Metabolic engineering of b-lactam production

Metabolic Engineering 5 (2003) 56–69

Metabolic engineering of b-lactam production Jette Thykaer and Jens Nielsen Center for Process Biotechnology, BioCentrum, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark Received 4 October 2002; accepted 30 December 2002

Abstract Metabolic engineering has become a rational alternative to classical strain improvement in optimisation of b-lactam production. In metabolic engineering directed genetic modification are introduced to improve the cellular properties of the production strains. This has resulted in substantial increases in the existing b-lactam production processes. Furthermore, pathway extension, by heterologous expression of novel genes in well-characterised strains, has led to introduction of new fermentation processes that replace environmentally damaging chemical methods. This minireview discusses the recent developments in metabolic engineering and the applications of this approach for improving b-lactam production. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Metabolic engineering; b-Lactam production; Genetic engineering; Strain improvement

1. Introduction b-Lactams have been used extensively for treatment of various bacterial infections for more than half a century, and today penicillins are commodity-type products with annual production volumes exceeding 60,000 tons. Optimisation of the industrial production of b-lactams is therefore of great economical importance for the b-lactam producing companies. Traditionally, b-lactam production has been improved by classical strain improvement programs such as the Panlabs Penicillin Strain Improvement Program, which was initiated in 1972 (Lein, 1986). These programs have involved an iterative process where the production strains were exposed to random mutagenesis followed by analysis and selection of superior production strains. The selected strains were subsequently subjected to a detailed characterisation in which strain stability, optimal fermentation medium and processing conditions were investigated (Lein, 1986). Consequently, great effort was put into investigating and understanding the microbial physiology of the strains, in order to guide the selection of improved production strains. Despite the success of classical strain improvement, it is however an indirect approach, and only little

Corresponding author. E-mail address: [email protected] (J. Nielsen).

insight is obtained into the mechanisms underlying the improved properties of the new strains. Developments in molecular biology have enabled the introduction of directed genetic modifications through recombinant DNA technology, resulting in a more direct approach to strain improvement—generally referred to as metabolic engineering (Bailey, 1991; Cameron and Tong, 1993; Nielsen, 1998a,b, 2001; Stephanopoulos et al., 1998). The essence of metabolic engineering is to apply analytical techniques for detailed phenotypic characterisation of cells grown under industrial-like conditions to design directed genetic modifications that may be obtained through recombinant DNA technology, resulting in cells with improved properties. The principles of metabolic engineering are outlined in Fig. 1. When the flux through an existing pathway is to be improved, a detailed phenotypic characterisation (or analysis) of the currently applied strain is performed. This leads to design of a strategy for improvement of the properties of the strain using recombinant DNA technology, and subsequently the genetic modifications are introduced, i.e. the synthesis part that may involve gene deletion, gene overexpression or heterologous expression of new genes. The synthesis step is similar to the traditional mutagenesis step, but the important difference is that the genetic modifications are directed towards specific parts of the metabolism. The synthesis

1096-7176/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1096-7176(03)00003-X

J. Thykaer, J. Nielsen / Metabolic Engineering 5 (2003) 56–69

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Fig. 1. Principles of metabolic engineering.

step results in new strains with modified cellular properties, and these strains are then analysed in great detail using the same analytical tools as applied for the original strain (Fig. 1). The effects of the genetic alteration are investigated by comparing the modified metabolism of the constructed strains with the metabolism of the reference strain. Often, the analysis part points towards additional genetic alterations to further improve the cellular properties of the strains and metabolic engineering is therefore a cyclic operation with close interactions between the different parts of the cycle. Through comparison of the properties of the different strains constructed in the process, fundamental insight into the physiology of the microorganism is gained, and there is, therefore, a close integration of metabolic engineering and microbial physiology (Nielsen and Olsson, 2002). In cases where a heterologous gene is to be expressed in the production organism, e.g. to produce a novel product in a given host or to extend the substrate range, the metabolic engineering process is initiated with a pathway design. This can involve empirical design or can be done based on silico analysis applying detailed metabolic models of cells (Burgard and Maranas, 2001). After this initial pathway design follows construction of a strain with the suggested pathway, and the subsequent optimisation proceeds through the metabolic engineering cycle. With the rapid developments in molecular biology the synthesis step in the metabolic engineering cycle has in many cases become relatively trivial (even though genetic engineering of industrial strains can be cumbersome due to low transformation efficiency or lack of multiple markers etc.), whereas tools that enable a fast and efficient phenotypic characterisation of cells grown at industrial-like conditions are still lacking. The availability of complete genome sequences has resulted in new high throughput analytical techniques for the analysis of the transcriptome, the proteome, the metabolome etc. (Nielsen and Olsson, 2002), but these techniques need to be further refined in order to identify regulators that may play an important role in the control of flux through pathways leading to secondary

metabolites, particularly as these regulators tend to be expressed at very low levels. In this review, the applications of metabolic engineering for improving the production of b-lactams are discussed.

