INTRODUCTION

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The discovery of antibiotics for clinical use started with the discovery of the efficacy of a -lactam compound and is perhaps the most important discovery in the history of therapeutic medicine. The application of antibiotics to the therapy of infectious diseases may conceivably have saved more lives than any other medical development (135). It began in 1929, when Alexander Fleming published his observation about the inhibition of growth of Staphylococcus aureus on an agar plate contaminated with Penicillium notatum (117). Three years later, it was shown that the growth inhibition was due to penicillin (71). The first clinical trials with penicillin were undertaken in 1941 (reviewed in reference 2). In parallel with efforts to provide penicillin in large amounts, its structure was elucidated in 1945, when Hodgkin and Low showed by X-ray crystallography analysis that it is composed of a -lactam structure (reviewed in reference 1).

During the late 1940s, the fungus Cephalosporium acremonium (now renamed Acremonium chrysogenum) was isolated from the sea at Cagliari, Italy, by Guiseppi Brotzu (51). This fungus was first found to produce penicillin N; later, another antibiotic was discovered in the culture broth, which was found to consist of different derivatives of a -lactam compound designated cephalosporin (reviewed in reference 2). The structure of cephalosporin C was described in 1961 by Abraham and Newton (3) and confirmed by X-ray crystallography analysis (141). The discovery of cephalosporin C generated a whole new group of clinically significant -lactams.

The success of -lactams in the treatment of infectious disease is due to their high specificity and their low toxicity. Despite a growing number of antibiotics and the incidence of penicillin-resistant isolates, -lactams are still by far the most frequently used antibiotics (299).

However, it is only in the past 20 years that the biosynthetic pathways leading to penicillins (penams) and cephalosporins (ceph-3-ems) have been elucidated. This is partly because biosynthetic enzymes are often unstable and are present in the cell in only small amounts, making their purification difficult. In addition, industrial production of penicillin and cephalosporin was achieved with P. chrysogenum and A. chrysogenum, respectively. These fungi, however, belong to the deuteromycetes, which are in general difficult to analyze genetically. Currently, the greatest progress in elucidation of the molecular regulation of biosyntheses of -lactams in fungi has been made in the penicillin producer Aspergillus (Emericella) nidulans, since this fungus is an ascomycete with a sexual cycle. Therefore, classical genetic techniques can be applied to A. nidulans (262) and hence a detailed genetic map is available (70). Together with molecular techniques, this facilitated a thorough analysis of the genetic regulation of metabolic pathways, including that of penicillin biosynthesis (reviewed in references 20, 49, and 204).

Since the biochemistry of penicillin and cephalosporin biosynthesis is rather well understood and recombinant techniques have been developed for some filamentous fungi, recent research has aimed at elucidating the molecular regulation of -lactam biosynthesis. Within the last few years, several studies have indicated that the -lactam biosynthesis genes are controlled by a complex regulatory network. A comparison with known regulatory proteins and DNA elements of eukaryotes involved in the regulation of genes of primary metabolism is thus of great interest. Such investigations might also provide hints about both the evolution of secondary metabolism and the signals leading to the production of -lactams. Furthermore, the overexpression of regulatory genes will lead to higher yields of -lactams in the respective production strains and knowledge of biosynthesis genes will allow the production of new compounds by combinatorial biology.

The biosynthesis of -lactam compounds and their molecular genetics were the subject of several recent reviews (8, 45, 49, 152, 211, 299). In particular, the regulation of -lactam biosynthesis in fungi has seen a tremendous increase in knowledge within the last years, and it is this aspect which is considered in most of the present review.

