-specificity Another area of environmental protection is the Pseudomonas enzyme, toluene dioxygenase, has been application of biological controls to supplant the use of cloned, expressed and shown to be active in D. radio- chemical pesticides. There are commercial products durans, even in the presence of high fluxes of ionizing that are currently on the market, for example, Bacillus radiation. thuringiensis has been used to selectively control certain insect pests. In this case, the whole organism is mar- Novel biocatalysis and biomaterials keted. Another biotechnology market is in transgenic The hydrocarbon-dihydroxylating dioxygenases of plants that biosynthesize the B. thuringiensis insecticidal the type cloned into D. radiodurans have been studied protein in situ. This has proven to be highly effective, for 20 years and interest is increasing to exploit the for example, against the cotton bollworm3. enzyme-generated chiral centers for use in synthesiz- The killing action of B. thuringiensis parasporal pro- ing complex chiral molecules6. Only recently, however, tein against insects has been known for decades. More has a model of the active site been available, based on recently, it has been discovered that certain Pseudomonas the X-ray structure derived for naphthalene dioxygen- strains enzymatically generate hydrogen cyanide and ase7. This should spur interest in protein and metabolic this trait is linked to the organism’s ability to suppress engineering using this broad class of dioxygenases.20 TIBTECH JANUARY 2000 (Vol. 18)
  • 3. FEATURES Many important commercial organic chemicals andsynthetic intermediates contain halogen substituents.Halogens are not typically thought of as being biologi-cally relevant functional groups, but they are found innaturally occurring antibiotics and other natural prod-ucts. The biosynthesis of these compounds has largelybeen attributed to enzymes known as haloperoxidases,but many natural halogenated compounds could not beexplained as coming from a haloperoxidase-type reactionmechanism. Recently, a new mechanism for biohalo-genation has been proposed and this also explains theoccurrence of novel fluorinated compounds that derivefrom biological systems8. When important new biochemical activities aredetected, it is often of interest to modify a naturalenzyme to achieve a desired substrate specificity, heatstability or higher activity. Rational protein design isstill a very difficult task if one wishes to obtain an‘improved’ enzyme. In this context, biologists are tak-ing a cue from the chemists and expanding the use ofcombinatorial methods to modify enzymes. One exam-ple of this approach generates genetic diversity by a pro-cess known as DNA shuffling. DNA shuffling producesmany enzyme variants that can be screened or putthrough a biological selection procedure to obtain oneor more desired traits. Recently, it has been found to Figure 2be powerful to start the DNA-shuffling process with Plants as potential expression systems for the production of desirablenatural genetic diversity9. The idea is to use several proteins (illustration courtesy of Photodisc).natural variant genes because these divergent sequenceshave already been selected for their ability to produce and plant biology, will be spurred on by widespreadproteins that fold correctly and have reasonable enzyme genome sequencing. The discovery of new gene prod-activity, thus enhancing one’s overall success in obtaining ucts will continue to lead to new commercial products.the improved enzyme. ReferencesPlant biotechnology 1 Konishi, J.Y. et al. (1997) Thermophilic carbon-sulfur-bond- Plants (Fig. 2) are potentially marvelous expression targeted biodesulfurization. Appl. Environ. Microbiol. 63, 3164–3169systems for the production of desirable gene products, 2 McTavish, H. (1998) Hydrogen evolution by direct electron transfersuch as natural plant products and foreign proteins. It from photosystem I to hydrogenases. J. Biochem. 123, 644–649may be possible to use the energy of sunlight to drive 3 Estruch, J.J. et al. (1997) Transgenic plants: an emerging approach to pest control. Nat. Biotechnol. 15, 137–141biomass production and product generation but in 4 Laville, C.B. et al. (1998) Characterization of the hcnABC gene clusterorder to become more widespread, this will require a encoding hydrogen cyanide synthase and anaerobic regulation bygreater knowledge of biosynthesis in plant systems. The ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescensmost abundant natural polymer of plants is cellulose and CHAO. J. Bacteriol. 180, 3187–3196recently, Arioli et al.10 reported new insights into the 5 Lange, C.C. et al. (1998) Engineering a recombinant Deinococcusbiosynthesis of cellulose by Arabidopsis, an important radiodurans for organopollutant degradation in radioactive mixedplant-model system. waste environments. Nat. Biotechnol. 16, 929–933 Another polymer class, not naturally found in plants, 6 Hudlicky, T. et al. (1999) Enzymatic dihydroxylation of aromaticsare the polyhydroxyalkanoates. These bacterial poly- in enantiomeric synthesis: expanding asymmetric methodology.mers are of considerable interest because of their unique Aldrichimica Acta 32, 35–62 7 Kauppi, B. et al. (1998) Structure of an aromatic-ring-hydroxylatingproperties and biodegradability features, both of which dioxygenase – naphthalene 1,2-dioxygenase. Structure 6, 571–586can be manipulated by the length of the alkanoate chain 8 Hohaus, K. et al. (1997) NADH-dependent halogenases areused in biosynthesis. There have been several advances more likely to be involved in halometabolite biosynthesis thanin the production of medium-chain-length polyhy- haloperoxidases. Angew. Chem., Int. Ed. Engl. 36, 2012–2013droxyalkanoates (PHAs) in plants11,12. These studies 9 Crameri, A. et al. (1998) DNA shuffling of genes from diverse specieshave been augmented with investigations into the accelerates directed evolution. Nature 391, 288–291reaction mechanism of PHA synthase13. Site-directed 10 Arioli, T. et al. (1998) Molecular analysis of cellulose biosynthesis inmutagenesis and radiolabel-trapping experiments sug- Arabidopsis. Science 30, 717–720gested the role of at least one of the enzyme’s cysteine 11 Mittendorf, V. et al. (1998) Synthesis of medium-chain-lengthresidues in covalent attachment of the growing PHA polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid ␤-oxidation. Proc. Natl. Acad. Sci. U. S. A.chain. 95, 13397–13402 12 Klinke, S. et al. (1999) Production of medium-chain-length poly(3-Conclusions hydroxyalkanoates) from gluconate by recombinant Escherichia coli. Environmental biotechnology is expanding, driven Appl. Environ. Microbiol. 65, 540–548by the needs of society and emerging developments in 13 Muh, U. et al. (1999) PHA synthase from Chromatium vinosum:research. The entire enterprise, predicated on microbial cysteine 149 is involved in covalent catalysis. Biochemistry 38, 826–837TIBTECH JANUARY 2000 (Vol. 18) 21