2. Biosynthesis of b-lactams b-Lactams are classified into the following five groups based on their chemical structure (Brakhage, 1998): penams, ceph-3-ems, clavams, carbapenems and monolactams. A wide variety of microorganisms are known to produce b-lactams ranging from filamentous fungi to both gram positive and gram negative bacteria. The two first groups, penams and ceph-3-ems, including penicillins, cephalosporins and cephamycins, are far the best characterised group of b-lactams and these groups are also considered to be the main b-lactam families (Diez et al., 1997). The clavams includes clavulanic acid, which in itself only possesses weakly antibiotic activity, but is a strong inhibitor of b-lactamases and is therefore used in combination with other b-lactams (typically penicillins) to fight b-lactam resistant infections (Demain and Elander, 1999). The products of the remaining two groups are all produced by bacteria, but only little is known about the molecular structure of the compounds and the microorganisms producing them, and thus knowledge of their biosynthesis is also very limited. For a more detailed description of these b-lactams, refer to the recent review by Demain and Elander, (1999). As mentioned above, the production of penicillins, cephalosporins and cephamycins are the best characterised processes. The main producers of these products are Penicillium chrysogenum, Acremonium chrysogenum, Norcardia lactamdurans and Streptomyces clavuligerus. An overview of the biosynthetic pathways of the blactams produced by these organisms is given in Fig. 2. The biosyntheses of penicillins, cephalosporins and cephamycins have the two first steps in common. The initial step in the biosynthesis of b-lactams is the condensation of the three amino acids l-a-aminoadipate (l-a-AAA), l-cysteine and l-valine to form the tripeptide a-aminoadipyl-cysteinyl-valine (ACV) catalysed by

58


Fig. 2. Biosyntheses of the b-lactams penicillin V, cephalosporin C and cephamycin C. Abreviations: lld-ACV, a-l-aminoadipyl-l-cysteinyl-dvaline; IPN, isopenicillin N; PenN, penicillin N; DAOC, deacetoxycephalosporin C; DAC, deacetylcephalosporin C; OCDAC, O-carbamoyldeacetylcephalosporin C; HOCDAC, 7-a-hydroxy-O-carbamoyl-deacetylcephalosporin C.


the enzyme ACV synthetase, which also performs an epimerisation of l-valine to d-valine. (Banko et al., 1987; Theilgaard et al., 1997; van Liempt et al., 1989; reviewed by Kleinkauf and von Do¨hren, 1996). The second step in the biosynthesis of b-lactams is the ring closure of lld-ACV to form isopenicillin N (IPN), catalysed by isopenicillin N synthase and in this reaction, the characteristic penam ring structure is formed (Fawcett et al., 1975; Konomi et al, 1979; O’Sullivan et al., 1979; White et al., 1982; reviewed by Kreisberg-Zakarin et al., 1999). Isopenicllin N is the branch point at which the biosynthesis of penicillin is separated from the biosyntheses of cephalosporins and cephamycins. In the third step of the penicillin pathway, the hydrophilic l-a-AAA side chain of IPN is exchanged with the CoA-thioester activated form of a hydrophobic acyl group such as phenylacetic acid or phenoxyacetic acid, leading to penicillin G or penicillin V, respectively. This side chain replacement is carried out by the enzyme acyl-CoA:IPN acyltranferase (IAT), but the conversion proceeds in two steps (Alvarez et al. 1993; Queener, 1990; Queener and Neuss, 1982). Initially, l-a-AAA is cleaved off followed by acetylation of 6-aminopenicillinanic acid (6-APA). The intermediate, 6-APA, remains bound to IAT when the enzyme is saturated with appropriate acyl-CoA substrates, but in the absence of acyl-CoA substrates, 6-APA is released to the medium, where it is subject to spontaneous carboxylation to 8hydroxy-penicillic acid (8-HPA) (Henriksen et al., 1997). In the biosyntheses of cephalosporin C and cephamycin C, IPN is epimerised to penicillin N by isopenicillin N epimerase (IPNE) (Jayatilake et al., 1981). Deacetoxycephalosporin C synthase (expandase) converts penicillin N to deacetoxycephalosporin C (DAOC) via an oxidative ring expansion that involves oxygen and 2-oxoglutarate (Kohsaka and Demain, 1976). DAOC is then hydroxylated to deacetylcephalosporin C (DAC) by another 2-oxoglutarate linked dioxygenase, deacetylcephalosporin C synthase (DACS) (Dotzlaf and Yeh, 1987; Scheidegger et al., 1984). In S. clavuligerus, the expandase and hydroxylase activities are performed by distinct enzymes (Jensen et al., 1985), whereas one bifunctional enzyme is responsible for both activities in A. chrysogenum (Dotzlaf and Yeh, 1987; Samson et al., 1987; Scheidegger et al., 1984). DACS constitutes the second branch point in the biosynthesis of b-lactams, dividing the pathways for cephalosporin C and cephamycin C biosynthesis. Acetyl-CoA:DAC acetyltransferase (DAT) is responsible for the last step in the formation of cephalosporin C, where an acetyl group is added to the –OH group of DAC by acetylCoA:DAC acetyltransferase (Felix et al., 1980; Fujisawa and Kanzaki, 1975). In the biosynthesis of cephamycin C, the C-3 acetoxy group of DAC is replaced by a carbamyl group by DAC-carbaoyl transferase

