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 antibi-otics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the b-lactam antibiotics, particularly penicillins and cephalos-porins, represent the world’s major biotechnology prod-ucts 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. Ceph-alosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation pro-ducers 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 hydrolysisprocess technology for 6-aminopenicillanic acid or7-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.

R.P. Elander is a former employee of Bristol-Myers Squibb (retired)

R. P. Elander ())Biotechnology Consultant,318 Gravilla Street, La Jolla, CA 92037-6006, USA e-mail: relan10731@aol.comTel.: +1-858-5514146Fax: +1-858-5514146

Introduction

The total world market for b-lactam antibiotics is now estimated to be ~US$ (hereafter $) 15 billion withcephalosporin dosage form sales at ~$9.9 billion andpenicillin 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 antibac-terial 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). Incontrast, 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, cepha-losporins 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 excep-tion of the isopenicillin N epimerase (cef D) gene(Brakhage 1998). Amplification of certain of the genes, in particular the deacetoxycephalosporin C synthase (cefE) gene, has been reported to increase cephalosporin C production and to decrease the levels of deacetoxy-cephalosporin C (DAOC) in production fermentationbroths (Skatrud et al.1989; Basch and Chiang 1998). The transfer to and expression of the cef E gene from Streptomyces clavuligerus in Penicillium chrysogenum resulted in the formation of adipyl-7-aminodeacetoxy-cephalosporanic acid (adipyl-7-ADCA) when the altered P. chrysogenum strain was fed adipic acid (Crawford et al. 1995). In this manner, the inherent greater biosynthetic capacity of P. chrysogenum may be used to produce DAOCs, molecules—with expanding markets—that are far more expensive to manufacture than penicillins

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Table 1 Marketed and experimental b-lactam antibiotics. Antibiotics italicized are major commercial antibiotics

Subclass Marketed b-lactam antibiotics

Penicillins

Penicillin-resistant penicillins Antipseudomonal penicillins First-generation cephalosporins Second-generation cephalosporins Third-generation cephalosporins

Fourth-generation cephalosporins OxycephamsCefamCarbapenemsMonobactamsClavams (b-lactamase-inhibitors) Penicillins/b-lactamase inhibitors

Ampicillina, amoxicillina, bacampicillin, cloxacillin, floxacillin, mezlocillin, nafcillin, oxacillin,penicillin Ga, penicillin Va

Methicillin, dicloxacillinCarbenicillin, indanyl piperacillin, ticarcillinCefalothin, cephradinea, cefadroxyla, cefazolin, cephalexina

Cefuroxine, cefaclora, cefotetam, cefmetazole, cefonicidCefiximea, ceftibuten, cefizoxime, ceftriaxone, cefamandol cefoperazone, cefotaxime, proxetil,

cefprozila, ceftazidime, cefuroxime axetil, cefpodeximeCefepimeFlomoxef, latamoxefCefoxitinLoracarbefa, imipenem, meropenem, panipenemAztreonam, carumonamClavulanate, sulbactam, tazobactamAmoxicillin/clavulanate, ampicillin/sulbactam, pipericillin/tazobactam, ticarcillin/clavulanate,

cefoperazone/sulbactam

a Orally administered b-lactams

(Cantwell et al. 1990, 1992). This interesting technologyhas 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. Thefermentation 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 fed-batch 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 antibi-otic 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 forcellular 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 phenyl-acetic 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 proce-dure can be repeated several times during the fermenta-

Table 2 Changes in penicillin manufacturing technology

Fermentation 1950 2000

Carbon source Lactose Glucose/sucroseOperational mode Batch Fed-batchMedium sterilization Batch ContinuousAir filtration Depth filters Membrane filtersFeeds None ManyMorphology Filamentous PelletedCycle time 120 h 120-200 hTank volume 10-20 20-60

(1,000 gallons)Assay Bio-assay HPLCControl Temperature only ComputerizedTiter (g/l) 0.5-1.0 >40Recovery and purificationMycelium removal Filtration Whole brothOperational mode Batch Semi-continuousExtraction stages Many Single to fewPrecursor recovery Discarded Recovered

and re-use and re-usedEfficiency (%) 70-80 >90Environmental issues Few ManyBulk cost ~US$275-350/kg ~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 overallextraction 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

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Fig. 1 Biosynthetic pathway for penicillins, cephalosporins and cephamycins

of potassium acetate and is isolated as a crystallinepotassium 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 down-stream processing over the past five decades.