-Lactams can be classified into five groups on the basis of their chemical structures (Fig. 1). All of these compounds have in common the four-membered -lactam ring. Apart from the monolactams, which have a single ring only, -lactams consist of a bicyclic ring system. The ability to synthesize -lactams is widespread in nature. It has been found in some fungi but also in some gram-positive and gram-negative bacteria (Fig. 1). However, although organisms belonging to all three groups can synthesize the hydrophilic cephalosporin compounds (ceph-3-ems), the hydrophobic penicillins are produced as end products only by filamentous fungi (Fig. 1). For the remaining groups of -lactams listed in Fig. 1, only bacterial producers have been reported so far. The number of prokaryotic and eukaryotic microorganisms recognized as being able to synthesize -lactam antibiotics is continuously increasing (152).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Naturally occurring classes of -lactam antibiotics essentially as compiled by O'Sullivan and Sykes (249). Modified from reference 8 with permission of the publisher.

The biosyntheses of penicillins and cephalosporins have the first two steps in common (Fig. 2). All naturally occurring penicillins and cephalosporins are formed from the same three amino acids: L--aminoadipic acid (L--AAA), L-cysteine, and L-valine. L--AAA is a nonproteinogenic amino acid and is derived from the fungus-specific aminoadipate pathway which leads to the formation of L-lysine. It can also be provided, at least in A. nidulans and P. chrysogenum, by catabolic degradation of L-lysine, although the contribution of this pathway to penicillin biosynthesis in these fungi has not been clarified yet (see "Amino acids as precursors and mediators of regulation," below).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Biosynthesis of penicillin, cephalosporin C, and cephamycin C. Gene and organism names are in italics, names of enzymes are in capital letters. L--AAA is an intermediate of the L-lysine biosynthetic pathway but can also be provided by catabolic degradation of L-lysine. The question mark indicates that the contribution of the latter to -lactam production has not been clarified yet. The penicillin biosynthesis occurs in fungi only, whereas cephalosporins are synthesized in both fungi (e.g., cephalosporin C by A. chrysogenum) and bacteria (e.g., cephamycin C by S. clavuligerus) (Fig. 1). See the text for details.

In the first reaction cycle, the amino acid precursors are condensed to the tripeptide -(L--aminoadipyl)-L-cysteinyl-D-valine (ACV). All reactions which are required for the formation of this tripeptide, e.g., specific recognition of amino acids, their activation via the formation of aminoacyl adenylates, and formation of peptide bonds, are catalyzed by a single multifunctional enzyme designated according to the product formed, ACV synthetase (ACVS) (Fig. 2). ACVS is encoded by a single structural gene designated acvA (pcbAB) (Fig. 2; Table 1).

In the second step, oxidative ring closure of the linear tripeptide leads to the formation of a bicyclic ring structure, i.e., the four-membered -lactam ring fused to the five-membered thiazolidine ring, which is characteristic of all penicillins. The resulting compound, isopenicillin N (IPN), possesses weak antibiotic activity and is thus the first bioactive intermediate of both the penicillin and cephalosporin pathways. This reaction is catalyzed by isopenicillin N synthase (IPNS), encoded by the ipnA (pcbC) gene (Table 2). IPN is the branch point of the penicillin and cephalosporin biosyntheses.

In the third and final step of penicillin biosynthesis, the hydrophilic L--AAA side chain of IPN is exchanged for a hydrophobic acyl group; the exchange is catalyzed by acyl coenzyme A (CoA):isopenicillin N acyltransferase (IAT). The corresponding gene has been designated aatA (penDE) (Table 3). In their natural habitats, penicillins DF, F, and K, which contain hexenoic acid, 3-hexenoic acid, and octenoic acid as side chains, respectively, are synthesized. By supplying the cultivation medium with phenoxyacetic or phenylacetic acid, the synthesis can be directed mainly toward penicillin V or penicillin G, respectively (Fig. 2, shown for penicillin G). The side chain precursors must be activated before they become substrates for the IAT. It is generally believed that the activated forms of the side chains consist of their CoA thioesters, but the mechanism behind this activation has still not been fully elucidated.