59

(DACCT) (Brewer et al. 1977; Brewer et al., 1980), followed by hydroxylation and methylation of the C-7 catalysed by 30 -carbamoyl-DAC 7-hydroxylase and 7hydroxy-30 -carbamoyl-DAC methyltransferase, a twoprotein system named P7 and P8 (Martin, 1998; Xiao et al., 1991; Xiao et al., 1993). A summary of the biochemical data for the enzymes in the b-lactam biosynthesis pathways is given in Table 1. In addition to the biosyntheses of b-lactams, Fig. 2 also shows the genes encoding the different enzymes in the pathways. The majority of these genes have been cloned within the last 15 years (Table 1) and this has been the basis for applying genetic engineering of the blactam producing strains. In P. chrysogenum all three, key genes involved in the biosynthesis of penicillin are clustered on the same chromosome in the genome. This is also the case for the genes involved in biosynthesis of cephamycin C by S. clavuligerus (Ward and Hogdson, 1993). However, in A chrysogenum two genes, pcbC and cefEF, are located on a different chromosome than the other genes (Skatrud and Queener, 1989).

3. Applications of metabolic engineering The detailed information about the molecular organisation of the b-lactam genes has led to intensive studies of applying genetic engineering in order to improve b-lactam production. The first use of recombinant DNA for improvement of an antibiotic producing strain was described by Skatrud et al. (1989) and since then recombinant DNA technology has been used extensively for construction of improved antibiotic producers. The most natural choice for genetic engineering is to overexpress single b-lactam genes or entire clusters, e.g. the penicillin biosynthetic cluster. This approach has indeed led to substantial increases in the production of b-lactams, which is described in more detail below. Some b-lactam products are produced by organisms which have low productivities, and other types of blactams are produced by chemical modification of fermentation end products. In both cases it is often beneficial to extend the pathway in well-characterised production organisms, hereby increasing the yields of the product or eliminating environmentally damaging chemical steps. However, modifying these strains genetically often results in new problems, e.g. precursor requirements and strain instability. Thus, these aspects have also become in focus in the field of metabolic engineering of b-lactam production. Therefore, in the following sections, each of the above-mentioned aspects are described in detail, illustrating the broad range of applications of metabolic engineering in the production of b-lactams.

pcbAB

pcbAB

acvA pcbAB pcbAB

pcbC

pcbC

pcbC

pcbC pcbC

penDE

acyA

nd cefD cefD

cefEF

cefE cefE

cefF cefF

cefG cefG cefG

cmcH cmcH

cmcI cmcI

cmcJ cmcJ

P. chr

A. chr

A. nid N. lac S. cla

P. chr

A. chr

A. nid

N. lac S. cla

P. chr

A. nid

A. chr N. lac S. cla

A. chr

N. lac S. cla

N. lac S. cla

A. chr

N. lac S. cla

N. lac S. cla

N. lac S. cla

ACVS

IPNE

DAOCS/DACS

DAOCS

DACS

DAT

DACCT

P7

P8

876 933

711 711

1563 1566

1332 1299 1300

933 954

942 933

996

nd 1194 1194

1217

1274

984 1316

993

1014

993

11310 10947 nd

11376 11328 11136

Gene size (bp)

32 nd

27 32

57.1 nd

49.3 nd 41

34.4 34.6

34.5 34.5

36.5

nd 43.6 43.5

39.2

39.9

37.5 36.9

37.5

38.4

38

422.5 404.1 nd

426 424 414.8

Protein size(kD)

Coque et al. (1995a) Alexander and Jensen (1998)

Coque et al. (1995a) Xiao et al. (1991) Alexander and Jensen (1998)

nd nd

nd 720 (CephC)

nd nd

nd

Gutie´rrez et al. (1992) Matsuda et al. (1992) Mathison et al. (1993) Coque et al. (1995b) Alexander and Jensen (1998)

nd 25 (DAOC), 14 (2-oxo)

