FEATURES

FEATURES
10 Hoogendoorn, B. et al. (1999) Genotyping single nucleotide
polymorphisms by primer extension and high performance
liquid chromatography. Hum. Genet. 104, 8993
11 Lander, E.S. (1999) Array of hope. Nat. Genet. 21, 34
12 Syvanen, A-C. et al. (1993) Identification of individuals by
analysis of bi-allelic DNA markers, using PCR and solid-phase
minisequencing.

Am. J. Hum. Genet. 52, 4659

13 Griffin, T.J. et al. (1999) Direct genetic analysis by matrixassisted laser desorption/ionization mass spectrometry. Proc. Natl.
Acad. Sci. U. S. A. 96, 63016306
14 Gibbs, R.A. et al. (1989) Detection of single DNA base differences
by competitive oligonucleotide priming. Nucleic Acids Res. 17, 2437
2448

Microbial biotechnology
Arnold L. Demain
For thousands of years, microorganisms have been used to supply products such as bread, beer and wine. A second phase
of traditional microbial biotechnology began during World War I and resulted in the development of the acetone-butanol and
glycerol fermentations, followed by processes yielding, for example, citric acid, vitamins and antibiotics. In the early
1970s, traditional industrial microbiology was merged with molecular biology to yield more than 40 biopharmaceutical
products, such as erythropoietin, human growth hormone and interferons. Today, microbiology is a major participant in global
industry, especially in the pharmaceutical, food and chemical industries.

icroorganisms are important for many

reasons, particularly because they produce
things that are of value to us1. These can be
very large materials
(e.g. proteins, nucleic acids, carbohydrate
polymers, even cells) or smaller molecules and are
usually divided into metabolites that are essential for
vegetative growth (primary) and those that are
inessential (secondary).
Although microbes are extremely good at
producing an amazing array of valuable products,
they usually produce these compounds in small
amounts that are needed for their own benefit.
Regulatory mechanisms have evolved that enable a
strain to avoid excessive pro- duction of its
metabolites so that it can compete effi- ciently with
other forms of life and survive in nature. By
contrast, the industrial microbiologist screens for a
wasteful strain that will overproduce a particular
com- pound that can be isolated and marketed.
After a desired strain has been found, a development
program is initiated to improve titers by modification
of culture conditions using mutation
and
recombinant DNA techniques. The main reason for
the use of microor- ganisms to produce compounds
that can otherwise be isolated from plants and
animals, or synthesized by chemists, is the ease of
increasing production by envi- ronmental and genetic
manipulation; 1000-fold increases have been recorded
for small metabolites2.

Traditional microbial biotechnology

Primary metabolites
Primary metabolites are the small molecules of living cells; they are intermediates or end products of
the
pathways of intermediary metabolism, building
blocks
for essential macromolecules, or are converted into

A.L. Demain (demain@mit.edu) is at the

Biology Department, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.

FEATURESPrimary metabolites used in the food and feed

coenzymes.
industries include: alcohols (ethanol), amino acids (monosodium
glutamate, lysine, threonine, phenylala- nine, tryptophan), flavor
nucleotides (59-guanylic acid,
59-inosinic acid), organic acids (acetic, propionic, suc- cinic,
fumaric, lactic), polyols (glycerol, mannitol, erythritol, xylitol),
polysaccharides (xanthan, gellan), sugars (fructose, ribose, sorbose)
and vitamins [riboflavin
(B2), cyanocobalamin (B12), biotin].
Mutants

During amino acid production, feedback regulation

is bypassed by isolating auxotrophic mutants and
partially starving them of their requirements. Another
method is to produce mutants that are resistant to a toxic
analog of the desired metabolite, that is, an antimetabolite. Combinations of auxotrophic and antimetabolite
resistance mutations are common in primary metaboliteproducing microorganisms.
Fermentation
Another factor is the increase in outward permeability, which is very important in the production of

26

FEATURES
acid, the major commercial
amino
acid.
Approximately 1.2 billion pounds of monosodium
glutamate are made annually by fermentation using
various
species of the genera Corynebacterium (e.g. C.
glutamicum)
and Brevibacterium (e.g.
B. flavum and B.
lactofermentum).
Molar yields of glutamate from sugar are 5060%
and
broth concentrations reach over 100 g
l21.
L-glutamic