The first step that commits the pathway to the production of cephalosporins is the isomerization of the L--AAA side chain of IPN to the D enantiomer to give penicillin N. This reaction is catalyzed by IPN epimerase (Fig. 2). Penicillin N is the precursor of antibiotics containing the ceph-3-em nucleus, i.e., cephalosporins and cephamycins (7-methoxycephalosporins), produced by fungi and bacteria, respectively (Fig. 1 and 2). Penicillin N is converted to deacetoxycephalosporin C (DAOC) by DAOC synthetase (expandase) activity (Fig. 2). This ring expansion step involves the oxidative opening of the penam thiazolidine ring to give, upon ring closure, the six-membered dihydrothiazine ring, which is characteristic of all ceph-3-ems. In the next step, the methyl group at C-3 of DAOC is hydroxylated and oxidized to form deacetylcephalosporin C (DAC) (Fig. 2). In A. chrysogenum, both reactions are catalyzed by a single enzyme, DAOC synthetase (expandase)/DAC hydroxylase, encoded by the cefEF gene, whereas in the bacterial cephalosporin producer Streptomyces clavuligerus, one enzyme has been found for each reaction, encoded by the two genes cefE and cefF (Table 4).

In the last step of cephalosporin C biosynthesis, which is best studied in the fungus A. chrysogenum, an acetyl moiety from acetyl-CoA is transferred to the -OH group of DAC; this step is catalyzed by the product of cefG, acetyl CoA:DAC acetyltransferase (Fig. 2; Table 5). Several cephalosporins that differ from cephalosporin C in the substituent attached to the 3' C oxygen have been isolated from a variety of microorganisms (152).

In addition, cephalosporins carrying a methoxy group at C-7 (7-methoxycephalosporin, or cephamycin) are produced by both S. clavuligerus (compound A-16884A [138]) and S. lipmanii (239, 265) (Fig. 2). The specific reactions leading to the formation of cephamycin C, which have been studied best in S. clavuligerus, start from the intermediate DAC (Fig. 2). A carbamoyl group is attached to DAC to give O-carbamoyl-DAC (OCDAC). This reaction is catalyzed by 3-hydroxymethyl ceph-3-em O-carbamoyltransferase, which is encoded by the cmcH gene (79) (Fig. 2). Then, C-7 is hydroxylated by the action of OCDAC hydroxylase, encoded by cmcI (343) (Fig. 2). In the final step of cephamycin biosynthesis, the hydroxy group at C-7 is methylated to form cephamycin C (7-methoxycephalosporin C); the reaction is catalyzed by cephamycin C synthetase, whose corresponding gene has been designated cmcJ (79).

Before their identification, the putative genes encoding ACVS were designated pcbA (penicillin cephalosporin biosynthesis) and pcbB, because it was believed that two enzymes were involved in the formation of an L-(-aminoadipyl)-L-cysteine (AC) dipeptide and the final ACV tripeptide, respectively (Fig. 1) (reviewed in references 147 and 245). Cloning and sequencing of the corresponding gene revealed, however, that a single polypeptide encoded by a single gene is responsible for the formation of the ACV tripeptide. Publications reporting the DNA sequence of the P. chrysogenum, A. nidulans, and A. chrysogenum genes named the gene acvA, which reflected the involvement of one genetic locus in the synthesis of ACVS (201, 203, 305, 307), or pcbAB, derived from the combination of pcbA and pcbB (91, 130). In the following discussion, the term acvA is used because it indicates that a single gene encodes ACVS. The same is relevant for the gene encoding IAT, which has been named penDE or aat. In this review, the gene is designated aatA, reflecting both the correct genetic nomenclature and the fact that one genetic locus encodes the enzyme. The IPNS gene has been named ipnA. The alternative names are shown in parentheses at the beginning of the relevant sections.

As far as we know, in bacteria and fungi all structural genes of -lactam biosyntheses are clustered (Fig. 3). In fungi, first the penicillin biosynthesis genes of both A. nidulans and P. chrysogenum are tightly clustered (201, 305). In the following, in A. chrysogenum, two clusters containing the cephalosporin biosynthesis genes have been identified (130, 131, 219, 220), whereas in cephamycin C-producing bacteria, the genes are organized into a single cluster (Fig. 3).