52 (PenN), 3 (2-oxo) 35 (PenN), 22 (2-oxo)

29 (PenN),22 (2-oxo)/ 18 (DAOQ, 20(2-oxo)

nd 270 (IPN) nd

4000 (IPN), 23 (IPN+PA-CoA) 9,3 (6-APA+PA-CoA) 6 (PA-CoA), 2000 (PenV) nd

180 (ACV) 320 (ACV)

170 (ACV) 300 (ACV) nd

130 (ACV)

46 (Aad), 80 (Cys), 83 (Val) 170 (Aad), 26 (Cys), 340 (Val) 120 (Aad), 90 (Cys), 320 (Val) nd nd 560 (Aad), 70 (Cys), 1140 (Val) 630 (Aad), 120 (Cys), 300 (Val) 630 (Aad), 430 (Cys), 380 (Val)

Km (mM)

Coque et al. (1996) Kovacevic and Miller (1991)

Coque et al. (1993) Kovacevic et al. (1989)

Samson et al. (1987)

Coque et al. (1993) Kovacevic et al. (1989)

Montenegro et al. (1990)

Barredo et al. (1989)

Ramoń et al. (1987) Weigel et al. (1988) Coque et al. (1991) Leskiw et al. (1988)

Barredo et al. (1989) Carr et al. (1986) Samson et al. (1985)

MacCabe et al. (1991) Coque et al. (1991) Tobin et al. (1991)

Diez et al. (1990) Smith et al. (1990) Gutie´rrez et al. (1991)

Reference

nd nd

nd 7.3–7.7

nd nd

7.0–7.5

nd 7.0–7.4

(5–11) 7.4

7.5–7.8/7.0–7.5

nd 7.0 nd

nd

8.0–8.5

nd 7.0

nd

7.8 nd

8.5

8.4 7.5 8.3 nd nd

pH opt

Xiao et al. (1991)

Fujisawa and Kanzaki (1975) Felix et al. (1980)

Yeh et al. (1991)

Corte`s et al. (1987) Yeh et al. (1991)

Yeh et al. (1991)

Laiz et al. (1990) Usui and Yu (1989)

Alvarez et al. (1987, 1993)

Castro et al. (1988) Jensen et al. (1986)

Ramos et al. (1985) Baldwin et al. (1985a, b, 1987) Kupka et al., 1983; Pang et al., 1984

Theilgaard et al. (1997) Banko et al. (1987), Baldwin et al. (1990) Kallow et al. (1998) Van Liempt et al. (1989) Coque et al. (1996) Jensen et al. (1988) Zhang et al. (1992) Kadima et al. (1995)

Reference

Proteins: ACVS, a-l-aminoadipyl-l-cysteinyl-d-valine synthase; DACS, deacetylcephalosporin C synthase; DAOCS, deacetoxycephalosporin C synthase; DAT, acetyl-CoA: deacetylcephalosporin C acetyltransferase; IAT, acyl-CoA:IPN acyltranferase; IPNE, isopenicillin N epimerase; IPNS, isopenicillin N synthase. Organisms: A. chr, Acremonium chrysogenum; A. nid, Aspergillus nidulans; N. lac, Norcardia lactamdurans; P. chr, Penicillium chrysogenum; S. cla, Streptomyces clavuligerus. Affinity constants: Aad, l-a-aminoadipate; ACV, a-l-aminoadipyl-l-cysteinyl-d-valine; 6-APA, 6aminopenicillanic acid; Cys, cysteine; DAOC deacetoxycephalosporin C; IPN, isopenicillin N; 2-oxo, 2-oxoglutarate; PenN, penicillin N; PenV, penicillin V; PA-CoA, phenylacetic acid-CoA; Val, valine.

IAT

IPNS

Gene

Organism

Protein

Table 1 Genes encoding the enzymes in the b-lactam biosynthesis pathways and kinetic properties of the enzymes