Glutamic acid
Normally, glutamic acid overproduction would not
occur because of feedback regulation. However,
modification of the cell membrane can cause glutamate to
be pumped out of the cell, thus allowing its
biosynthesis

0167-7799/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01400-6

TIBTECH JANUARY 2000 (Vol. 18)

to proceed unabated. This membrane alteration is

intentionally effected by biotin limitation (all
glutamic acid bacteria are biotin auxotrophs),
glycerol limitation of glycerol auxotrophs, oleate
limitation of oleate auxotrophs, or addition of
penicillin or fatty acid derivatives to exponentially
growing cells. Apparently, all of
these
manipulations result in a phospholipid- deficient
cytoplasmic membrane. The excretion is carried out by
a specific efflux system involving a carrier that is
dependent on membrane potential.
Lysin
e
Most cereals are deficient in the essential amino acid
L-lysine. Lysine is a member of the aspartate family
of
amino acids and is produced in bacteria by a
branched
pathway that also produces methionine, threonine
and
isoleucine. This pathway is controlled very tightly in
an
organism such as Escherichia coli, which includes
three
aspartate kinases that are each regulated by a
different
end product. In addition, after each branch point,
the
initial enzymes are inhibited by their respective end
products and no overproduction occurs. However,
in
lysine-fermentation organisms (e.g. various mutants
of
C. glutamicum and its relatives), there is only a
single
aspartate kinase, which is regulated via concerted
feedback inhibition by threonine and lysine. By the
genetic removal of homoserine dehydrogenase,
a
glutamate-producing wild-type Corynebacterium is converted into a lysine-overproducing mutant that
cannot
grow unless methionine and threonine are added to
the
medium. As long as the threonine supplement is
maintained at a limiting concentration, the intracellular
concentration of threonine is the limiting factor and
feedback inhibition of aspartate kinase is bypassed.
In addition to the difference in the mode of aspartate
kinase
feedback
inhibition,
lysine
overproducers
differ from E. coli in the following ways: (1) no
feedback repression of aspartate kinase or aspartate
semialdehyde
dehydrogenase
occurs
in
lysine
overproducers;
(2) the first and second enzymes of the lysine
branch
(dihydrodipicolinate
synthetase
and
dihydrodipicolinate
reductase) are neither inhibited nor repressed by
lysine
in lysine overproducers;
and (3) L-lysine
decarboxylase

is absent in lysine overproducers. Lysine excretion is

via active transport involving a carrier and is driven
by membrane potential, the lysine gradient and the
proton gradient. Excretion uses a (2OH2)lysine symporter and is catalysed by a dipeptide-uptake system
dependent on electromotive force, not on ATP. World
markets for amino acids amount to US$915 million for
L-glutamate, US$450 million for L-lysine, US$198 million
for L-phenylalanine and US$43 million for L-aspartate.
Recombinant DNA technology
Recombinant DNA technology is beginning to have
a major impact on amino acid production3,4. The major
manipulation for lysine production is aimed at increasing the levels of feedback-resistant aspartate kinase and
dihydrodipicolinate synthase. As a result, lysine industrial production yields 120 g l21 and 0.250.35 g
L-lysine HCl g21 glucose used (molar yield of 0.250.35
mol of L-lysine mol21 of glucose used). Recombinant
technology and traditional mutagenesis, plus selection,
have been major influences in constructing bacterial
strains capable of producing these levels (in g l21)

of amino acids: L-threonine, 100; Lisoleucine, 40; L-leucine, 34; Lvaline, 31; L-phenylalanine, 28; Ltryptophan, 55; L-tyrosine, 26; Lproline, 100; L-arginine, 100; and Lhistidine, 40.
Commercial interest in nucleotide
fermentations is due to the activity
of two purine ribonucleoside
59-monophosphates, namely guanylic
acid (GMP) and inosinic acid (IMP), as
flavor enhancers. Approximately
2500 tons of GMP and IMP are
produced annually in Japan alone, with
a world market of US$350 million.
Techniques similar to those described
above for amino acid fermentations
have yielded IMP titers of 27 g l21.
V
i
t
a
m
i
n
s
Riboflavin
(vitamin
B2)
overproducers include two yeast-like
molds, Eremothecium
ashbyii and
Ashbya gossypii, which synthesize
riboflavin in concentrations greater
than 20 g l21. New processes using
Candida sp. or recombinant Bacillus
subtilis strains that produce up to 30 g
l21 riboflavin have been developed in
recent
years. Vitamin B12 is produced on an
industrial scale by Propionibacterium
shermanii or Pseudomonas denitrificans.
The key to the fermentation is to
avoid feedback repression by vitamin
B12. The early stage of the P. sher- manii
fermentation is conducted under
anaerobic con- ditions in the absence
of the
precursor 5,6-dimethylbenzimidazole.
These
conditions
prevent vitamin B12 synthesis and allow
for the accumulation of the intermediate, cobinamide. The culture is
TIBTECH JANUARY 2000 (VOL 18)