View larger version (32K): [in this window] [in a new window]   FIG. 3.   -Lactam biosynthesis gene cluster in fungi and bacteria. References to each fungal gene are listed in Tables 1 to 5 or in the text. The A. chrysogenum cefD gene has not been identified yet. The whole gene cluster of N. lactamdurans has been characterized (76-79). The organization of the L. lactamgenus gene cluster was taken from references 166 and 167. Roman numerals indicate the chromosomes (in fungi) on which the genes are localized (115, 201, 235, 300). The intergenic regions between acvA and ipnA of P. chrysogenum, A. nidulans, and A. chrysogenum are 1,107 bp (91, 307), 872 bp (203) and 1,233 bp (230), respectively. Bacterial genes with fungal homologs are boxed. The transcriptional orientation and the transcript units (Bacteria), as far as it has been determined, are indicated by arrows above or below the boxes. Arrows between boxes (Bacteria) and arrows with broken lines below boxes mark the orientation of genes. The function of ORF is unknown. Abbreviations (76-79, 258): cmcT, transmembrane protein; pbp, penicillin binding protein; bla, -lactamase; blp, showing similarity to the extracellular -lactamase inhibitory protein BLIP.

The linkage of antibiotic biosynthesis genes is a well-known phenomenon in many antibiotic-producing organisms. It has been speculated that linkage occurred during evolution owing to an ecological selective advantage (212). Seno and Baltz (294) suggested that coordinated regulation of antibiotic biosynthesis genes could be achieved by organizing the genes into large operons controlled by a single promoter. For example, genes of the actinorhodin biosynthetic pathway in Streptomyces coelicolor are clustered and expressed in several polycistronic mRNAs (207). In eukaryotic fungi, however, -lactam biosynthesis genes are transcribed separately and are expressed from different promoters (reviewed in reference 49). Hence, in fungi, there is no obvious need for clustering, and it thus seems more likely that linkage reflects a common ancestral origin (see "Origin of -lactam biosynthesis genes in fungi"). However, there is no evidence that the IAT gene (aatA) has a close relative in modern prokaryotes, even though it is part of the cluster. This fact supports the hypothesis that linkage might also confer an ecological advantage to the eukaryotic fungi in their natural habitat, although the reason for this is not yet understood.

STRUCTURAL GENES AND DEDUCED PROTEINS

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Genes Common to Penicillin- and Cephalosporin-Producing Fungi

acvA (pcbAB), encoding ACVS. The first reaction of the cephalosporin and penicillin biosynthesis is the formation of the ACV tripeptide, which was first found in 1960 as an intracellular component of P. chrysogenum (16). All the reactions required for synthesis of the tripeptide are catalyzed by a single enzyme, ACVS, which is encoded by the acvA (pcbAB) gene (reviewed in reference 169) (Fig. 2; Table 1). Thus, the ACV tripeptide is formed via a nonribosomal enzyme thiotemplate mechanism from its amino acid precursors. This is similar in many aspects to the synthesis of other microbial peptides (reviewed in reference 169).

ipnA (pcbC), encoding IPNS. The second step of the penicillin and cephalosporin biosynthesis, i.e., cyclization of the linear ACV tripeptide to the bicyclic IPN, is catalyzed by IPNS (cyclase), a nonheme-Fe(II)-dependent oxidase (106, 107, 174, 248, 340) (Fig. 2; Table 2). The enzyme has a molecular mass of about 38 kDa and formally catalyzes the removal of four hydrogen equivalents of the ACV tripeptide in a desaturative ring closure with concomitant reduction of dioxygen to water (reviewed in reference 279).

