FEATURES

10 Hoogendoorn, B. et al. (1999) Genotyping single nucleotide Am. J. Hum. Genet. 52, 46–59
polymorphisms by primer extension and high performance liquid 13 Griffin, T.J. et al. (1999) Direct genetic analysis by matrix-assisted
chromatography. Hum. Genet. 104, 89–93 laser desorption/ionization mass spectrometry. Proc. Natl. Acad. Sci.
11 Lander, E.S. (1999) Array of hope. Nat. Genet. 21, 3–4 U. S. A. 96, 6301–6306
12 Syvanen, A-C. et al. (1993) Identification of individuals by analysis 14 Gibbs, R.A. et al. (1989) Detection of single DNA base differences by
of bi-allelic DNA markers, using PCR and solid-phase minisequencing. 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, coenzymes. Primary metabolites used in the food and

M 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 liv-
ing 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.
feed 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 antimetab-
olite. Combinations of auxotrophic and antimetabolite
resistance mutations are common in primary metabolite-
producing microorganisms.
Fermentation
Another factor is the increase in outward permeabil-
ity, which is very important in the production of
L-glutamic acid, the major commercial amino acid.
Approximately 1.2 billion pounds of monosodium glu-
tamate 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 50–60% and
broth concentrations reach over 100 g l21.
Glutamic acid
Normally, glutamic acid overproduction would not
occur because of feedback regulation. However, modi-
fication of the cell membrane can cause glutamate to
be pumped out of the cell, thus allowing its biosynthesis

