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10 Hoogendoorn, B. et al. (1999) Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography. Hum. Genet. 104, 89–93

11 Lander, E.S. (1999) Array of hope. Nat. Genet. 21, 3–412 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, 46–5913 Griffin, T.J. et al. (1999) Direct genetic analysis by matrix-

assisted laser desorption/ionization mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 96, 6301–6306

14 Gibbs, R.A. et al. (1989) Detection of single DNA base differences by competitive oligonucleotide priming. Nucleic Acids Res. 17, 2437–2448

Microbial biotechnologyArnold 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 biotechnologyPrimary metabolites

Primary metabolites are the small molecules of liv-ing cells; they are intermediates or end products of thepathways of intermediary metabolism, building blocksfor 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.

F Fcoenzymes. Primary metabolites used in the food and 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].

MutantsDuring amino acid production, feedback regulation

is bypassed by isolating auxotrophic mutants andpartially starving them of their requirements. Anothermethod is to produce mutants that are resistant to a toxicanalog of the desired metabolite, that is, an antimetab-olite. Combinations of auxotrophic and antimetaboliteresistance mutations are common in primary metabolite-producing microorganisms.

FermentationAnother 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 variousspecies 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% andbroth concentrations reach over 100 g l21.

Glutamic acidNormally, glutamic acid overproduction would not

occur because of feedback regulation. However, modi-fication of the cell membrane can cause glutamate tobe 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)

F F

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 acidL-lysine. Lysine is a member of the aspartate family ofamino acids and is produced in bacteria by a branchedpathway that also produces methionine, threonine andisoleucine. This pathway is controlled very tightly in anorganism such as Escherichia coli, which includes threeaspartate kinases that are each regulated by a differentend product. In addition, after each branch point, theinitial enzymes are inhibited by their respective endproducts and no overproduction occurs. However, inlysine-fermentation organisms (e.g. various mutants ofC. glutamicum and its relatives), there is only a singleaspartate kinase, which is regulated via concerted feed-back inhibition by threonine and lysine. By thegenetic removal of homoserine dehydrogenase, aglutamate-producing wild-type Corynebacterium is con-verted into a lysine-overproducing mutant that cannotgrow unless methionine and threonine are added to themedium. As long as the threonine supplement is main-tained at a limiting concentration, the intracellularconcentration of threonine is the limiting factor andfeedback inhibition of aspartate kinase is bypassed.

In addition to the difference in the mode of aspar-tate kinase feedback inhibition, lysine overproducersdiffer 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 dihydrodipicolinatereductase) are neither inhibited nor repressed by lysinein lysine overproducers; and (3) L-lysine decarboxylase

is absent in lysine overproducers. Lysine excretion isvia active transport involving a carrier and is drivenby membrane potential, the lysine gradient and theproton gradient. Excretion uses a (2OH2)–lysine sym-porter and is catalysed by a dipeptide-uptake systemdependent on electromotive force, not on ATP. Worldmarkets for amino acids amount to US$915 million forL-glutamate, US$450 million for L-lysine, US$198 millionfor L-phenylalanine and US$43 million for L-aspartate.

Recombinant DNA technologyRecombinant DNA technology is beginning to have

a major impact on amino acid production3,4. The majormanipulation for lysine production is aimed at increas-ing the levels of feedback-resistant aspartate kinase anddihydrodipicolinate synthase. As a result, lysine indus-trial production yields 120 g l21 and 0.25–0.35 gL-lysine•HCl g21 glucose used (molar yield of 0.25–0.35mol of L-lysine mol21 of glucose used). Recombinanttechnology and traditional mutagenesis, plus selection,have been major influences in constructing bacterialstrains capable of producing these levels (in g l21)

F Fof 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 ribonucleoside59-monophosphates, namely guanylic acid (GMP) and inosinic acid (IMP), as flavor enhancers. Approximately2500 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 recentyears. 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 biotin5-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.