aatA (penDE), encoding IAT. The third and final reaction of penicillin biosynthesis, which does not occur in cephalosporin biosynthesis and has been found in fungi only, is catalyzed by IAT (10, 11, 54, 98, 123, 199, 263, 265, 311). The hydrophilic L--AAA side chain is exchanged for a hydrophobic acyl group, e.g., phenylacetyl in penicillin G (Fig. 2). IAT shows a broad substrate specificity (reviewed in references 198 and 211). By addition of appropriate precursor molecules, the fermentation can be directed toward a specific penicillin; e.g., for production of penicillin G, phenylacetic acid is added, whereas for production of penicillin V, phenoxyacetic acid is added. Once the precursor has been taken up, it must be activated to its CoA thioester. This reaction seems to be carried out by phenylacetyl-CoA ligase (53, 171). It is unclear, however, whether a specific enzyme is needed for this reaction, because a possible candidate is the acetyl-CoA synthetase (ACS), which has been purified from P. chrysogenum and whose structural gene, acuA, has been cloned (215, 216). It was shown that the ACS enzymes of both P. chrysogenum and A. nidulans have the capability to catalyze in vitro the activation (to their CoA thioesters) of some of the side chain precursors required for the production of several penicillins by these fungi (216).

cefD, encoding IPN epimerase. The reaction catalyzed by IPN epimerase directs the pathway (Fig. 2) to the production of cephalosporins. IPN epimerase catalyzes the epimerization of the L--AAA side chain of IPN to the D enantiomer to give penicillin N, which is the precursor of antibiotics containing the ceph-3-em nucleus, i.e., cephalosporins and cephamycins (7-methoxycephalosporins) (151, 174) (Fig. 2). The purification of A. chrysogenum IPN epimerase proved to be difficult because the enzyme was extremely labile in cell-free preparations (174). So far, no further data on the fungal protein or gene are available. Therefore, it is still unknown whether the putative cefD gene is part of one of the A. chrysogenum clusters (Fig. 3).

cefEF, encoding DAOC synthetase (ring expandase)/DAC hydroxylase. In cephalosporin biosynthesis, penicillin N is converted to DAOC by expansion of the five-membered thiazolidine ring to give the six-membered dihydrothiazine ring of DAOC (Fig. 2). This reaction is catalyzed by DAOC synthetase, which possesses the required expandase function (172). The enzyme was purified from A. chrysogenum (93, 182), S. clavuligerus (155), and S. lactamdurans (81). Fungal and bacterial expandases are stimulated by reducing agents, like dithiothreitol or glutathione, and show an absolute requirement for Fe²⁺, molecular oxygen, -ketoglutarate, ascorbate, and possibly ATP. These unusual cofactor requirements are characteristic of the class of intermolecular dioxygenases which activate oxygen in the decomposition of equimolar amounts of -ketoglutarate to form carbon dioxide and succinate (182; reviewed in reference 245).

cefG, encoding acetyl-CoA:DAC acetyltransferase. In the last step of the cephalosporin C biosynthesis, which is best studied in the fungus A. chrysogenum, an acetyl moiety from acetyl-CoA is transferred to the -OH group of DAC; this step is catalyzed by acetyl-CoA:DAC acetyltransferase (108, 122) (Fig. 2). The corresponding structural gene (cefG) of A. chrysogenum was cloned and sequenced independently by three groups (Table 5). The cefG gene was cloned based on its close linkage to cefEF. Sequencing of the region adjacent to cefEF led to the identification of an ORF, and a DNA fragment encoding this ORF allowed complementation of the A. chrysogenum mutant M40, which is deficient in acetyl-CoA:DAC acetyltransferase activity. Thus, the identified ORF most probably contained the cefG gene, which was confirmed by overexpression of the gene in A. niger, which naturally lacks such an activity (219). Gutiérrez et al. (131) screened an A. chrysogenum bacteriophage lambda library with a probe specific for the cefEF gene. Northern blotting and DNA sequence analysis revealed the existence of the cefG gene close to the cefEF gene. The authenticity of the cefG gene was proved by complementation of A. chrysogenum ATCC 20371, which lacks acetyl-CoA:DAC acetyltransferase activity. Matsuda et al. (220) cloned the gene by screening a cDNA library with oligonucleotides based on the N-terminal sequence of the enzyme. In addition, they proved the cloning of cefG by performing a gene disruption (replacement) experiment. The cefG-disrupted strains lacked the ability to produce cephalosporin C and accumulated its precursor, DAC, in the culture broth (221).

