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Appl Microbiol Biotechnol (2003) 61:385-392 DOI 10.

1007/s00253-003-1274-y

MINI-REVIEW

R. P. Elander

Industrial production of b-lactam antibiotics

Received: 27 November 2002 / Revised: 28 January 2003 / Accepted: 31 January 2003 / Published online: 3 April 2003 Springer-Verlag 2003

Abstract The industrial production of b-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the blactam antibiotics, particularly penicillins and cephalosporins, represent the worlds major biotechnology products with worldwide dosage form sales of ~US$ 15 billion or ~65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (~80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.

Introduction

The total world market for b-lactam antibiotics is now estimated to be ~US$ (hereafter $) 15 billion with cephalosporin dosage form sales at ~$9.9 billion and penicillin dosage form sales at ~$5 billion (Barber 1996, 2000). In 1996, the total world antibiotic market at the dosage form level was estimated to be ~$23 billion (Demain and Elander 1999). The United States antibacterial market was >$8 billion with cephalosporins ($3.6 billion), penicillins ($1.2 billion), fluoroquinolones ($0.9 billion), tetracyclines ($0.5 billion) and macrolides ($0.4 billion) reported recently by Strohl (1999). In contrast, the total world sales for antifungal products was ~$3 billion and growing and the antiviral market was ~$2.6 billion with a market forecast of >$5 billion for 2000. The b-lactam antibiotics now account for over 65% of the world antibiotic market. There are now more than 50 marketed cephalosporins. Many of these are listed in Table 1. The biosynthetic pathways for the penicillins, cephalosporins and cephamycins are well characterized both genetically and biochemically (Demain et al. 1998). A generalized pathway is shown in Fig. 1. Most of the important genes have now been cloned, with the exception of the isopenicillin N epimerase (cef D) gene (Brakhage 1998). Amplification of certain of the genes, in particular the deacetoxycephalosporin C synthase (cef E) gene, has been reported to increase cephalosporin C production and to decrease the levels of deacetoxycephalosporin C (DAOC) in production fermentation broths (Skatrud et al.1989; Basch and Chiang 1998). The transfer to and expression of the cef E gene from Streptomyces clavuligerus in Penicillium chrysogenum R.P. Elander is a former employee of Bristol-Myers Squibb resulted in the formation of adipyl-7-aminodeacetoxy(retired) cephalosporanic acid (adipyl-7-ADCA) when the altered P. chrysogenum strain was fed adipic acid (Crawford et R. P. Elander ()) al. 1995). In this manner, the inherent greater biosynthetic Biotechnology Consultant, capacity of P. chrysogenum may be used to produce 318 Gravilla Street, La Jolla, CA 92037-6006, USA e-mail: relan10731@aol.com DAOCs, moleculeswith expanding marketsthat are Tel.: +1-858-5514146 far more expensive to manufacture than penicillins
Fax: +1-858-5514146

386 Table 1 Marketed and experimental b-lactam antibiotics. Antibiotics italicized are major commercial antibiotics Subclass Penicillins Penicillin-resistant penicillins Antipseudomonal penicillins First-generation cephalosporins Second-generation cephalosporins Third-generation cephalosporins Fourth-generation cephalosporins Oxycephams Cefam Carbapenems Monobactams Clavams (b-lactamase-inhibitors) Penicillins/b-lactamase inhibitors
a Orally

Marketed b-lactam antibiotics Ampicillina, amoxicillina, bacampicillin, cloxacillin, floxacillin, mezlocillin, nafcillin, oxacillin, penicillin Ga, penicillin Va Methicillin, dicloxacillin Carbenicillin, indanyl piperacillin, ticarcillin Cefalothin, cephradinea, cefadroxyla, cefazolin, cephalexina Cefuroxine, cefaclora, cefotetam, cefmetazole, cefonicid Cefiximea, ceftibuten, cefizoxime, ceftriaxone, cefamandol cefoperazone, cefotaxime, proxetil, cefprozila, ceftazidime, cefuroxime axetil, cefpodexime Cefepime Flomoxef, latamoxef Cefoxitin Loracarbefa, imipenem, meropenem, panipenem Aztreonam, carumonam Clavulanate, sulbactam, tazobactam Amoxicillin/clavulanate, ampicillin/sulbactam, pipericillin/tazobactam, ticarcillin/clavulanate, cefoperazone/sulbactam

administered b-lactams Table 2 Changes in penicillin manufacturing technology Fermentation Carbon source Operational mode Medium sterilization Air filtration Feeds Morphology Cycle time Tank volume (1,000 gallons) Assay Control Titer (g/l) Recovery and purification Mycelium removal Operational mode Extraction stages Precursor recovery and re-use Efficiency (%) Environmental issues Bulk cost 1950 Lactose Batch Batch Depth filters None Filamentous 120 h 10-20 2000 Glucose/sucrose Fed-batch Continuous Membrane filters Many Pelleted 120-200 h 20-60