60 J. Thykaer, J. Nielsen / Metabolic Engineering 5 (2003) 56–69


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3.1. Improvement of productivity and yield 3.1.1. Penicillin production The production of penicillin is one of the oldest and largest biotechnological industries with a world market of more than 60,000 tons in 2000, of which 25,000 tons were bulk penicillin (Bruggink and Roy, 2001). Despite more than 50 years of medical use this antibiotic is still heavily used for treating bacterial infections, and it is probably the most cost effective drug existing. However, development of microbial resistance to penicillin and the lack of activity against gram-negative bacteria have intensified the search for penicillins with improved properties. Today, a major part of the commercially available penicillins are semi-synthetic, being synthesised from the penicillin nucleus, 6-APA (van de Sandt and de Vroom, 2000). As described above the traditional optimisation of industrial production of penicillin has been achieved through random mutagenesis followed by selection of superior production strains as illustrated in Fig. 3. The first use of molecular biology to improve penicillin production was reported in 1991 (Veenstra et al. 1991). By introducing additional copies of both the pcbC and penDE genes, the penicillin production was increased by up to 40% in the single copy strain Wis 54-1255 of P. chrysogenum. A similar study was performed by Theilgaard et al. (2001) where different combinations of the three structural genes for the penicillin biosynthesis were introduced in Wis 54-1255. The largest increase of 176% in the specific penicillin productivity was observed in transformants with the whole penicillin gene cluster amplified. Comparing this major increase in the productivity with increases measured during the strain improvement program of DSM (formerly Gist Brocades) in the period between 1962 and 1987 (Fig. 3), it is observed that an increase in productivity of 176% corresponds to about 5 years (1972–1977) of strain

Fig. 3. Increase in productivity (output rate/unit volume, arbitary units) of pencillin G production by Gist Brocades, Delft, in the period between 1962 and 1987. The introduction of new productions strains is indicated with arrows. Based on Nielsen, 1997.

Fig. 4. Results of the SmithKline Beecham strain improvement series. Penicillin titre in percentage of the SmithKline Beecham strain BW 1901 versus penicillin cluster copy number. Modified from Newbert et al. (1997).

improvement and introduction of two new production strains, illustrating the power of metabolic engineering. Several research groups have investigated the molecular basis of commercially improved strains of P. chrysogenum resulting from the strain improvement programs. The main explanation for the high b-lactam production in these strains was an elevated copy number and high transcription levels of the entire penicillin biosynthesis gene cluster (Barredo et al., 1989; Christensen et al., 1995; Fierro et al., 1995; Newbert, 1997). However, as indicated in Fig. 4, there seems to be an upper limit to how many copies of the gene cluster can be inserted and still result in an increased yield, indicating that increasing the gene dosage is not effective in industrial strains of P. chrysogenum because several copies of the structural genes are already present (Smith et al., 1989). Thus, the potential of the additional genes were not exploited. Therefore, overexpression and regulation studies have been performed in single copy strains of both P. chrysogenum and Aspergillus nidulans. The results of the SmithKline Beecham strain improvement series shown in Fig. 4 indicate that there is a linear relationship between the cluster copy number and penicillin titre for cluster copy numbers below five. Assuming that there is proportionality between cluster copy number and enzyme activity levels, this means that all flux control for penicillin production is collected in the penicillin biosynthetic pathway, when the cluster copy number is below five. In other words, this indicates that for low cluster copy numbers, there are no limitations in the supply of precursors or co-factors required for penicillin biosynthesis. However, for high cluster copy numbers, there seem to be limitations in the metabolism causing a shift to a more complex flux control, with either a more complex regulation of flux through the penicillin biosynthetic pathways or a shift of

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part of the flux control to other branches of the cellular metabolism. A valuable analytical tool for investigating the details in regulation and control of different enzymes in a given pathway is metabolic control analysis (MCA) (Kascer and Burns, 1973; Fell, 1992). In MCA the control exerted by a specific enzyme in a pathway is given by the so-called flux control coefficient FCC. By applying MCA on kinetic data from fermentations of a high yielding strain of P. chrysogenum it was shown that flux control in the penicillin biosynthetic pathway was only exerted by the two first enzymes, ACVS and INPS, and that there was a shift in the flux control from ACVS to IPNS during a typical fed-batch cultivation (Nielsen and J^rgensen, 1995; Pissarra et al., 1996). Overexpression of the different genes in the penicillin biosynthetic pathway in A. nidulans by the inducible ethanol dehydrogenase promoter showed that ACV synthase, as in P. chrysogenum, is also rate controlling in the biosynthesis of penicillin by A. nidulans (Kennedy and Turner, 1996). Therefore, focus should be on overexpression pcbAB or pcbC for improving the penicillin production, as illustrated recently by Theilgaard et al. (2001). 3.2. Cephalosporin production Cephalosporin C is produced by the filamentous fungus A. chrysogenum, and this compound has a broader spectrum of antibiotic activity, as it is active against both the gram-positive and gram-negative bacteria (Weil et al., 1995). The disadvantage on the other hand is that because of the low potency, this compound cannot be used clinically (Demain and Elander, 1999). However, the nucleus of cephalosporin C, named 7-aminocephalosporanic acid (7-ACA), has shown to be an attractive precursor in the production of active semi-synthetic cephalosporins. The industrial production of cephalosporin C by A. chrysogenum is therefore an important process in the overall production of cephalosporins. Despite intensive strain improvement programs for the cephalosporin C production, the productivity of the cephalosporin C producing strains is still significantly lower than the corresponding productivity achieved in P. chrysogenum for penicillin production (Brakhage, 1998). Therefore, the effect of gene dosage has also been investigated in A. chrysogenum in order to further improve the cephalosporin C production. By introducing an extra copy of the cefEF, encoding DAOC synthase/DAC hydroxylase cephalosporin C production was increased by 15% (Skatrud et al., 1989). However, at the time of the work it was not known that the plasmid used for the transformation also contained the cefG gene, encoding acetyl-CoA:DAC acetyltransferase. It was later demonstrated that overexpression of the cefG gene alone resulted in a 2–3-fold increase in the cephalosporin C production in A.