then aerated and dimethylbenzimidazole is added,

converting cobinamide to the vitamin. In the P.
denitrificans fermentation, the entire process is carried
out under low oxygen. A high level of oxygen results
in an oxidizing intracellular envi- ronment that
represses the formation of the early enzymes in
the pathway. Production of vitamin B12 has reached
levels of 150 mg l21 and a world market value of
US$71 million.
During production of biotin, feedback repression
is caused by the enzyme protein acetyl-coenzyme A
car- boxylase biotin holoenzyme synthetase, with
biotin
5-adenylate acting as corepressor. Strains of Serratia
marcescens obtained by mutagenesis, selection for
resist- ance to biotin antimetabolites, and molecular
cloning can produce 600 mg l21 in the presence of
high con- centrations of sulfur and ferrous iron. Such a
titer is high enough to economically compete with
the traditional chemical production process.
Fungi
Filamentous fungi are widely used for the commercial production of organic acids, for example, 1
billion
pounds of citric acid are produced per year with a
market value of US$1.4 billion. Citric acid is produced
via
the Embden-Meyerhof pathway and the first step of
the
tricarboxylic acid cycle; the control of the
process
involves the inhibition of phosphofructokinase by citric acid. The commercial process uses Aspergillus
niger
in media deficient in iron and manganese. A high
level
of citric acid production is also associated with a
high
intracellular concentration of fructose 2,6biphosphate,
an
activator
of
glycolysis.
Other factors contributing to high citric acid production
are the
inhibition
of isocitrate
dehydrogenase
by citric acid and the low optimum pH (1.72.0).
Higher pH values (e.g. 3.0) lead to the production
of

27

oxalic and gluconic acids, instead of citric acid. The

low pH inactivates glucose oxidase, which would
normally yield gluconic acid. In approximately 45
days, the major proportion (80%) of the sugar is
converted to citric acid, with titers reaching about
100 g l21.
Alternative processes have been developed for
the production of citric acid by Candida yeasts,
especially from hydrocarbons. Such yeasts are able
to convert n-paraffins to citric and isocitric acids in
extremely high yields [150170% (w/w)
of
substrate used]; titers as high as 225 g l21 have been
reached.
Alcohol
Ethyl alcohol is a primary metabolite produced by
fermentation of sugar, or a polysaccharide that can be
depolymerized to a fermentable sugar. Saccharomyces
cerevisiae is used for the fermentation of hexoses,
whereas Kluyveromyces fragilis or Candida species can be
used if lactose or a pentose, respectively, is the
substrate.
Under optimum conditions, approximately 10
12%
ethanol by volume is obtained within five days. Such
a
high concentration slows down growth and the
fermentation ceases. With special yeasts, the fermentation
can be continued to produce alcohol concentrations
of
20% by volume, but these concentrations are
attained
only after months or years of
fermentation.
At present, all beverage alcohol is made by
fermentation. Industrial ethanol is mainly manufactured by
fermentation, but some is produced from ethylene
by
the petrochemical industry. Bacteria such as
clostridia
and Zymomonas are being re-examined for ethanol
production after years of neglect.
Clostridium
thermocellum,
an anaerobic thermophile, can convert waste
cellulose
(i.e. biomass) and crystalline cellulose directly to
ethanol.
Other clostridia produce acetate, lactate, acetone
and
butanol, and will be used to produce these
chemicals
when the gobal petroleum supplies begin to become
depleted. Fuel ethanol produced from biomass would
provide relief from air pollution caused by the use
of
gasoline and would not contribute to the greenhouse
effect. E. coli has been converted into an excellent
ethanol producer (43% yield, v/v) by recombinant
DNA techniques.