(Cantwell et al. 1990, 1992). This interesting technology has not been scaled to production levels at this time.

Commercial production of penicillin


The fermentation production of penicillin-G or -V is a fed-batch process carried out aseptically in stainless steel tank reactors of 30,000-100,000 gallon capacity. The fermentation usually involves two to three initial seed growth phases followed by a fermentation production phase having a time cycle ranging from 120 to 200 h. High dissolved oxygen levels are critical, especially during peak growth periods that often occur at the 40-50 h time-period of the cycle. The fermentation mode is fedbatch and crude sugar and precursor are fed throughout the cycle. Current penicillin fermentations are highly computerized and automated. Temperature, pH, dissolved oxygen, carbon dioxide, sugar, precursor, ammonia, etc. are closely monitored and controlled for optimal antibiotic production (Waites et al. 2001). Various carbon sources have been adopted for the fermentation including glucose, sucrose and other crude sugars. About 65% of the carbon is metabolized for cellular maintenance, 20-25% for growth and 10-12% for penicillin production (Van Nistelrooij et al. 1998). Sugar and precursor are fed continuously and the sugar is also used to help regulate the pH of the fermentation to between 6.4-6.8 during the active penicillin production phase. Corn steep liquor and cottonseed or soybean meal, ammonia and ammonium sulfate represent major nitrogen sources. The essential precursor substances are phenylacetic acid (for penicillin G) or phenoxyacetic acid (for penicillin V) that are either fed or batched. Mini-harvest protocols are often used in penicillin fermentations. This "batch-fill and withdraw" system involves the removal of 20-40% of the fermentor contents with replacement with fresh sterile medium. This procedure can be repeated several times during the fermenta-

Bio-assay HPLC Temperature only Computerized 0.5-1.0 >40 Filtration Batch Many Discarded 70-80 Few ~US$275-350/kg Whole broth Semi-continuous Single to few Recovered and re-used >90 Many ~US$15-20/kg

tion without yield reduction and, in reality, can enhance the total penicillin yield per fermentor. Penicillin G titers of ~100,000 U/ml have been reported by researchers at Panlabs (Rowlands 1991). Penicillin is excreted into the medium and is recovered at the end of the fermentation. Whole broth extraction is usually performed at acidic pH by most manufacturers and has resulted in a 2-5% improvement in overall extraction efficiency by the elimination of the rotary vacuum filtration step. Solvent extraction of chilled acidified broth is carried out with amyl, butyl or isobutyl acetate. Multiple back-extractions into buffer and solvent at varying pH using countercurrent contactors has led to considerable penicillin concentration in the early recovery stages of the purification process. Pigments and other broth impurities are removed by the use of activated charcoal. The penicillin is crystallized upon the addition

387 Fig. 1 Biosynthetic pathway for penicillins, cephalosporins and cephamycins

of potassium acetate and is isolated as a crystalline potassium salt. Additional carbon treatments and solvent washes results in a highly purified final product. Usually, conversion grade product used for 6-aminopenicillanic acid (6-APA) has lower purity and lower final product cost. Table 2 shows a number of the major production changes that have occurred in both upstream and downstream processing over the past five decades. In 1949, the United States was the major manufacturing country, with penicillin production amounting to ~83 tons of sodium penicillin G. In 1982, penicillin production was carried out throughout the world, with a total production of >12,000 tons and Europe being the

major manufacturing sector (Van der Beck and Roels 1984). In 1995, the total world production was reported to be ~33,000 tons, a five-fold increase since the late 1960s (Barber 1996). The tremendous increase in penicillin fermentation productivity (Elander and Chiang 1991) and corresponding high (>90%) recovery yield has led to significant cost reduction despite increasing labor, energy and raw material costs. In 1953, the bulk cost for penicillin G was ~$300/kg (Sylvester and Coghill 1954). In 1980, the bulk price for penicillin was ~$35/kg. In the late 1990s, bulk penicillin cost ranged from $10 to $20/kg and bulk marketed costs for 6-APA have been estimated to range