chrysogenum (Mathison et al., 1993; Gutie´rrez et al., 1997). Thus, from these results it is not possible to conclude whether DAOC synthase/DAC hydroxylase alone results in increasing cephalosporin C production. However, gene dosage seems to have a positive effect on cephalosporin C production. 3.3. Cephamycin C production The cephamycins are characterised as cephalosporins that are modified at C7 and/or have a side chain attached to the C3. The cephamycins are only produced by actinomycetes, and the advantage of these compounds compared to the other b-lactams described is that they are more active against gram-negative bacteria and more resistant to gram-negative b-lactamases (Demain and Elander, 1999). Cephamycin C is produced by the actinomycetes S. clavuligerus and N. lactamdurans, and strain improvement has been achieved through random mutagenesis. Unlike the other b-lactam producing organisms described so far, the cephamycin C producing strains have not been subjected to gene dosage studies although most of the genes responsible for cephamycin C biosynthesis have been cloned, Table 1.

4. Extension of product spectrum All medically used cephalosporins are semisynthetic, being produced by chemical modifications of 7-aminocephalosporanic acid (7-ACA) or 7-aminodeacetoxycephalosporanic acid (7-ADCA) (Demain and Elander, 1999). Traditionally, the production processes for 7ACA and 7-ADCA have involved complex and expensive chemical methods, that employed non-environmentally compatible organic solvents. 7-ACA has been produced by multistep chemical removal of the d-aaminoadipyl side chain from cephalosporin C (Isogai et al., 1991), whereas 7-ADCA has been produced chemically by oxidative ring expansion of penicillin G and subsequent side chain removal penicillin acylase catalysed enzymatic deacylation (Van de Sandt and de Vroom, 2000). From an economical and environmental point of view, replacement of these chemical processes by biological fermentations or enzymatic bioconversions would be desirable, as higher specificity and milder operation conditions are obtained using enzymes. Therefore, development of bioprocesses for production of semi-synthetic antibiotics has been of very high priority during the last 10–15 years (Bruggink and Roy, 2001). As described above, the productivity achieved in strain improvement of P. chrysogenum is much higher than the productivity of cephalosporin producing strains. Thus, P. chrysogenum has a high potential for


b-lactam production and it would therefore be interesting to genetically engineer this organism to produce cephalosporins directly by fermentation (Nielsen, 2000). By introducing the genes cefD of S. lipmanii and cefE of S. clavuligerus encoding IPN epimerase and DAOC synthase, respectively (see Fig. 2), into P. chrysogenum, the resulting recombinant strain was capable of producing detectable amount of DAOC simultaneously with the production of penicillin V (Cantwell et al., 1992). Based on these results, Crawford et al. (1995) suggested that feeding adipic acid to a P. chrysogenum strain expressing S. clavuligerus expandase or A. chrysogenum expandase-hydroxylase activity would result in the production of cephalosporins with adipoyl side chains. Subsequent enzymatic removal of the adipoyl side chains would then lead to the production of 7-ADCA and 7-ACA, respectively, Fig. 5 (Crawford et al., 1995). Using this method, chemical modifications applying organic solvents could be avoided resulting in a more environmentally friendly production of 7-ADCA and 7ACA. Physiological studies of the P. chrysogenum strain expressing S. clavuligerus expandase activity showed slightly lower productivity compared to the penicillin producing host strain during chemostat cultivations (Robin et al., 2003). Furthermore, there was a shift in the optimal productivity from a dilution rate of 0.03 h1 in the host strain (van Gulik et al., 2000) to 0.065 h1 in the recombinant strain (Robin et al., 2003), and this was speculated to be mainly due to the introduction of the heterologous expandase gene. The major advantages of this new process compared to the old chemical process recently resulted in the start up of a new production plant in the Netherlands for production of 7-ADCA by P. chrysogenum (van de Sandt and de Vroom, 2000). This process is one of the best examples for illustrating the power of metabolic engineering for introducing novel fermentation processes that replace chemical synthesis routes. A. chrysogenum has also been exploited for developing bioprocesses for the production of 7-ACA and 7ADCA. In an attempt to produce 7-ACA directly during fermentation, Isogai et al. (1991) constructed an A. chrysogenum strain expressing activity of d-amino acid oxidase (DAO) from Fusarium solani and glutaryl acylase (GLA) from Pseudomona diminuta (Fig. 5). The problem with this process was that in addition to 7ACA, two by-products 7-aminodeacetylcephalosporanic acid (7-ADACA) and 7-aminodeacetoxycephalosporanic acid (7-ADOCA) were also produced. Furthermore, the amount of 7-ACA produced was not commercially significant (Isogai et al. 1991). However, the study did show that it was possible to express bacterial genes in a fungal host. In a recent study, an alternative bioprocess for production of 7-ADCA using A. chrysogenum was introduced (Velasco et al., 2000). The concept of this