compound is converted into a

structurally related product by one or a small number of
enzymes contained
in the cells5. Bioconverting-organisms
are known for
practically every type of chemical
reaction. The reactions are stereospecific; the ultimate in
specificity is
exemplified by steroid bioconversions.
Bioconversions
are characterized by extremely high
yields, approximately 90100%. Other attributes
include mild reaction
conditions and the coupling of
reactions, using a
microorganism
containing
several
enzymes working in
s
e
r
i
e
s
.
Seconda
ry
metabol
ites
Microbially produced secondary
metabolites6 are
extremely important for health and
nutrition. As a
group that includes antibiotics, other
medicinals, toxins,
biopesticides and animal and plant
growth factors, they

Bioconverting-organisms
In addition to the multireaction sequences of fermentations, microorganisms are extremely useful in
carrying out biotransformation processes in which a
28

TIBTECH JANUARY 2000 (Vol. 18)

have tremendous economic importance. Secondary metabolites

have no function in the growth of the pro- ducing cultures
(although, in nature, they are essential for the survival of the
producing organism), are pro- duced by certain restricted
taxonomic groups of organ- isms and are usually formed as
mixtures of closely related members of a chemical family.
Antibiotics
The best-known group of the secondary metabolites
are the antibiotics7. Their targets include DNA replication (actinomycin, bleomycin and griseofulvin), transcription (rifamycin), translation by 70-S ribosomes
(chloramphenicol, tetracycline, lincomycin, erythromycin and streptomycin), transcription by 80-S ribosomes (cyclohexamide), transcription by 70- and 80-S
ribosomes (puromycin and fusidic acid), cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin
and vancomycin) and cell membranes (surfactants
including: polymyxin and amphotericin; channelforming ionophores, such as linear gramicidin; and
mobile carrier ionophores, such as monensin).
Since 1940, there has been a virtual explosion of new
and potent antibiotic molecules that have been of great
use in medicine, agriculture and basic research. In
1996, the antibiotic market was composed of 160 antibiotics and amounted to a world market value of
~US$23 billion. The search for new antibiotics continues,
in order to: combat evolving pathogens, naturally resistant bacteria and fungi, and previously susceptible
microbes that have developed resistance; improve pharmacological properties; combat tumors, viruses and
parasites; and discover safer, more potent and broaderspectrum compounds. In the search for new antibiotics,
many of the new products are made chemically by
modification of natural antibiotics via semisynthesis.
Antibiotics are used not only for chemotherapy in human
and veterinary medicine, but also for growth promo-

29

tion in farm animals and for the protection of

plants.
Non-antibiotic agents
In nature, secondary metabolites are important to the
organisms that produce them, functioning as: (1) sex
hormones; (2) ionophores; (3) competitive
weapons
against other bacteria, fungi, amoebae, insects and
plants; (4) agents of symbiosis; and (5) effectors of differentiation. For years, most pharmaceuticals that
were
used for non-infectious diseases were strictly
synthetic
products8. Similarly, most therapeutics for nonmicrobial
parasitic diseases in animals (e.g.
coccidiostats
and antihelminthics) came from the screening of
synthesized compounds followed by molecular modification. Despite the testing of thousands of
synthetic
compounds, only a few promising structures were
found. As new lead compounds became more and
more difficult to find, microbial broths filled the
void
and microbial products increased in importance in
the
therapy of non-microbial diseases9. Today,
microbially
produced polyethers such as monensin, lasalocid and
salinomycin dominate the coccidiostat market and
are also the chief growth promoters in use for
ruminant animals. The avermectins, another group of
streptomycete products with a market of more than
US$1 billion per year, have high activity
against
helminths and arthropods.