388 Table 3 Ranking of major blactam producers and world bulk production volumesa. 6APA 6-aminopenicillanic acid, 7-ACA 7-aminocephalosporanic acid, 7-ADCA 7-aminodeacetoxycephalosporanic acid Penicillins(1995) 1. Gist-Brocades 2. Antibioticos SpA 3. Biochemie 4. North China Pharma Works 5. Glaxo/SmithKline 6. Bristol-Myers Squibb World bulk production volumes (metric tons) Penicillins ~33,000 6-APA ~8,800 7-ADCA ~1,950 Intermediates ~2,130
a

Cephalosporins (1999) 1. Antibioticos SpA 2. Biochemie/Hoechst 3. Glaxo/Wellcome 4. Fujisawa 5. Cheil Jedang (Korea) 6. Bristol-Myers Squibb Cephalosporin C 7-ACA ~4,300 ~2,140

Adapted from Barber (1996, 2000)

from $35 to $40/kg. Table 3 lists the worlds major strated that penicillin sulfoxides could be rearranged to producers of penicillin and important bulk b-lactam form a variety of important cephalosporin derivatives. intermediates. When esters of penicillin sulfoxides are heated under acidic conditions, an acid-catalyzed chemical ring-expansion takes place (Morin et al. 1963). Eventually, a more efficient process was developed using silyl protection Semi-synthetic b-lactams during the ring expansion rearrangement. Silyl protection Approximately 75% of the total bulk penicillin volume chemistry has led to efficient chemical production of 7-ADCA and has led to highly efficient production of produced in 1995, ~33,000 tons, was used for the production of semi-synthetic penicillins and cephalospor- the oral cephalosporins, cephalexin and cephradine. ins (Barber 1996). A number of these important antibi- Cephadroxyl is synthesized after silylation of 7-aminootics are listed in Table 1. The penicillin nucleus (6-APA) cephalosporanic acid (7-ACA) followed by acylation with has enabled researchers to develop many excellent semi- a mixed anhydride prepared from a salt of r-hydroxsynthetic penicillins. 6-APA can also be chemically ring- yphenylglycine and ethylchloroformate. Amoxicillin is expanded to 7-ADCA to generate a number of important synthesized using a similar process. An important Lilly product, the cefaclor, involves a orally-active cephalosporins (cephalexin, cephradine, cefadroxyl, etc.). 6-APA has now grown to be the worlds ring enlargement of a penicillin V ester to an expanded cephalosporin-S oxide with an exocyclic double bond. largest selling b-lactam bulk intermediate. 6-APA was discovered in the late 1950s and although The product is a useful intermediate in that it can be microbial enzymes were soon discovered that hydrolyzed converted into 3-substituted cephalosporins and into penicillins to 6-APA, their use in production processes cefaclor, a highly prescribed oral cephalosporin with chlorine on the C-3 position. was abandoned in the late 1960s. Efficient chemical splitting technology was developed in Holland by Weissenburger and van der Hoeven (1970) and was soon adopted by many manufacturers. Environmental problems Cephalosporin C and important semi-synthetic and high solvent and energy costs prompted researchers to re- cephalosporins investigate enzymatic routes for 6-APA. When the reaction product is soluble in water, enzyme reuse is Cephalosporin C fermentation difficult since the enzyme was lost or degraded during the isolation of 6-APA. This problem was largely obviated in High-yielding strains of A. chrysogenum are used in largethe chemical splitting technology. Enzyme reuse and its scale, fed-batch fermentations. Major fermentation proassociated loss problems were soon eliminated by the ducers of cephalosporin C obtain harvest titers in the development of improved enzyme immobilization meth- range of 20-25 g/l. Production-scale fermentations are odology enabling long-term enzyme reuse. The immobi- fed-batch with carbon supplied as simple or complex lized enzyme can now be used in modified fluidized-bed carbohydrate feeds during the growth phase of the modules (Matsumoto 1993). Recombinant Escherichia fermentation. As the fermentation progresses, sugar feeds coli strains are used as extremely efficient producers of are reduced and are usually replaced by higher energy oils penicillin G acylase (Savidge 1984) and recombinant such as soybean oil or peanut oil. Energy conservation strains of Fusarium oxysporum can be used as highly from oil as a substrate is considerably less efficient and efficient producers of penicillin V acylase (Chiang and leads to slower growth, with the vegetative mycelium Basch 1999). becoming largely transformed into multicellular arthrosThe chemical relationship of the penicillin (thiazoli- pores. The arthrospore stage leads to greater oxygen dine) and the cephalosporin (dihydrothiazine) skeletons availability to the organism and results in rapid cephalowas established in the early 1960s, when it was demonsporin production.