26 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.
Lysine
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 feed-
back inhibition by threonine and lysine. By the
genetic removal of homoserine dehydrogenase, a
glutamate-producing wild-type Corynebacterium is con-
verted into a lysine-overproducing mutant that cannot
grow unless methionine and threonine are added to the
medium. As long as the threonine supplement is main-
tained 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 aspar-
tate kinase feedback inhibition, lysine overproducers
differ from E. coli in the following ways: (1) no feed-
back repression of aspartate kinase or aspartate semi-
aldehyde 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 sym-
porter 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 increas-
ing the levels of feedback-resistant aspartate kinase and
dihydrodipicolinate synthase. As a result, lysine indus-
trial production yields 120 g l21 and 0.25–0.35 g
L-lysine • HCl g21 glucose used (molar yield of 0.25–0.35
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)
TIBTECH JANUARY 2000 (VOL 18)
FEATURES
of amino acids: L-threonine, 100; L-isoleucine, 40;
L-leucine, 34; L-valine, 31; L-phenylalanine, 28;
L-tryptophan, 55; L-tyrosine, 26; L-proline, 100;
L-arginine, 100; and L-histidine, 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.
Vitamins
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-dimethyl-
benzimidazole. These conditions prevent vitamin B12
synthesis and allow for the accumulation of the inter-
mediate, cobinamide. The culture is 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 commer-
cial production of organic acids, for example, 1 billion
pounds of citric acid are produced per year with a mar-
ket 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 cit-
ric 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 2,6-biphosphate,
an activator of glycolysis.
Other factors contributing to high citric acid pro-
duction are the inhibition of isocitrate dehydrogenase
by citric acid and the low optimum pH (1.7–2.0).
Higher pH values (e.g. 3.0) lead to the production of
27
FEATURES
oxalic and gluconic acids, instead of citric acid. The low
pH inactivates glucose oxidase, which would normally
yield gluconic acid. In approximately 4–5 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 [150–170% (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 fer-
mentation 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 fermen-
tation. 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.
Bioconverting-organisms
In addition to the multireaction sequences of fer-
mentations, microorganisms are extremely useful in
carrying out biotransformation processes in which a
compound is converted into a structurally related prod-
uct by one or a small number of enzymes contained
in the cells5. Bioconverting-organisms are known for
practically every type of chemical reaction. The reac-
tions are stereospecific; the ultimate in specificity is
exemplified by steroid bioconversions. Bioconversions
are characterized by extremely high yields, approxi-
mately 90–100%. Other attributes include mild reaction
conditions and the coupling of reactions, using a
microorganism containing several enzymes working in
series.
Secondary metabolites
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
28
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 repli-
cation (actinomycin, bleomycin and griseofulvin), tran-
scription (rifamycin), translation by 70-S ribosomes
(chloramphenicol, tetracycline, lincomycin, erythro-
mycin and streptomycin), transcription by 80-S ribo-
somes (cyclohexamide), transcription by 70- and 80-S
ribosomes (puromycin and fusidic acid), cell wall syn-
thesis (cycloserine, bacitracin, penicillin, cephalosporin
and vancomycin) and cell membranes (surfactants
including: polymyxin and amphotericin; channel-
forming 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 anti-
biotics and amounted to a world market value of
~US$23 billion. The search for new antibiotics continues,
in order to: combat evolving pathogens, naturally resist-
ant bacteria and fungi, and previously susceptible
microbes that have developed resistance; improve phar-
macological properties; combat tumors, viruses and
parasites; and discover safer, more potent and broader-
spectrum 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-
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 dif-
ferentiation. For years, most pharmaceuticals that were
used for non-infectious diseases were strictly synthetic
products8. Similarly, most therapeutics for non-
microbial parasitic diseases in animals (e.g. coccidiostats
and antihelminthics) came from the screening of
synthesized compounds followed by molecular modi-
fication. 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 rumi-
nant 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-methyl-
glutaryl-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 (gib-
berellins), immunosuppressants for organ transplants
(cyclosporin A, FK-506, rapamycin), anabolic agents
in farm animals (zearelanone), uterocontractants (ergot
alkaloids) and antitumor agents (doxorubicin, daunoru-
bicin, 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 enzy-
matic 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 devel-
opment program often involves the testing of hundreds
of additives as possible limiting precursors of the desired
product. Occasionally, a precursor that increases pro-
duction of the secondary metabolite is found. The pre-
cursor may also direct the fermentation towards the for-
mation 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 benzylpeni-
cillin, 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 trypto-
phan for ergot-alkaloid production. In many fermen-
tations, however, precursors show no activity because
their syntheses are not rate-limiting. In such cases,
TIBTECH JANUARY 2000 (Vol. 18)
FEATURES
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 fermen-
tation 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
propelled biotechnology to new heights and led to the
establishment of a new industry. In addition to recom-
binant DNA technology, modern microbial biotech-
nology 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 micro-
biology, 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 micro-
organisms (Fig. 1) provided the techniques and experi-
ence 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 invest-
ment activity in new companies, mainly dedicated to
innovation via genetic approaches. Newer companies
entered the scene in various niches such as biochemi-
cal 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
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. Japan’s 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 geneti-
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, granulocyte-
macrophage-colony stimulating factor, epidermal
growth factor and other growth factors, interleukin-2,
a-, b- and g-interferons, and bovine somatotropin.
Vaccines
Vaccine production is another important part of the
new technology; the first subunit vaccine on the market
30
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.
Combinatorial biosynthesis
As mentioned previously, recombinant DNA tech-
niques have made a significant impact on the produc-
tion of vitamins, amino acids, nucleotides, bioconversion
and secondary metabolites. Most microbial biosynthetic
pathways are encoded by clustered genes, which fa-
cilitates the transfer of an entire pathway in a single
manipulation. Even in fungi, pathway genes are some-
times 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).
Enzyme production
The production of enzymes by fermentation was an
established business before modern microbial biotech-
nology. However, recombinant DNA methodology
was so perfectly suited to the improvement of enzyme-
production technology that it was almost immediately
used by companies involved in manufacturing enzymes.
Industrial enzymes have now reached an annual mar-
ket 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 rev-
olution in agriculture. Two bacteria have had a major
influence: Agrobacterium tumefaciens, a bacterium that
normally produces crown gall tumors on dicotyledo-
nous 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 (nutri-
tional factors required by the bacterium that it cannot
produce by itself ). The Ti vector has been exceedingly
valuable for introducing foreign genes into dicotyledo-
nous plants for production of transgenic plants. How-
ever, the Ti plasmid is not very successful for transfer-
ring genes into monocotyledonous plants, a problem
bypassed by, for example, the development of a particle-
acceleration 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), antibiotic-
resistance 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 agen-
cies, as well as an understanding and supportive public.
References
1 Demain, A.L. (1990) Achievements in microbial technology. Biotechnol.
Adv. 8, 291–301
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
M humankind, and the past few decades have seen
enormous advances in our ability to treat, man-
age 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.
TIBTECH JANUARY 2000 (Vol. 18) 0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(99)01393-1
FEATURES
2 Demain, A.L. (1988) Contributions of genetics to the production
and discovery of microbial pharmaceuticals. Pure Appl. Chem. 60,
833–836
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, 73–103
4 Sahm, H. et al. (1995) Metabolic design in amino acid producing
bacterium Corynebacterium glutamicum. FEMS Microbiol. Rev. 16,
243–252
5 Kieslich, K. (1997) Biotransformations. In Fungal Biotechnology
(Anke, T., ed.), pp. 297–399, Chapman & Hall
6 Demain, A.L. (1992) Microbial secondary metabolism: a new
theoretical frontier for academia, a new opportunity for industry.
In Secondary Metabolites: Their Function and Evolution (Chadwick, D.J.
and Whelan, J., eds), pp. 3–23, John Wiley & Sons
7 Strohl, W.R. (1997) Biotechnology of Antibiotics, 2nd edn, Marcel
Dekker
8 Demain, A.L. (1996) Fungal secondary metabolism: regulation and
functions. In A Century of Mycology (Sutton, B., ed.), pp. 233–254,
Cambridge University Press
9 Demain, A.L. (1998) Microbial natural products: alive and well in
1998. Nat. Biotechnol. 16, 3–4
10 Cohen, S.N. (1979) The transplantation and manipulation of genes
in microorganisms. The Harvey Lectures 74, 173–204
11 Swartz, J.R. (1996) Escherichia coli recombinant DNA technology.
In Escherichia coli and Salmonella: Cellular and Molecular Biology 2nd
edn (Neidhardt, F.C., ed.), pp. 1693–1711, American Society of
Microbiology (ASM) Press
12 Demain, A.L. et al. (1994) Contributions of recombinant microbes
and their potential. In Recombinant Microbes for Industrial and Agricul-
tural Applications (Murooka, T. and Imanaka, T., eds), pp. 27–46,
Marcel Dekker
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
31