TIBTECH JANUARY 2000 (Vol. 18)

Many microbial products with important

pharmaco- logical activities were discovered by
screening for inhibitors using simple enzymatic
assays. One huge success has been the statins,
including lovastatin (also known as mevinolin) and
pravastatin: fungal products that are used as
cholesterol-lowering agents in humans and animals.
In its hydroxy acid form, lovastatin is a potent
competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from liver. Other
well- known enzyme inhibitors include: clavulanic
acid, a penicillinase-inhibitor that
protects
penicillin from inactivation by resistant pathogens;
and acarbose, a natural inhibitor of intestinal
glucosidase, which is produced by an actinomycete
of the genus Actinoplanes. Acarbose decreases
hyperglycemia and triglyceride syn- thesis in adipose
tissue, the liver and the intestinal wall of patients
suffering from diabetes, obesity and type IV
hyperlipidemia.
Biopesticides
Also in commercial or near-commercial use are
biopesticides, including biofungicides (e.g.
kasugamycin,
polyoxins), bioinsecticides (nikkomycin,
spinosyns),
bioherbicides (bialaphos), antihelminthics (avermectin),
coccidiostats, ruminant-growth promoters (monensin,
lasalocid, salinomycin), plant-growth regulators (gibberellins), immunosuppressants for organ transplants
(cyclosporin A, FK-506, rapamycin), anabolic agents
in farm animals (zearelanone), uterocontractants (ergot
alkaloids) and antitumor agents (doxorubicin,
daunorubicin, mitomycin, bleomycin).
Tropophase and idiophase
In batch culture, most secondary metabolite processes
have a distinct growth phase (trophophase) followed
by
a production phase (idiophase). In other
fermentations,
the two phases overlap; the timing depends on the
nutritional environment presented to the culture, the
growth rate, or both. A delay in antibiotic production
until after trophophase helps the producing organism
because the microbe is sometimes sensitive to its own
antibiotic during growth. Resistance mechanisms that
develop in producing microorganisms include enzymatic modification of the antibiotic, alteration of the
cellular target of the antibiotic and decreased uptake
of
the excreted antibiotic.
Directed biosynthesis
The manipulation of the culture media in any
development program often involves the testing of
hundreds
of additives as possible limiting precursors of the
desired
product. Occasionally, a precursor that increases production of the secondary metabolite is found. The precursor may also direct the fermentation towards the
formation of one specific desirable product: this is known
as directed biosynthesis.
Examples of directed biosynthesis include the use of

phenylacetic acid in the fermentation of benzylpenicillin, and specific amino acids in the production of
actinomycins and tyrocidins. Stimulatory precursors
include: methionine, as an inducer in cephalosporin C
formation; valine, in tylosin production; and tryptophan for ergot-alkaloid production. In many fermentations, however, precursors show no activity because
their syntheses are not rate-limiting. In such cases,

screening of additives has often revealed

dramatic effects, both stimulatory and
inhibitory, of non-precursor
molecules on the production of
secondary metabolites. These effects are
usually due to the interaction of these
compounds with the regulatory
mechanisms existing in the
fermentation organism. Antibiotic
biosynthesis ends via the decay of
antibiotic synthetases or because of
feedback inhibition and repression of
these enzymes. Because the regulatory
mechanisms are genetically
determined, mutations have had a
major effect on the production of
secondary metabolites. Indeed, it is the
chief factor responsible for the 100
1000-fold increases obtained in the
production of antibiotics from their
initial discovery to the present time.
These tremendous increases in
fermentation productivity and the
result- ing decreases in costs have come
about mainly by ran- dom mutagenesis
and screening for higher-producing
microbial strains. Mutation has also
served to: (1) shift the proportion of
metabolites produced in a fermentation broth to a more favorable
distribution; (2) elucidate the
pathways of secondary metabolism;
and
(3) yield new compounds.
Modern microbial biotechnology
Modern biotechnology is now over
25 years old10.
In 1972, the birth of recombinant DNA technology
TIBTECH JANUARY 2000 (Vol. 18)