In 1949, the United States was the major manufactur-ing country, with penicillin production amounting to ~83 tons of sodium penicillin G. In 1982, penicillinproduction 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 Roels1984). 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 correspond-ing 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

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Table 3 Ranking of major b-lactam producers and world bulk production volumesa. 6-APA 6-aminopenicillanic acid, 7-ACA 7-aminocephalosporanic acid, 7-ADCA 7-aminodeace-toxycephalosporanic acid

Penicillins(1995)

1. Gist-Brocades2. Antibioticos SpA3. Biochemie4. North China Pharma Works5. Glaxo/SmithKline6. Bristol-Myers SquibbWorld bulk production volumes (metric tons)Penicillins ~33,0006-APA ~8,8007-ADCA ~1,950Intermediates ~2,130

Cephalosporins (1999)

1. Antibioticos SpA2. Biochemie/Hoechst3. Glaxo/Wellcome4. Fujisawa5. Cheil Jedang (Korea)6. Bristol-Myers Squibb

Cephalosporin C ~4,3007-ACA ~2,140

a Adapted from Barber (1996, 2000)

from $35 to $40/kg. Table 3 lists the world’s majorproducers of penicillin and important bulk b-lactam intermediates.

Semi-synthetic b-lactams

Approximately 75% of the total bulk penicillin volume produced in 1995, ~33,000 tons, was used for theproduction of semi-synthetic penicillins and cephalospor-ins (Barber 1996). A number of these important antibi-otics are listed in Table 1. The penicillin nucleus (6-APA) has enabled researchers to develop many excellent semi-synthetic penicillins. 6-APA can also be chemically ring-expanded to 7-ADCA to generate a number of important orally-active cephalosporins (cephalexin, cephradine, ce-fadroxyl, etc.). 6-APA has now grown to be the world’s largest selling b-lactam bulk intermediate.

6-APA was discovered in the late 1950s and although microbial enzymes were soon discovered that hydrolyzed penicillins to 6-APA, their use in production processes was abandoned in the late 1960s. Efficient chemicalsplitting technology was developed in Holland by Weis-senburger and van der Hoeven (1970) and was soon adopted by many manufacturers. Environmental problems and high solvent and energy costs prompted researchers to re-investigate enzymatic routes for 6-APA. When thereaction product is soluble in water, enzyme reuse is difficult since the enzyme was lost or degraded during the isolation of 6-APA. This problem was largely obviated in the chemical splitting technology. Enzyme reuse and its associated loss problems were soon eliminated by the development of improved enzyme immobilization meth-odology enabling long-term enzyme reuse. The immobi-lized enzyme can now be used in modified fluidized-bed modules (Matsumoto 1993). Recombinant Escherichia coli strains are used as extremely efficient producers of penicillin G acylase (Savidge 1984) and recombinantstrains of Fusarium oxysporum can be used as highly efficient producers of penicillin V acylase (Chiang and Basch 1999).

The chemical relationship of the penicillin (thiazoli-dine) and the cephalosporin (dihydrothiazine) skeletons was established in the early 1960s, when it was demon-

strated that penicillin sulfoxides could be rearranged toform a variety of important cephalosporin derivatives. When esters of penicillin sulfoxides are heated under acidic conditions, an acid-catalyzed chemical ring-expan-sion takes place (Morin et al. 1963). Eventually, a more efficient process was developed using silyl protection during the ring expansion rearrangement. Silyl protection chemistry has led to efficient chemical production of 7-ADCA and has led to highly efficient production of the oral cephalosporins, cephalexin and cephradine. Cephadroxyl is synthesized after silylation of 7-amino-cephalosporanic acid (7-ACA) followed by acylation with a mixed anhydride prepared from a salt of r-hydrox-yphenylglycine and ethylchloroformate. Amoxicillin is synthesized using a similar process.

An important Lilly product, the cefaclor, involves a ring enlargement of a penicillin V ester to an expanded cephalosporin-S oxide with an exocyclic double bond. The product is a useful intermediate in that it can be converted into 3-substituted cephalosporins and intocefaclor, a highly prescribed oral cephalosporin with chlorine on the C-3 position.

Cephalosporin C and important semi-synthetic cephalosporins

Cephalosporin C fermentation

High-yielding strains of A. chrysogenum are used in large-scale, fed-batch fermentations. Major fermentation pro-ducers of cephalosporin C obtain harvest titers in the range of 20-25 g/l. Production-scale fermentations are fed-batch with carbon supplied as simple or complex carbohydrate feeds during the growth phase of the fermentation. As the fermentation progresses, sugar feeds are reduced and are usually replaced by higher energy oils such as soybean oil or peanut oil. Energy conservation from oil as a substrate is considerably less efficient and leads to slower growth, with the vegetative mycelium becoming largely transformed into multicellular arthros-pores. The arthrospore stage leads to greater oxygen availability to the organism and results in rapid cephalo-sporin production.