FungiFilamentous fungi are widely used for the commer-

cial production of organic acids, for example, 1 billionpounds of citric acid are produced per year with a mar-ket value of US$1.4 billion. Citric acid is produced viathe Embden-Meyerhof pathway and the first step of thetricarboxylic acid cycle; the control of the processinvolves the inhibition of phosphofructokinase by cit-ric acid. The commercial process uses Aspergillus nigerin media deficient in iron and manganese. A high levelof citric acid production is also associated with a highintracellular concentration of fructose 2,6-biphosphate,an activator of glycolysis.

Other factors contributing to high citric acid pro-duction are the inhibition of isocitrate dehydrogenaseby citric acid and the low optimum pH (1.7–2.0).Higher pH values (e.g. 3.0) lead to the production of

TIBTECH JANUARY 2000 (VOL 18) 27

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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.

AlcoholEthyl alcohol is a primary metabolite produced by

fermentation of sugar, or a polysaccharide that can bedepolymerized to a fermentable sugar. Saccharomycescerevisiae is used for the fermentation of hexoses,whereas Kluyveromyces fragilis or Candida species can beused if lactose or a pentose, respectively, is the substrate.Under optimum conditions, approximately 10–12%ethanol by volume is obtained within five days. Such ahigh concentration slows down growth and the fer-mentation ceases. With special yeasts, the fermentationcan be continued to produce alcohol concentrations of20% by volume, but these concentrations are attainedonly after months or years of fermentation.

At present, all beverage alcohol is made by fermen-

tation. Industrial ethanol is mainly manufactured byfermentation, but some is produced from ethylene bythe petrochemical industry. Bacteria such as clostridiaand Zymomonas are being re-examined for ethanolproduction 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 andbutanol, and will be used to produce these chemicalswhen the gobal petroleum supplies begin to becomedepleted. Fuel ethanol produced from biomass wouldprovide relief from air pollution caused by the use ofgasoline and would not contribute to the greenhouseeffect. E. coli has been converted into an excellentethanol producer (43% yield, v/v) by recombinantDNA techniques.

Bioconverting-organismsIn addition to the multireaction sequences of fer-

mentations, microorganisms are extremely useful incarrying out biotransformation processes in which a

compound is converted into a structurally related prod-uct by one or a small number of enzymes containedin the cells5. Bioconverting-organisms are known forpractically every type of chemical reaction. The reac-tions are stereospecific; the ultimate in specificity isexemplified by steroid bioconversions. Bioconversionsare characterized by extremely high yields, approxi-mately 90–100%. Other attributes include mild reactionconditions and the coupling of reactions, using amicroorganism containing several enzymes working inseries.

Secondary metabolites

Microbially produced secondary metabolites6 are

extremely important for health and nutrition. As agroup that includes antibiotics, other medicinals, toxins,biopesticides and animal and plant growth factors, they

2 TIBTECH JANUARY 2000 (Vol.

F Fhave 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.

AntibioticsThe 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-Sribosomes (puromycin and fusidic acid), cell wall syn-thesis (cycloserine, bacitracin, penicillin, cephalosporinand vancomycin) and cell membranes (surfactantsincluding: polymyxin and amphotericin; channel-forming ionophores, such as linear gramicidin; andmobile carrier ionophores, such as monensin).

Since 1940, there has been a virtual explosion of newand potent antibiotic molecules that have been of greatuse in medicine, agriculture and basic research. In1996, 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 susceptiblemicrobes that have developed resistance; improve phar-macological properties; combat tumors, viruses andparasites; and discover safer, more potent and broader-spectrum compounds. In the search for new antibiotics,many of the new products are made chemically bymodification of natural antibiotics via semisynthesis.Antibiotics are used not only for chemotherapy in humanand veterinary medicine, but also for growth promo-tion in farm animals and for the protection of plants.