propelled biotechnology to new heights and led to the

establishment of a new industry. In addition to recombinant DNA technology, modern microbial biotechnology encompasses fermentation, microbial physiology,
high-throughput screening for novel metabolites and
strain improvement, bioreactor design and downstream
processing, cell immobilization (enzyme engineering),
cell fusion, metabolic engineering, bioreactor design,
downstream processing, in vitro mutagenesis (protein
engineering) and directed evolution of enzymes
(applied molecular evolution).
Recombinant microorganisms
The revolutionary exploitation of microbial genetic
discoveries in the 1970s, 1980s and 1990s depended
heavily upon the solid structure of industrial microbiology, described above. The major microbial hosts
for production of recombinant proteins are E. coli11, B.
subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorpha
and Aspergillus niger. The use of recombinant microorganisms (Fig. 1) provided the techniques and experience necessary for the successful application of higher
organisms, such as mammalian and insect cell culture,
and transgenic animals and plants as hosts for the
production of glycosylated recombinant proteins.
Progress
The progress in biotechnology has been truly
remarkable. Within four years of the discovery of
recombinant DNA technology, genetically engineered
bacteria were making human insulin and human
growth hormone. This led to an explosion of investment activity in new companies, mainly dedicated to
innovation via genetic approaches. Newer companies
entered the scene in various niches such as biochemical engineering and downstream processing. Today,
biotechnology in the USA is represented by some
1300 companies with revenues of US$19.6 billion, of
which sales represent US$13.4 billion, and there are

29

FEATURES

FEATURES

was that of hepatitis B virus surface antigen

produced in yeast. The great contribution made by
recombinant vaccines is the elimination of the tragic
problems asso- ciated with conventional vaccines.
Through reversion of the attenuated pathogen, some
individuals receiving the conventional vaccine not only
failed to be protected, but also came down with the
disease.

Figure 1
Escherichia coli: the workhorse of modern microbial biotechnology. (Electron
micrograph taken by Erika Hartweig, bar = 1 mm.)

approximately 153 000 employees. The number of

biotechnology companies in Canada reached 282 in
1998, employing 10 000 workers and with revenues
of approximately US$1.1
billion.
Japans
biotechnology sales were approximately US$10
billion, mainly by established pharmaceutical, food
and beverage compa- nies. European biotechnology
moved rapidly in the
1990s, after years of lagging behind and, in 1998,
1178 biotechnology companies existed with 45 000
employees, and revenues of US$3.7
billion.
The major thrust of recombinant DNA technology
has been in the area of rare mammalian peptides,
such
as hormones, growth factors, enzymes, antibodies and
biological response modifiers12. Among those geneti30

Combinatorial biosynthesis
As mentioned previously, recombinant DNA techniques have made a significant impact on the
production of vitamins, amino acids, nucleotides,
bioconversion
and secondary metabolites. Most microbial
biosynthetic
pathways are encoded by clustered genes, which facilitates the transfer of an entire pathway in a
single
manipulation. Even in fungi, pathway genes are
sometimes clustered, such as the penicillin genes in
Penicillium
or the aflatoxin genes in Aspergillus. For the
discovery
of new or modified secondary products, recombinant
DNA techniques are being used to introduce genes
for
the synthesis of one product into producers of other
antibiotics
or
into
non-producing
strains
(combinatorial
biosynthesis).
cally engineered products that have
been approved
for use in the USA are human insulin,
human growth
hormone,
erythropoietin,
antihemophelia factor,
granulocyte-colony stimulating factor,
granulocytemacrophage-colony
stimulating
factor, epidermal
growth factor and other growth factors,
interleukin-2,
a-, b- and g-interferons, and bovine
somatotropin.
V
a
c
c
i
n
e
s

Vaccine production is another

important part of the
new technology; the first subunit
vaccine on the market

TIBTECH JANUARY 2000 (Vol. 18)

FEATURES
Enzyme
production
The production of enzymes by fermentation was an
established business before modern microbial biotechnology. However, recombinant DNA methodology
was so perfectly suited to the improvement of enzymeproduction technology that it was almost immediately
used by companies involved in manufacturing enzymes.
Industrial enzymes have now reached an annual market of US$1.6 billion. Important enzymes are proteases,
lipases, carbohydrases, recombinant chymosin for
cheese manufacture and recombinant lipase for use in
detergents. Recombinant therapeutic enzymes already
have a market value of over US$2 billion, being used
for thromboses, gastrointestinal and rheumatic disorders,
metabolic diseases and cancer. They include tissue
plasminogen activator, human DNAase and Cerozyme.
Agriculture
Industrial microbiology through genetic engineering
and its associated disciplines has brought about a revolution in agriculture. Two bacteria have had a major