The penicillin biosynthesis pathway occurs in different compartments of the cell (Fig. 4). For localization of the ACVS protein, subcellular fractions obtained from protoplasts of a high-penicillin-producing P. chrysogenum strain (BC 1505) were analyzed. Because ACVS protein was detected in both the membrane and the soluble fraction of purified vacuoles, it was concluded that it is located either within or bound to the vacuolar membrane (187). In addition, a large portion of cellular L--AAA, which is most probably used for -lactam synthesis, is also contained in the vacuoles and thus is sequestered from the cytosol (5, 143) (Fig. 4).

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Compartmentalization of penicillin biosynthesis gene products. Enzyme names are boxed. Reactions which are hypothetical are labelled by two question marks. Most of the transport processes (indicated by a single question mark) which seem likely to exist because of the compartmentalization of the different enzymes have not been elucidated yet. The ACVS seems to be located within or bound to the vacuolar membrane. IPNS and IAT are located in the cytoplasm and in microbodies, respectively. See the text for details. The stage at which the processing of IAT occurs remains to be determined. Abbreviations: Aa, amino acids; cA, cyclized L--AAA (6-oxo-piperdine-2-carboxylic acid [Fig. 6]); CV, L-cysteinyl-D-valine; Mito, mitochondrion.

P. chrysogenum IPNS protein was found in the cytoplasm, whereas IAT was detected in organelles with a diameter of 200 to 800 nm, which were assumed to be microbodies (238) (Fig. 4). The latter result has been supported by the finding that the P. chrysogenum IAT contains a putative targeting signal sequence, a C-terminal alanine-arginine-leucine (34, 125). The importance of this sequence was proved by deleting it in vitro. After transformation of the P. chrysogenum npe6 strain lacking IAT specific activity, the mutated enzyme was located in vacuoles and the neighboring cytoplasm. Although IAT was produced in vivo, as shown by Western blot analysis and by measurement of IAT specific activity in vitro, the mutants did not produce penicillin (237), indicating that targeting of the enzyme to microbodies is essential for penicillin biosynthesis. Furthermore, a positive correlation between the capacity for penicillin production and the number of organelles per cell was observed when different P. chrysogenum strains were compared (238). Hence, the biogenesis of organelles and the genes responsible for this process might have an impact on the penicillin production. The localization of the penicillin biosynthesis in three different cellular compartments reflects the complexity of this biosynthetic pathway. Several transport steps are thus required to bring intermediates of the penicillin biosynthesis pathway together with the enzymes.

Although the presence in P. chrysogenum Wis54-1255 of a transport system for the side chain precursor phenylacetic acid was reported (113), subsequent investigations showed that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species (140).

-Lactam biosynthesis genes have been found both in bacterial species and in fungi. The availability of sequence information about bacterial and fungal genes led to speculations about their evolutionary relationship. Based on several observations, a horizontal transfer of -lactam biosynthesis genes from bacteria to fungi during evolution has been proposed by several authors (63, 184, 254, 339). This hypothesis was recently questioned by Smith et al. (308). The arguments in favor of a horizontal gene transfer are as follows. (i) ipnA genes of fungi and bacteria show high sequence similarities. More than 60% of the nucleotide bases and 50% of the deduced amino acids are identical. (ii) Bacterial as well as fungal -lactam genes are organized in clusters. The -lactam biosynthesis genes in bacteria are organized into a single cluster, as are the penicillin biosynthesis genes in fungi (Fig. 3). The cephalosporin biosynthesis genes in A. chrysogenum are organized into two clusters located on different chromosomes (Fig. 3). This finding led to the assumption that the -lactam biosynthesis genes were transferred as a single cluster from an ancestral prokaryote to a common ancestor of the -lactam-synthesizing fungi. In the eukaryotic ancestor, the biosynthesis genes were split between two chromosomes. One part encodes the early genes of -lactam biosynthesis, and the other encodes the late genes. Later in the lineage, an ancestor of A. nidulans and P. chrysogenum diverged from A. chrysogenum and has presumably lost the second cluster with the genes for the late stage of cephalosporin biosynthesis (299) (Fig. 3). (iii) The G+C content in the third position of codons containing the ipnA gene of A. nidulans and P. chrysogenum is unusually high and could indicate an evolutionary origin from streptomycetes, which show G+C contents of greater than 70% (8). (iv) Fungal acvA and ipnA genes do not contain introns, indicating a bacterial origin of the genes.