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dl-Methionine addition, which also results in the onset of arthrospore formation, is often added to the medium during the early growth phase of the fermentation. The formation of arthrospores is also correlated with improved dissolved oxygen concentration in the broth and is critical for maximal expression of the important biosynthetic cyclase and expandase enzymes. Organic nitrogen is often supplied as a combination of soybean and cottonseed meals supplemented with ammonium sulfate and ammonia that is also used to help control the pH throughout the fermentation. Corn steep liquor is also supplied as a cheap nitrogen source and is rich in amino acids, vitamins, organic acids and trace elements. The pH of the fermentation is maintained between 6.2 and 7.0 and the temperature range is controlled between 24 and 28 C. A major problem associated with cephalosporin C fermentation is the inherent chemical instability of the cephalosporin C molecule. This is probably one of the major reasons why long-cycle cephalosporin C fermentations often result in reduced cephalosporin production compared to typical long-cycle penicillin fermentations. Cephalosporin C is readily degraded to compound X (2-(d-4-amino-4-carboxybutyl)-thiazole-4-carboxylic acid), which can account for as much as a ~40% loss of the cephalosporin C produced (Usher et al. 1988). The biosynthetic precursor molecules of cephalosporin C, deacetylcephalosporin C and DAOC have much more chemical stability. Strains of the yeast, Rhodosporidium toruloides possess a potent acetyl esterase and, when the organism is added to active cephalosporin C fermentations, result in increased levels of deacetylcephalosporin C with an increase in total cephalosporin nucleus levels of ~40% (Chiang and Basch 1999). Over the past decade, the cloning of many of the genes involved in the biosynthetic pathway of cephalosporins has resulted in more productive strains. Researchers at Lilly demonstrated that the expandase/hydroxylase activity could be rate-limiting in certain production strains (Skatrud et al. 1989). Transformants with an extra copy of the cef EF gene had twice the expandase/hydroxylase activity of the non-transformed strain, a reduction of penicillin N levels and an increase in cephalosporin C titer. The increase in cephalosporin C titer varied from 47% in laboratory scale fermentations to 15% in pilot plant fermentors (Skatrud et al. 1989). There have been no further reports from Lilly regarding the productivity of the engineered strains in large production fermentors. Basch and Chiang (1998) reported on the use of genetic engineering strategies to reduce the levels of undesirable byproducts in cephalosporin C fermentations at Bristol-Myers Squibb. They showed that using a recombinant strain of A. chrysogenum with an increased copy number of the bifunctional expandase/hydroxylase (cef EF) gene resulted in a reduced level of DAOC in large production fermentors. The recovery and purification of these broths and subsequent chemical conversion to 7-ACA resulted in significant reduction of contaminating 7-ADCA.