31

FEATURES
influence: Agrobacterium tumefaciens, a bacterium
that
normally produces crown gall tumors on dicotyledonous plants; and Bacillus thuringiensis, an insecticidal
bacterium. The tumor-forming genes of A. tumefaciens
are present on its tumor-inducing (Ti) plasmid, along
with genes directing the plant to form opines (nutritional factors required by the bacterium that it cannot
produce by itself ). The Ti vector has been exceedingly
valuable for introducing foreign genes into dicotyledonous plants for production of transgenic plants. However, the Ti plasmid is not very successful for transferring genes into monocotyledonous plants, a problem
bypassed by, for example, the development of a
particleacceleration gun, which shoots DNA-coated metal
particles into plant cells. The activity of the insecticidal
bacterium, B. thuringiensis, is caused by its crystal protein
produced during sporulation. Crystals and spores have
been applied to plants for many years to protect them
against lepidopteran insects. B. thuringiensis preparations
are highly potent, approximately 300 times more
active

TIBTECH JANUARY 2000 (Vol. 18)

on a molar basis than synthetic pyrethroids and 80

000 times more active than organophosphate
insecticides.
In the modern biotechnology era, plants resistant to
insects have been produced by expressing forms of
the B. thuringiensis toxin gene in the plant. Recently
devel- oped bioinsecticides include insect viruses, such
as bac- uloviruses, that are engineered to produce
arthropod toxins. Transgenic plants, resistant to
herbicides, are also available, as are virus-resistant
plants produced by ex- pressing viral-coat-protein
genes in plants. Interestingly, chemical pesticides
against plant viruses were never available.
Conclusion
Although most of the early promises of biotechnology
have been achieved, major challenges remain. We
must
use our brains, technology, drive and dedication to
solve
the problems of evolving diseases (e.g. AIDS),
established
diseases (cancer and parasitic infection), antibioticresistance development and environmental pollution,
by
converting urban, industrial and agricultural wastes
into
resources such as liquid fuel. These efforts will
require
continued interaction between different
disciplines,
major support by governments and international
agencies, as well as an understanding and supportive
public.

2 Demain, A.L. (1988) Contributions of genetics to the production

and discovery of microbial pharmaceuticals. Pure Appl. Chem. 60,
833836
3 Jetten, M.S. and Sinskey, A.J. (1995) Recent advances in the
physiology and genetics of amino acid-producing bacteria. Crit.
Rev. Biotechnol. 15, 73103
4 Sahm, H. et al. (1995) Metabolic design in amino acid producing
bacterium Corynebacterium glutamicum. FEMS Microbiol. Rev. 16,
243252
5 Kieslich, K. (1997) Biotransformations. In Fungal Biotechnology
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Biotechnol. Adv. 8, 291301

Drug discovery in the new millennium: the

pivotal role of biotechnology
Graham J. Boulnois
Biotechnology has made, and will continue to make, a major contribution to the health of mankind by providing new insights
into disease and acting as a pivotal enabler for the drug-discovery process. The available techniques are diverse and
changing rapidly: selecting and integrating the best approach is the key to success.

odern medicines have improved the life of

humankind, and the past few decades have
seen enormous advances in our ability to treat,
manage and prevent a large number of diseases. Successful
therapies range from the treatment of acute
infections with powerful antibiotics and the
availability of anaesthetics for surgery and
management, through risk reduction in cardiovascular
disease to the prevention of a range of infectious
diseases via vaccination.

Biotechnology has already played a major role in modern

medicine discovery, delivering a range of new treatments and
vaccines in its own right, and gene ther- apy is just round the
corner. Despite these impressive
G.J. Boulnois (graham.boulnois@astrazeneca.com) is at AstraZeneca
Pharmaceuticals, Alderley Park, Macclesfield, UK SK10 4TG.

advances, there are many diseases for

which treatments are lacking or are
imperfect, and we are a long way from
eradicating or even reducing the
prevalence of many debilitating
conditions. In short, the opportunities
for new treatments is huge.
We have also seen a change in the
way new medi- cines are discovered,
driven by biotechnology in gen- eral

but particularly by molecular biology. The reliance on

testing new synthetic organic molecules in animals or
in whole-organ preparations, as an early part of the
discovery process, has changed to a molecular
target approach in which in vitro screening of
compounds against purified, recombinant proteins
or genetically modified cell lines is carried out with a
high throughput. This change has come about as a
consequence of better and ever-improving knowledge
of the molecular basis

TIBTECH JANUARY 2000 (Vol. 18)

0167-7799/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01393-1

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