In addition, Aharonowitz et al. (8) proposed that during the evolution of -lactam biosynthesis genes, Streptomyces spp. must have evolved specific resistance mechanisms to avoid self-toxification. If the transfer had occurred from fungi to bacteria, it would have been lethal for bacteria. Hence, the transfer is more likely to have occurred from bacteria to fungi, which would not have been forced to evolve resistance mechanisms because of their lack of susceptibility. In contrast to the other penicillin biosynthesis genes, the aatA genes contain introns. On the basis of linkage of the aatA and ipnA genes (Fig. 3), Skatrud (299) suggested that a sequence functionally related to aatA was transferred together with the -lactam genes and was later modified to its current functional form. Since IAT possesses amidohydrolase activity to deacylate IPN to 6-APA (11) (Fig. 2), which shows a weak antibiotic activity only, an ancestral amidohydrolase activity in the prokaryotic ancestor might have had a resistance function. Its corresponding gene might have been fused in fungi with a eukaryotic gene (299). Genetic linkage of antibiotic synthesis genes and resistance genes is common in prokaryotes (294). Based on the DNA sequences of ipnA genes from gram-positive streptomycetes and from fungi and a rate of nucleotide substitution of 10⁹ nucleotide change per site per year (188), Weigel et al. (339) proposed that the transfer occurred 370 million years ago. The cloning and sequencing of an ipnA gene from a gram-negative bacterium, Flavobacterium sp., however, led to an extension and modification of the hypothesis of horizontal gene transfer. The ipnA gene of Flavobacterium sp. is 69% identical to the streptomycete gene and 64 to 65% identical to the fungal genes (A. chrysogenum and P. chrysogenum) (73). A recent reevaluation of the divergence times of organisms by using a protein clock suggested that gram-positive and gram-negative bacteria split about 2 billion years ago and that prokaryotes and a eukaryotic ancestor split about 3.2 to 3.8 billion years ago (111). If the gene transfer from streptomycetes to fungi had occurred only 370 million years ago, as proposed by Weigel et al. (339), it could be expected that the fungal and streptomycete genes would show a greater similarity than the gram-positive (streptomycete) and gram-negative (Flavobacterium sp.) bacterial genes. As outlined above, this is not the case (73). Hence, Aharonowitz et al. (8) suggested that multiple gene transfer events might have occurred from bacteria to fungi. It is difficult to imagine, however, why these multiple gene transfers then happened at about the same time as would be expected from the degree of similarity among the proteins of the various organisms. In addition, Smith et al. (308) argued against a horizontal transfer. The authors pointed out that the hypothesis of a horizontal gene transfer, e.g., of the ipnA gene, was made on the basis of a very limited data set and was based solely on assumptions about rates of change. They compared the similarity of both IPNS of A. nidulans, P. chrysogenum, A. chrysogenum, S. clavuligerus, S. anulatus, and Flavobacterium sp. and DAOC synthetase of S. clavuligerus and A. chrysogenum. Based on these similarities, they constructed a tree with conventional evolutionary descent. The authors argued that the simplest interpretation is that the genes for the two enzymes resulted from a duplication that occurred before the prokaryote-eukaryote divergence. The topology of the tree rooted with the duplicated enzymes, the depth of the bacterial branches, and the different orientations of the genes in fungi and eubacteria all appear to be consistent with an ordinary evolution for IPNS. However, if the genes appeared very early in the evolution, why have most of the eukaryotes and fungi lost the gene cluster? This question cannot be satisfactorily answered at the moment. Thus, the evolutionary origin of -lactam biosynthesis genes remains speculative.

