1. FEATURES
References 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 using
10 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 from
11 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, 68–73
Environmental biotechnology
Lawrence P. Wackett
There 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. Plants
can be more readily engineered for resistances that enhance yield or produce new products whereas microorganisms are
exploited for their catalytic diversity and ease of genetic engineering.
harmaceutical biotechnology has matured; several dealing with environmental protection, biocatalysis
P 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 protection
margin. 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 make
products and services deal with the agricultural, spe- energy generation cleaner. The burning of fossil fuels
ciality 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 sulfur
the overall value of the market for food products is dioxide. This can be prevented by removing the sulfur
trillions of US dollars. This provides more than enough from fossil fuels. However, it is particularly difficult to
incentive to develop new biotechnological products. remove the sulfur ‘tied up’ in organic compounds while
This 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 removing
L.P. Wackett (wackett@biosci.cbs.umn.edu) is at the Department sulfur from coal and oil by microorganisms containing
of Biochemistry, Molecular Biology and Biophysics and BioProcess enzymes that selectively cleave the carbon-to-sulfur
Technology Institute, University of Minnesota, MN 55108, USA. bonds in the fuel. Recently, the use of thermophilic
TIBTECH 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 and
synthetic intermediates contain halogen substituents.
Halogens are not typically thought of as being biologi-
cally relevant functional groups, but they are found in
naturally occurring antibiotics and other natural prod-
ucts. The biosynthesis of these compounds has largely
been attributed to enzymes known as haloperoxidases,
but many natural halogenated compounds could not be
explained as coming from a haloperoxidase-type reaction
mechanism. Recently, a new mechanism for biohalo-
genation has been proposed and this also explains the
occurrence of novel fluorinated compounds that derive
from biological systems8.
When important new biochemical activities are
detected, it is often of interest to modify a natural
enzyme to achieve a desired substrate specificity, heat
stability or higher activity. Rational protein design is
still 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 of
combinatorial methods to modify enzymes. One exam-
ple of this approach generates genetic diversity by a pro-
cess known as DNA shuffling. DNA shuffling produces
many enzyme variants that can be screened or put
through a biological selection procedure to obtain one
or more desired traits. Recently, it has been found to Figure 2
be powerful to start the DNA-shuffling process with Plants as potential expression systems for the production of desirable
natural genetic diversity9. The idea is to use several proteins (illustration courtesy of Photodisc).
natural variant genes because these divergent sequences
have already been selected for their ability to produce and plant biology, will be spurred on by widespread
proteins 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.
References
Plant 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, 3164–3169
systems for the production of desirable gene products, 2 McTavish, H. (1998) Hydrogen evolution by direct electron transfer
such as natural plant products and foreign proteins. It from photosystem I to hydrogenases. J. Biochem. 123, 644–649
may 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, 137–141
biomass production and product generation but in 4 Laville, C.B. et al. (1998) Characterization of the hcnABC gene cluster
order to become more widespread, this will require a encoding hydrogen cyanide synthase and anaerobic regulation by
greater knowledge of biosynthesis in plant systems. The ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens
most abundant natural polymer of plants is cellulose and CHAO. J. Bacteriol. 180, 3187–3196
recently, Arioli et al.10 reported new insights into the 5 Lange, C.C. et al. (1998) Engineering a recombinant Deinococcus
biosynthesis of cellulose by Arabidopsis, an important radiodurans for organopollutant degradation in radioactive mixed
plant-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 aromatics
are 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-hydroxylating
properties and biodegradability features, both of which dioxygenase – naphthalene 1,2-dioxygenase. Structure 6, 571–586
can be manipulated by the length of the alkanoate chain 8 Hohaus, K. et al. (1997) NADH-dependent halogenases are
used in biosynthesis. There have been several advances more likely to be involved in halometabolite biosynthesis than
in the production of medium-chain-length polyhy- haloperoxidases. Angew. Chem., Int. Ed. Engl. 36, 2012–2013
droxyalkanoates (PHAs) in plants11,12. These studies 9 Crameri, A. et al. (1998) DNA shuffling of genes from diverse species
have been augmented with investigations into the accelerates directed evolution. Nature 391, 288–291
reaction mechanism of PHA synthase13. Site-directed 10 Arioli, T. et al. (1998) Molecular analysis of cellulose biosynthesis in
mutagenesis and radiolabel-trapping experiments sug- Arabidopsis. Science 30, 717–720
gested the role of at least one of the enzyme’s cysteine 11 Mittendorf, V. et al. (1998) Synthesis of medium-chain-length
residues 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, 540–548
by 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, 826–837
TIBTECH JANUARY 2000 (Vol. 18) 21