Non-antibiotic agentsIn nature, secondary metabolites are important to the

organisms that produce them, functioning as: (1) sexhormones; (2) ionophores; (3) competitive weaponsagainst other bacteria, fungi, amoebae, insects andplants; (4) agents of symbiosis; and (5) effectors of dif-ferentiation. For years, most pharmaceuticals that wereused for non-infectious diseases were strictly syntheticproducts8. Similarly, most therapeutics for non-microbial parasitic diseases in animals (e.g. coccidiostatsand antihelminthics) came from the screening ofsynthesized compounds followed by molecular modi-fication. Despite the testing of thousands of syntheticcompounds, only a few promising structures werefound. As new lead compounds became more andmore difficult to find, microbial broths filled the voidand microbial products increased in importance in thetherapy of non-microbial diseases9. Today, microbiallyproduced polyethers such as monensin, lasalocid andsalinomycin dominate the coccidiostat market andare also the chief growth promoters in use for rumi-nant animals. The avermectins, another group ofstreptomycete products with a market of more thanUS$1 billion per year, have high activity againsthelminths and arthropods.

F F

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.

BiopesticidesAlso 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 agentsin farm animals (zearelanone), uterocontractants (ergotalkaloids) and antitumor agents (doxorubicin, daunoru-bicin, mitomycin, bleomycin).

Tropophase and idiophaseIn batch culture, most secondary metabolite processes

have a distinct growth phase (trophophase) followed bya production phase (idiophase). In other fermentations,the two phases overlap; the timing depends on thenutritional environment presented to the culture, thegrowth rate, or both. A delay in antibiotic productionuntil after trophophase helps the producing organismbecause the microbe is sometimes sensitive to its ownantibiotic during growth. Resistance mechanisms thatdevelop in producing microorganisms include enzy-matic modification of the antibiotic, alteration of thecellular target of the antibiotic and decreased uptake ofthe excreted antibiotic.

Directed biosynthesisThe manipulation of the culture media in any

devel-opment program often involves the testing of hundredsof additives as possible limiting precursors of the desiredproduct. 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 knownas directed biosynthesis.

Examples of directed biosynthesis include the use ofphenylacetic acid in the fermentation of benzylpeni-cillin, and specific amino acids in the production ofactinomycins and tyrocidins. Stimulatory precursorsinclude: methionine, as an inducer in cephalosporin Cformation; valine, in tylosin production; and trypto-phan for ergot-alkaloid production. In many fermen-tations, however, precursors show no activity becausetheir syntheses are not rate-limiting. In such cases,

F Fscreening 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 biotechnologyModern biotechnology is now over

25 years old10.In 1972, the birth of recombinant DNA technology

propelled biotechnology to new heights and led to theestablishment 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 andstrain improvement, bioreactor design and downstreamprocessing, cell immobilization (enzyme engineering),cell fusion, metabolic engineering, bioreactor design,downstream processing, in vitro mutagenesis (proteinengineering) and directed evolution of enzymes(applied molecular evolution).

Recombinant microorganismsThe revolutionary exploitation of microbial genetic

discoveries in the 1970s, 1980s and 1990s dependedheavily upon the solid structure of industrial micro-biology, described above. The major microbial hostsfor production of recombinant proteins are E. coli11, B.subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorphaand Aspergillus niger. The use of recombinant micro-organisms (Fig. 1) provided the techniques and experi-ence necessary for the successful application of higherorganisms, such as mammalian and insect cell culture,and transgenic animals and plants as hosts for theproduction of glycosylated recombinant proteins.

ProgressThe progress in biotechnology has been truly

remarkable. Within four years of the discovery ofrecombinant DNA technology, genetically engineeredbacteria were making human insulin and humangrowth hormone. This led to an explosion of invest-ment activity in new companies, mainly dedicated toinnovation via genetic approaches. Newer companiesentered the scene in various niches such as biochemi-cal engineering and downstream processing. Today,biotechnology in the USA is represented by some1300 companies with revenues of US$19.6 billion, ofwhich sales represent US$13.4 billion, and there are

TIBTECH JANUARY 2000 (Vol. 18) 29

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3 TIBTECH JANUARY 2000 (Vol.