Cephalosporin C recovery and purification The purification and recovery of harvest cephalosporin C broth begins with the rapid chilling of the active broth to 3-5 C followed by removal of the mycelial solids either by filtration or by centrifugation. The active broth contains not only the desired cephalosporin C component, but also small quantities of the biosynthetic precursors, penicillin N, DAOC, deacetylcephalosporin C and the degraded cephalosporin C product, compound X. Two major strategies can be used for the recovery and purification of cephalosporin C. One strategy involves the use of activated carbon or the use of a non-ionic resin. Because of the high selectivity of the resin, cephalosporin C is preferentially adsorbed over penicillin N or the contaminating biosynthetic precursor molecules. Most of the penicillin N is removed in the pH 2.0 acidification step. An additional anion- and cation-exchange step usually results in high quality cephalosporin C. A large fraction of the cephalosporin C is converted to 7-ACA and derivatized to semi-synthetic cephalosporins. A second purification strategy involves the substitution of the amine moiety on the a-aminoadipyl side-chain at C-7. Two substituted derivatives, N-2,4-dichlorobenzoyl cephalosporin C and tetrabromocarboxybenzoyl cephalosporin C, can be crystallized from acidic aqueous solution. Alternatively, salts can be formed between the N-substituted derivatives and an organic base such as dicyclohexylamine or dimethylbenzylamine results in cephalosporin salts that are solvent extractable. BristolMyers Squibb uses a solvent-extractable process resulting in the isochlorobutylformate (ICBF) ester of cephalosporin C, termed cephalosporin D. Several extraction steps are usually necessary to achieve the final desired purity. N-Substituted cephalosporin C salts containing small amounts of contaminants can be effectively converted to 7-ACA. Efficient enzymatic processes are now utilized for the conversion of cephalosporins to 7-ACA, which has resulted in dramatic cost reduction for this important bulk intermediate. Two key genetically engineered enzymes are involved. The initial step is reaction of the a-aminoadipyl group with d-amino acid oxidase to produce glutaryl-7-ACA. This reaction proceeds through a keto-7-ACA intermediate that undergoes an oxidative decarboxylation in the presence of hydrogen peroxide. A glutaryl acylase is used to remove the glutaryl side-chain to produce 7-ACA. About one-third of commercial cephalosporins are derived from 7-ADCA. Due to the lower cost of penicillin, 7-ADCA is usually produced from penicillin G by ring expansion of a penicillin sulfoxide ester to yield a cephalosporin ester. The ester group is removed, followed by removal of the phenylacetyl side-chain to give 7-ADCA. Two-thirds of the commercial cephalosporins are derived from 7-ACA that is produced from cephalosporin C by either chemical or enzymatic deacylation. In the chemical process, after protection of the amino and

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carboxyl groups, reaction with potassium pentachloride in the presence of base forms an iminochloride derivative. The iminoether is formed on the addition of alcohol. The iminoether is hydrolyzed to form 7-ACA. Enzymatic processes are now used by the major producers of 7-ACA. Recent bulk market costs for 7-ACA ranges from $115 to $130/kg. Antibioticos SpA and Biochemie/Hoechst produced nearly 50% of the worlds market needs in 1998 (Barber 2000). Other major producers of 7-ACA are Glaxo-Wellcome, Fujisawa and Bristol-Myers Squibb (Table 3).

Cephamycins
The cephamycins are derived from the cephalosporins by methoxylation at the C-7 position. An enzymatic system was identified in S. clavuligerus that converted cephalosporin C or O-carbamoyl-DAOC to the a-methoxy derivatives. The discovery of cephamycin C at Merck in the early 1970s led to considerable research and development on prokaryotic cephalosporins, since the presence of the methoxy group on the b-lactam ring made the molecule more active against Gram-negative and anaerobic pathogens and more resistant to Gram-negative blactamase (Stapley et al. 1979). Thus, for the first time, methoxylated cephalosporins were available that showed a high degree of stability to b-lactamase enzymes. Cephamycin C was never used clinically, but was employed for the semi-synthesis of many medically useful compounds. The early fermentation studies of 7-methoxycephalosporin antibiotics by Streptomyces lipmanii and S. clavuligerus were reported by Nagarajan (1972) and by Stapley et al. (1972). Extremely low titers of 130-170 mg/ ml were reported. The cephamycin broths are recovered and purified by inactivating penicillin N by adjusting the pH of the clarified broth to 2.0-2.5. The cephamycins are isolated by a combination of active carbon adsorption followed by adsorption on an anion-exchange resin. Small amounts of DAOC can be removed by passage through silica gel columns with 30% aqueous acetonitrile as the eluant (Nagarajan 1972). Cefoxitin is a marketed cephamycin-type cephalosporin that is now manufactured by chemical synthesis. Other marketed molecules in this class are cefmetazole, temocillin and cefotetan (Table 1).