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study resembles the work of Cantwell et al (1992) except that A. chrysogenum was employed. In the work of Velasco et al. (2000) the cefE gene of S. clavuligerus was overexpressed simultaneously with the disruption of the A. chrysogenum cefEF gene. The latter turned out to be the key aspect of that study because it ensured no contamination with other cephalosporins. Overexpression of cefE instead of cefEF resulted in the production of large amounts of DAOC, which constitutes the starting material for a two-step enzymatic bioconversion with DAO and GLA (Fig. 5). This process also illustrated an environmentally friendly alternative to the traditional chemical methods.

5. Precursor and co-factor requirements Overexpressing the b-lactam genes, in most cases as described above, results in increased yield or productivity of b-lactams. However, as shown in Fig. 4, there is an upper limit in productivity that can be achieved through gene dosage (Newbert et al., 1997). This indicates that there might be limitations somewhere else in the metabolism, e.g. in the precursor supply from the primary metabolism. It is therefore of interest in the field of metabolic engineering to investigate these limitations and to try to compensate for these by genetic engineering. In Fig. 6, the required precursors for the production of different b-lactams are shown. Metabolic network analysis can be used for obtaining information about which pathways are active and how the fluxes are distributed in the metabolic network (Christensen and Nielsen, 1999, 2000). This is essential for understanding how the primary and secondary metabolism interacts. For quantifying the metabolic fluxes either metabolite balancing alone or in combination with 13C-labelling experiments can be used (Christensen and Nielsen, 1999; Wiechert, 2001). The advantage of using 13C-labelling experiments is that this technique also enables identification of new pathways (Christensen and Nielsen, 2000; Thykaer et al., 2002) and that the metabolic fluxes can be estimated without any assumptions about co-factor requirements. Flux estimations based on metabolite balancing have indicated that the supply/regeneration of NADPH for l-cysteine biosynthesis is a limitation in the production of penicillin from glucose (J^rgensen et al., 1995a; Henriksen et al., 1996; van Gulik et al., 2000). However, 13 C-labelling analysis of the metabolic network of P. chrysogenum showed that up to 75% of the glucose is metabolised through the pentose phosphate pathway, which is the main supplier of NADPH (Christensen and Nielsen, 2000; Christensen et al., 2000). These results clearly showed an excess of NADPH, which indicated the presence of unknown NADPH consuming reactions. This does not support the hypothesis described above,

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Fig. 5. Different strategies for producing cephalosporins directly from fermentation with the aims of developing bioprocesses for direct production of 7-ADCA and 7-ACA. The traditional production process for 7-ADCA is shown in the upper left comer. Modified from Velasco et al. (2000).

but comparison of the flux through the PP-pathway of a high- and low-yielding strain of P. chrysogenum showed a slightly higher PP-pathway flux in the high producing strain (Christensen et al., 2000), again indicating that the formation of penicillin is dependent on the supply of NADPH from the PP-pathway. The biosynthesis of cephamycin C is very similar to the biosyntheses of penicillin and cephalosporin C. The main difference is found in the supply of the amino acid a-AAA. In filamentous fungi, a-AAA is an intermediate in the biosynthesis of lysine, whereas in actinomycetes this amino acid is synthesised by deamination of lysine by the enzyme lysine-6-aminotransferase (LAT), which is encoded by the lat gene (Liras, 1999), Fig. 6. The LAT enzyme has been reported to be rate controlling in the production of cephamycin C (Malmberg et al., 1993). Overexpression of the lat gene in N. lactamdurans also increased cephamycin C yields 2-fold (Chary et al., 2000). However, the results indicated that there was no

quantitative correlation between the expression of the lat gene and the increase in cephamycin C production. This was further substantiated by studies of the effect of exogenous lysine on the activity of the LAT enzyme. Despite a large increase in LAT activity no parallel increase in the cephamycin C yield was observed (Khetan et al., 1996; Letiaõ et al, 2001). An important factor in production of b-lactams is the availability of activated side-chain precursor. In the final step of b-lactam production in P. chrysogenum, the aAAA side-chain is exchanged with activated adipic acid (AA), phenoxyacetic acid (POA) or penaylacetic acid (PAA) for production of adipoyl-7-ADCA, penicillin V or penicillin G, respectively. In the process of adipoyl-7ADCA production by P. chrysogenum it was shown that adipate besides being used as side-chain precursor was degraded and used as carbon source in the primary metabolism (Robin et al., 2001). This degradation of adipate is undesirable because, from an economic point