REGULATORY CIRCUITS AND REGULATORY GENES

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Expression of Biosynthesis Genes under Standard Fermentation Conditions

Studies of the expression of penicillin biosynthesis genes were performed mainly with the E. coli reporter genes lacZ and uidA, encoding -galactosidase (-Gal) and -glucuronidase (-Glu), respectively (see, e.g., references 46, 124, and 194). Most of the results based on the analysis of gene fusions were supported by Northern or Western blot analysis or determination of enzyme specific activities and penicillin titers.

These studies led to the finding that the promoter strengths of penicillin biosynthesis genes are rather different. It was shown that in A. nidulans, aatA had lower expression than ipnA and threefold-higher expression than acvA (46, 194). A similar observation was made for the corresponding acvA and ipnA genes of both P. chrysogenum and A. chrysogenum. On the basis of reporter gene fusions, it became evident that in both fungi the expression of acvA was much weaker than that of ipnA (109, 230). The intergenic regions between acvA and ipnA thus seem to contain the information required for the remarkable difference in expression levels between acvA and ipnA. The low expression of acvA is, at least in wild-type strains of A. nidulans, rate limiting for penicillin production, because overexpression of acvA led to drastically increased production of penicillin (165) while similar overexpression of ipnA and aatA did not (112).

It seems reasonable to assume that the expression of penicillin biosynthesis genes is coordinated to ensure the synthesis of penicillin by the concomitant appearance of all gene products. But how is coordination achieved? Biosynthesis genes could be expressed simultaneously; i.e., the genes could be activated by the same regulatory factors. Alternatively, the expression of biosynthesis genes could be sequentially induced.

Ramos et al. (268) showed that a mutant of A. chrysogenum (N-2), incapable of producing the -lactam cephalosporin, lacked IPNS, IPN epimerase, and DAOC synthetase (expandase) activities (Fig. 2). Subsequent investigations revealed that strain N-2 encodes an inactive IPNS caused by a single C-to-T mutation within the coding region of the ipnA gene. It was postulated that a functional IPNS or its biosynthetic product IPN might be necessary for the regulation of the later stages of the biosynthesis, i.e., induction of the cefD and cefEF expression, respectively (Fig. 2 and 3) (269). Furthermore, Hoskins et al. (145) disrupted the acvA gene of A. chrysogenum. Although the predicted alterations of the target gene were not detected, the authors demonstrated IPNS activity in non-cephalosporin-producing transformants. This suggested that the ipnA gene can be expressed without the presence of precursor tripeptide molecules. Hence, in A. chrysogenum, the ipnA gene seems to be coordinatedly regulated whereas the later genes of the cephalosporin pathway (cefD and cefEF) (Fig. 3) appear to be sequentially induced.

To further study these observations, acvA was disrupted in a strain of A. nidulans (47, 305). This strain had a disrupted acvA gene on chromosome VI and, in addition, reporter gene fusions of the penicillin biosynthesis genes integrated in single copy at the chromosomal argB gene locus on chromosome III. acvA, ipnA, and aatA gene fusions were expressed at the same level in this strain as in the nondisrupted strain (47, 194). This was confirmed by determining IPNS and IAT specific activities and by Western blot analysis of IPNS, which showed the presence of both enzymes in an acvA-disrupted strain (47, 194). Hence, the genes were expressed despite the lack of precursor ACV and IPN molecules, indicating that in contrast to the cephalosporin biosynthesis genes, none of