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 biosynthesisAs mentioned previously, recombinant DNA tech-

niques have made a significant impact on the produc-tion of vitamins, amino acids, nucleotides, bioconversionand secondary metabolites. Most microbial biosyntheticpathways are encoded by clustered genes, which fa-cilitates the transfer of an entire pathway in a singlemanipulation. Even in fungi, pathway genes are some-times clustered, such as the penicillin genes in Penicilliumor the aflatoxin genes in Aspergillus. For the discoveryof new or modified secondary products, recombinantDNA techniques are being used to introduce genes forthe synthesis of one product into producers of otherantibiotics or into non-producing strains (combinatorialbiosynthesis).

Figure 1Escherichia 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 in1998, 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 the1990s, after years of lagging behind and, in 1998,1178 biotechnology companies existed with 45 000employees, and revenues of US$3.7 billion.

The major thrust of recombinant DNA technologyhas been in the area of rare mammalian peptides, suchas hormones, growth factors, enzymes, antibodies andbiological response modifiers12. Among those geneti-

cally engineered products that have been approvedfor use in the USA are human insulin, human growthhormone, erythropoietin, antihemophelia factor,granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, epidermalgrowth 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

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3 TIBTECH JANUARY 2000 (Vol.

Enzyme productionThe production of enzymes by fermentation was an

established business before modern microbial biotech-nology. However, recombinant DNA methodologywas so perfectly suited to the improvement of enzyme-production technology that it was almost immediatelyused 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 forcheese manufacture and recombinant lipase for use indetergents. Recombinant therapeutic enzymes alreadyhave a market value of over US$2 billion, being usedfor thromboses, gastrointestinal and rheumatic disorders,metabolic diseases and cancer. They include tissueplasminogen activator, human DNAase and Cerozyme.

AgricultureIndustrial microbiology through genetic engineering

and its associated disciplines has brought about a rev-olution in agriculture. Two bacteria have had a majorinfluence: Agrobacterium tumefaciens, a bacterium thatnormally produces crown gall tumors on dicotyledo-nous plants; and Bacillus thuringiensis, an insecticidalbacterium. The tumor-forming genes of A. tumefaciensare present on its tumor-inducing (Ti) plasmid, alongwith genes directing the plant to form opines (nutri-tional factors required by the bacterium that it cannotproduce by itself ). The Ti vector has been exceedinglyvaluable 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 problembypassed by, for example, the development of a particle-acceleration gun, which shoots DNA-coated metalparticles into plant cells. The activity of the insecticidalbacterium, B. thuringiensis, is caused by its crystal proteinproduced during sporulation. Crystals and spores havebeen applied to plants for many years to protect themagainst lepidopteran insects. B. thuringiensis preparationsare highly potent, approximately 300 times more active

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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.

ConclusionAlthough most of the early promises of biotechnology

have been achieved, major challenges remain. We mustuse our brains, technology, drive and dedication to solvethe problems of evolving diseases (e.g. AIDS), establisheddiseases (cancer and parasitic infection), antibiotic-resistance development and environmental pollution, byconverting urban, industrial and agricultural wastes intoresources such as liquid fuel. These efforts will requirecontinued interaction between different disciplines,major support by governments and international agen-cies, as well as an understanding and supportive public.

References1 Demain, A.L. (1990) Achievements in microbial technology.

Biotechnol. Adv. 8, 291–301

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, MarcelDekker

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 in1998. 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

Drug discovery in the new millennium: thepivotal role of biotechnologyGraham 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, 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 AstraZenecaPharmaceuticals, Alderley Park, Macclesfield, UK SK10 4TG.

F Fadvances, 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 31