oxazolidine ring (Howarth et al. 1976). The antibiotic is produced by strains of S. clavuligerus (Reading and Cole 1977). Clavulanic acid shows broad affinity for a number of penicillin-binding proteins and is currently used in combination with amoxicillin and is marketed under the trade name Augumentin for the treatment of infections caused by b-lactamase producing pathogenic bacteria. Strains of S. clavuligerus are propagated on a fermentation medium containing soy bean meal, soluble starch, glycerol and potassium phosphate at a pH of 6.5 at 26 C for 100 h (Lawrence and Lilly 1980). There is no recently reported production data for the clavulanic acid fermentation. The bulk of the clavulanic acid is found in culture filtrates. Adsorption of the antibiotic on active carbon followed by elution with aqueous acetone or solvent extraction at pH 2.0 using butanol with back-extraction into water at pH 7.0 proved to be effective primary purification protocols. Secondary purification has been achieved by conversion of the purified clavulanate to the benzyl ester, which was then dissolved in ethyl acetate and subjected to two chromatographic steps using Sephadex LH20 and silica gel. The purified benzyl ester was then hydrogenated over 10% Pd/C in the presence of sodium bicarbonate to yield sodium clavulanate tetrahydrate. Clavulanic acid possesses only weak antibiotic activity against a variety of Gram-positive and Gram-negative bacteria, but is an excellent inhibitor of a variety of blactamases. The molecule has been coformulated with a variety of broad-spectrum semi-synthetic penicillins with amoxicillin being one of the best known formulations. Augmentin had a world sales value of ~$1.3 billion in 1995 and was the second largest selling antibacterial that year.

Carbapenems

The carbapenems resemble the penicillins, having a blactam ring fused to a five-membered ring that does not contain sulfur. Sulfur is present in the molecule outside the ring in all carbapenems produced by streptomycetes. A large number of carbapenems have been discovered, but thienamycin produced by a unique strain of Streptomyces cattleya is the only carbapenem that is medically and industrially important at this time. Thienamycin is one of the most potent, broadspectrum, non-toxic antibacterial compounds ever disClavulanic acid covered. It was discovered at Merck by Kahan et al. Clavulanic acid has relatively weak antibacterial activity, (1979) by a highly sensitive screening protocol based on but has been shown to be a potent inhibitor of the b- the inhibition of peptidoglycan synthesis. The chemical lactamases produced by staphylococci and plasmid- structure of thienamycin was reported by Albers-Schoenmediated b-lactamases of Escherichia coli, Klebsiella, berg et al. (1978). The multi-component nature of carbapenem fermentaProteus, Shigella, Pseudomonas and Hemophilus (Brown tions, its extremely low titer and high chemical instability et al. 1976). The molecule is a naturally produced made recovery and purification of the antibiotic extremecompound consisting of a b-lactam ring fused to an ly difficult. These many difficulties led Mercks chemists

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to employ organic synthesis rather than fermentation as the preferred route for its commercial production (Miller 1981). Thienamycin decomposes in dilute aqueous solution and its decomposition accelerates as its concentration is increased. A more stable and more potent derivative, Nformimidoylthienamycin was developed and marketed as imipenem. The molecule has high resistance to bacterial b-lactamases, but is readily degraded by human renal peptidase. A renal peptidase inhibitor, cilastatin, was synthesized and formulated with the product. Imipenem is a highly successful antibiotic and its world market, which had grown to <$500 million by 1995, is among the top ten marketed b-lactam antibiotics.