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Fig. 6. Different precursors in the production of b-lactams.

of view, adipate is much more expensive than glucose and from a metabolic point of view, less activated sidechain precursor is available for b-lactam biosynthesis. In order to eliminate this undesired consumption of adipate, the first step was to identify how and where the degradation takes place. Using metabolic network analysis (Christensen and Nielsen, 1999, 2000, 2002), it was shown that adipate was degraded by b-oxidation to succinyl-CoA and acetyl-CoA, and that the degradation of adipate replaced the anaplerotic reaction from pyruvate to oxaloacetate. Furthermore, the labelling data indicated that adipate was degraded in the microbodies and the formed acetyl-CoA was metabolised in the glyoxylate shunt (Thykaer et al., 2002). This study constituted the analytical part of metabolic engineering. Future experiments should therefore be focused on deleting the enzymes responsible for adipate degradation. A similar study of penicillin V producing strain of P. chrysogenum showed that the side-chain precursor in this process, phenoxyacetic acid, was also degraded during cultivation. However, the mechanism for the degradation was not elucidated (Christensen and Nielsen, 2000). An alternative approach for making more activated side chain precursor available for penicillin G formation in P. chrysogenum was to overexpress the genes encoding acetyltransferase and phenylacetic acid activating CoA ligase (PCL). Strains expressing additional copies of PCL from Pseudomonas putida showed improved penicillin production (Minambreset et al.,

1996), illustrating the importance of a high concentration of activated side-chain precursor. A key element in the production of adipoyl-7-ADCA, penicillin V and penicillin G is the reuse of a-AAA, when the side chain of isopenicillin N is replaced. This is general for b-lactam biosynthesis in P. chrysogenum, whereas no recycling of a-AAA is observed in the biosynthesis of cephalosporin C in A. chrysogenum. During both continuous cultivations and fed-batch cultivations the by-product 6-oxo-piperdine-2-carboxylic acid (OPC), the cyclic form of a-AAA has been found in substantial amounts (Henriksen et al., 1998; J^rgensen et al., 1995b). The mechanism of biosynthesis of OPC is still unknown but it is likely to be closely related to the biosynthesis of penicillin (Brundidge et al., 1980). This hypothesis was supported by fed-batch results indicating that OPC is formed in an almost constant ratio to penicillin V (J^rgensen et al., 1995b) and by data from continuous cultivations, which showed that formation of OPC increases with increased concentrations of the side chain precursor, phenoxyacetic acid (Henriksen et al., 1998).

6. Summary and future directions Metabolic engineering has proven to be a rational alternative to classical strain improvement, and today the b-lactam industry applies the concept of metabolic engineering in parallel with classical strain

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improvement. This has been illustrated by the recent introduction of a completely new process for the direct production of adipoyl-7-ADCA by a fermentation route. The future will surely bring many other examples of new and improved processes for the production of b-lactams. As mentioned in the Introduction the key to success in metabolic engineering is efficient tools for detailed phenotypic characterisation of cells at industrial-like growth conditions. Even today, with many powerful analytical tools at hand, the analysis part of the metabolic engineering cycle is often the most timeconsuming step, and the limitation in our understanding of the complex mechanisms prevailing within the cells is, in many cases, hindering rational design of improved strains. Through several passages of the metabolic engineering cycle, however, a detailed insight into the physiology of the microorganism studied is gained, and metabolic engineering therefore overlaps with microbial physiology (Nielsen and Olsson, 2002). With the developments in functional genomics, where novel high-throughput techniques for analysis of the transcriptome, the proteome and the metabolome are being developed, it will become possible to perform a more detailed phenotypic characterisation of cells and thereby speed up the passage through the metabolic engineering cycle. Through the use of these high-throughput techniques insight into the function of specific genes in the genome is obtained, pointing out that metabolic engineering also overlaps with functional genomics. This is particularly the case for so-called inverse metabolic engineering, where different strains in a strain lineage are analysed in great detail in order to identify key regulatory elements, and subsequently using this information to construct high performing strains.

Acknowledgments The authors would like to acknowledge DSM antiinfectives for financially supporting the b-lactam research at Center for Process Biotechnology and for a close and inspiring collaboration. Furthermore, we would like to thank Mhairi McIntyre for carefully reading this manuscript and giving valuable comments.

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