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dl-Methionine addition, which also results in the onsetof 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 ammo-nium 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 fermen-tations 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). Thebiosynthetic 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 fermenta-tions, 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 activ-ity 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 ofgenetic 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 purifica-tion of these broths and subsequent chemical conversion to 7-ACA resulted in significant reduction of contami-nating 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 cephalo-sporin 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. Bristol-Myers Squibb uses a solvent-extractable process resulting in the isochlorobutylformate (ICBF) ester of cephalo-sporin 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 hasresulted 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 ofpenicillin, 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 inthe 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 andBiochemie/Hoechst produced nearly 50% of the world’s market needs in 1998 (Barber 2000). Other majorproducers 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 cephalo-sporin 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 devel-opment on prokaryotic cephalosporins, since the presence of the methoxy group on the b-lactam ring made the molecule more active against Gram-negative and anaer-obic pathogens and more resistant to Gram-negative b-lactamase (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-methoxy-cephalosporin 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 cephalospor-in that is now manufactured by chemical synthesis. Other marketed molecules in this class are cefmetazole, temocillin and cefotetan (Table 1).

Clavulanic acid

Clavulanic acid has relatively weak antibacterial activity, but has been shown to be a potent inhibitor of the b-lactamases produced by staphylococci and plasmid-mediated b-lactamases of Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Hemophilus (Brown et al. 1976). The molecule is a naturally producedcompound consisting of a b-lactam ring fused to an

oxazolidine ring (Howarth et al. 1976). The antibiotic isproduced 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 fermen-tation 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 fermen-tation.

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 primarypurification 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 tetrahy-drate.

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 b-lactamases. 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 b-lactam 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 Strepto-myces cattleya is the only carbapenem that is medically and industrially important at this time.

Thienamycin is one of the most potent, broad-spectrum, non-toxic antibacterial compounds ever dis-covered. It was discovered at Merck by Kahan et al. (1979) by a highly sensitive screening protocol based on the inhibition of peptidoglycan synthesis. The chemical structure of thienamycin was reported by Albers-Schoen-berg et al. (1978).

The multi-component nature of carbapenem fermenta-tions, its extremely low titer and high chemical instability made recovery and purification of the antibiotic extreme-ly difficult. These many difficulties led Merck’s chemists

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to employ organic synthesis rather than fermentation asthe 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, N-formimidoylthienamycin 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.

Future prospects regarding the industrial production of b-lactam antibiotics

The production efficiencies of the two major marketed b-lactam antibiotic classes, penicillins and cephalosporins, have now been under major development for over four decades. As a consequence, the bulk production costs for penicillin and its important bulk intermediate, 6-APA, can now almost be considered as commodity chemicals when compared to other major marketed pharmaceuticals. However, with increasing higher labor, energy and raw material costs, especially in Europe, the United States and Japan, more bulk penicillin manufacturing is moving to the Far East and other developing countries (Barber1996). Bristol-Myers Squibb has the only major manu-facturing plant producing bulk penicillin V and 6-APA in the United States.

Cephalosporins are continuing to gain market share, and their final product cost still justifies continued development and manufacture in Europe, Japan and the United States. New and effective cephalosporins continue to be introduced and there is still a major need to find more effective cephalosporins for methicillin-resistant (MRSA) and other resistant pathogens.

The development of new, fifth-generation, cephalos-porins will depend more on an increased understanding of b-lactam biosynthetic regulation and sophisticated meta-bolic engineering of the producing organisms.

Most of the improvement in productivity of penicillin and cephalosporin biosynthesis was obtained through intensive screening of variant strains following mutagen-esis and through recent applications of genetic engineer-ing (Chiang et al. 1991; Elander and Chiang 1991). With an increased knowledge of biosynthetic gene clustering and possible gene transfer between already efficient b-lactam organisms, this may lead to more rational approaches to new, improved b-lactam products and improved process technologies for more efficient b-lactam antibiotic manufacture. Researchers at Merck and Panlabs have reported on the introduction of the ex-pandase gene from S. clavuligerus to a high-producing strain of P. chrysogenum (Crawford et al. 1995). The recombinant penicillin-producing strain, when fed adipic

acid, produced adipyl-7-ADCA, which can be easilyhydrolyzed 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 7-ACA with a higher purity (Basch and Chiang 1998).

The future, and need, for new b-lactam antibiotics remains bright. Major pharmaceutical companies contin-ue to have research and development efforts for new and improved b-lactam biotechnology and chemistry because of the need to discover new molecules due to the ever increasing, continuing emergence of resistant bacterial pathogens. New types of b-lactamase enzymes continue to be discovered and the need for new semi-synthetic or chemically-synthesized inhibitors is great.

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