acid, produced adipyl-7-ADCA, which can be easily hydrolyzed to 7-ADCA, the major building block for many oral cephalosporins. In this way, the greater antibiotic-producing capability of P. chrysogenum could be used for the manufacture of 7-ADCA. This novel genetic engineering strategy could result in significant cost reduction for many oral cephalosporin products. Gene amplification of the bifunctional expandase/ hydroxylase gene in strains of C. acremonium has led to improved fermentation of cephalosporin C as well as decreased production of contaminating DAOC in large production fermentors at Bristol-Myers Squibb. The lower DAOC content resulted in more acceptable 7ACA with a higher purity (Basch and Chiang 1998). The future, and need, for new b-lactam antibiotics remains bright. Major pharmaceutical companies continFuture prospects regarding the industrial production ue to have research and development efforts for new and of b-lactam antibiotics improved b-lactam biotechnology and chemistry because of The production efficiencies of the two major marketed b- the need to discover new molecules due to the ever lactam antibiotic classes, penicillins and cephalosporins, increasing, continuing emergence of resistant bacterial have now been under major development for over four pathogens. New types of b-lactamase enzymes continue to decades. As a consequence, the bulk production costs for be discovered and the need for new semi-synthetic or penicillin and its important bulk intermediate, 6-APA, can chemically-synthesized inhibitors is great. now almost be considered as commodity chemicals when compared to other major marketed pharmaceuticals. However, with increasing higher labor, energy and raw References material costs, especially in Europe, the United States and Albers-Schoenberg G, Arison BH, Hensens OD, Hirshfield J, Japan, more bulk penicillin manufacturing is moving to Hoogsteen K, Kaczka EA, Rhodes RE, Kahan JJ, Kahan FM, the Far East and other developing countries (Barber Ratcliffe RW, Walton E, Ruswinkle LJ, Morin RB, Christiansen BG (1978) The structure and absolute configuration of 1996). Bristol-Myers Squibb has the only major manuthienamycin. J Am Chem Soc 100:6491-6499 facturing plant producing bulk penicillin V and 6-APA in Barber M (1996) The penicillins business. Michael Barber and the United States. Associates, 18 Croydon Road, Catterham, Surrey, UK Cephalosporins are continuing to gain market share, Barber M (2000) The cephalosporins business: 2000 and beyond. Michael Barber and Associates, 18 Croydon Road, Catterham, and their final product cost still justifies continued Surrey, UK development and manufacture in Europe, Japan and the J, Chiang S-JD (1998) Genetic engineering approach to United States. New and effective cephalosporins continue to Basch reduce undesirable products in cephalosporin C fermentation. J be introduced and there is still a major need to find Ind Microbiol Biotechnol 20:344-351 more effective cephalosporins for methicillin-resistant Brakhage AA (1998) Molecular regulation of b-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev 62:547-585 (MRSA) and other resistant pathogens. The development of new, fifth-generation, cephalos- Brown AG, Butterworth D, Cole M, Hanscomb G, Hood JD, Reading C, Rolinson CN (1976) Naturally occurring b-lactaporins will depend more on an increased understanding of bmase inhibitors without antibacterial activity. J Antibiot lactam biosynthetic regulation and sophisticated meta29:668-669 Cantwell CA, Beckman RJ, Dotzlaf JE, Fisher DL, Skatrud PL, bolic engineering of the producing organisms. Yeh WH, Queener SW (1990) Cloning and expression of a Most of the improvement in productivity of penicillin hybrid Streptomyces clavuligerus cef E gene in Penicillium and cephalosporin biosynthesis was obtained through chrysogenum. Curr Genet 17:213-221 intensive screening of variant strains following mutagen- Cantwell CA, Beckman R, Whiteman P, Queener SW, Abraham E esis and through recent applications of genetic engineer(1992) Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: coning (Chiang et al. 1991; Elander and Chiang 1991). With struction of a new biosynthetic pathway. Proc R Soc London B an increased knowledge of biosynthetic gene clustering 238:283-289 and possible gene transfer between already efficient b- Chiang SJ, Chang LT, Chen YS, Hou HH, Elander RP (1991) Strain lactam organisms, this may lead to more rational improvement in Penicillium chrysogenum, from classical genetics to genetic engineering. In: Kleinkauf H, von Dohren approaches to new, improved b-lactam products and H (eds) 50 years of penicillin applications: history and trends. improved process technologies for more efficient bPublic, Czech Republic, pp 245-257 lactam antibiotic manufacture. Researchers at Merck and Chiang S-JD, Basch J (1999) Cephalosporins. In: Flickinger MC, Panlabs have reported on the introduction of the exDrew SW (eds) Encyclopedia of bioprocess technology: fermentations, biocatalysis and bioseparations, vol 1. Wiley, pandase gene from S. clavuligerus to a high-producing New York, pp 560-570 strain of P. chrysogenum (Crawford et al. 1995). The Crawford L, Stepan AM, McAda PC, Rambosek JA, Conder MJ, recombinant penicillin-producing strain, when fed adipic Vinci VA, Reeves CD (1995) Production of cephalosporin

392 intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Biotechnology 13:58-62 Demain AL, Elander RP (1999) The b-lactam antibiotics: past, present and future. Antonie van Leeuwenhoek 75:5-19 Demain AL, Martin JF, Elander RP (1998) Penicillin biochemistry and genetics. In: Mateles RI (ed) Penicillin: a paradigm for biotechnology. Candida, Chicago, pp 98-114 Elander RP, Chiang S-J (1991) Genetics and antibiotic process improvement: from classical genetics to genetic engineering. In: Procop A, Bajpai RK, Ho C (eds) Recombinant DNA technology and applications. McGraw-Hill, New York, pp 153170 Howarth TT, Brown AG, King TJ (1976) Clavulanic acid, a novel b-lactam isolated from Streptomyces clavuligerus: X-ray crystal structure analysis. J Chem Soc Chem Commun 266-267 Kahan JS, Kahan FM, Goegelman R, Currie SA, Jackson M, Stapley EO, Miller TW, Hendlin D, Mochales S, Hernandez S, Woodrull HB, Birnbaum J (1979) Thienamycin, a new betalactam antibiotic. 1, discovery, taxonomy, isolation and physical properties. J Antibiot 32:1-12 Lawrence GC, Lilly G (1980) UK Patent 1,571,888 Matsumoto K (1993) Production of 6-APA, 7-ACA and 7-ADCA by immobilized cephalosporin amidase. In: Tanaka A, Tosa T, Kobayashi T (eds) Industrial application of immobilized biocatalysis. Dekker, New York, pp 67-88 Miller TW (1981) Crystalline N-formimidoylthienamycin. US Patent 4,260,543 Morin RB, Jackson BG, Mueller RA, Lavagnino ER, Scanlon WB, Andrews SL (1963) Chemistry of cephalosporin antibiotics. 3. Chemical correlation of penicillin and cephalosporin antibiotics. J Am Chem Soc 85:1896-1897 Nagarajan R (1972) b-Lactam antibiotics from Streptomyces. In: Flynn EH (ed) Cephalosporins and penicillins: chemistry and biology. Academic Press, New York, pp 636-661 Reading E, Cole M (1977) Clavulanic acid: a beta-lactamase inhibitor from Streptomyces clavuligerus. Antimicrob Agents Chemother 11:852-857 Rowlands RT (1991) Industrial strain improvement and the Panlabs penicillin club. In: Kleinkauf H, von Dohren H (eds) 50 years of penicillin applications: history and trends. Public, Czech Republic, pp 258-266 Savidge TA (1984) Enzymatic conversion used in the production of penicillins and cephalosporins. In: Vandamme E (ed) Biotechnology of industrial antibiotics. Dekker, New York, pp 191-224 Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DC, Chapman JL, Queener SW (1989) Use of recombinant DNA to improve the production of cephalosporin C by Cephalosporium acremonium. Biotechnology 7:477-485 Stapley EO, Jackson M, Hernandez S, Zimmerman SB, Currie SA, Mochalis S, Mahta JM, Woodruff HB, Hendlin D (1972) Cephamycins, a new family of b-lactam antibiotics. 1. Production by actinomycetes, including Streptomyces lactamdurans sp. n. Antimicrob Agents Chemother 2:122-131 Stapley EO, Birnbaum J, Miller AK, Wallick H, Hendlin D, Woodruff HB (1979) Cefoxitin and cephamycins: microbiological studies. Ref Infect Dis 1:73-87 Strohl WR (1999) Secondary metabolites; antibiotics. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparations, vol 5. Wiley, New York, pp 2348-2365 Sylvester JC, Coghill RD (1954) The penicillin fermentation. In: Underkofler LA, Hickey RJ (eds) Industrial fermentations, vol 2. Chemical Publishing, New York, pp 219-263 Usher JJ, Lewis MA, Hughes DW, Compton BJ (1988) Development of the cephalosporin C fermentation taking into account the instability of cephalosporin C. Biotechnol Lett 10:543-548 Van der Beck CP, Roels JA (1984) Penicillin production: biotechnology at its best. Antonie van Leeuwenhoek 50:625639 Van Nistelrooij, Krijgsman J, DeVroom E, Oldenhof C (1998) Penicillin update. In: Mateles RI (ed) Penicillin: a paradigm for biotechnology. Candida, Chicago, pp 85-91 Waites MJ, Morgan NL, Rockey JS, Higton G (2001) Industrial microbiology: an introduction. Blackwell, Oxford, UK Weissenberger HWO, van der Hoeven MB (1970) An efficient nonenzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid. Recl Trav Chim Pays-Bas 89:1081-1084