Microbiology and Molecular Biology Reviews, September 1998, p. 547-585, Vol. 62, No. 3
1092-2172/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
-Lactam Biosynthesis in
Filamentous Fungi
Lehrstuhl für Mikrobiologie, Universität München, D-80638 Munich, Germany
SUMMARY
INTRODUCTION
GENERAL ASPECTS OF-LACTAM COMPOUNDS
BIOSYNTHESIS OF PENICILLINS AND CEPHALOSPORINS: AN OUTLINE
GENETIC NOMENCLATURE
CLUSTERING OF BIOSYNTHESIS GENES
STRUCTURAL GENES AND DEDUCED PROTEINS
Genes Common to Penicillin- and Cephalosporin-Producing Fungi
acvA (pcbAB), encoding ACVS.
ipnA (pcbC), encoding IPNS.
Gene Specific for Penicillin Biosynthesis
aatA (penDE), encoding IAT.
Genes Specific for Cephalosporin Biosynthesis
cefD, encoding IPN epimerase.
cefEF, encoding DAOC synthetase (ring expandase)/DAC hydroxylase.
Gene Specific for Cephalosporin C Biosynthesis in Fungi
cefG, encoding acetyl-CoA:DAC acetyltransferase.
COMPARTMENTALIZATION OF GENE PRODUCTS
ORIGIN OF-LACTAM BIOSYNTHESIS GENES IN FUNGI
REGULATORY CIRCUITS AND REGULATORY GENES
Expression of Biosynthesis Genes under Standard Fermentation Conditions
Promoter Structures of the A. nidulans Genes acvA and ipnA
Carbon Source Regulation
pH Regulation Mediated by the Transcriptional Factor PACC
Nitrogen Regulation
Amino Acids as Precursors and Mediators of Regulation
Influence of Phosphate and Oxygen
The CCAAT Box Binding Protein Complex PENR1
trans-Acting Mutations Affecting the Expression of Penicillin Biosynthesis Genes
Posttranscriptional Regulation
REGULATION OF-LACTAM BIOSYNTHESIS IN FUNGAL PRODUCTION STRAINS
APPLICATIONS
Increase of Expression of-Lactam Biosynthesis Genes
Genetic Engineering of-Lactam Biosynthetic Pathways
Generation of Novel Compounds by Genetic Engineering of Peptide Synthetases
CONCLUSIONS AND FUTURE DIRECTIONS
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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The most commonly used
-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as an end product by some fungi, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesized by both bacteria and fungi, e.g., by the fungus Acremonium chrysogenum (Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. Penicillin biosynthesis is catalyzed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC), and aatA (penDE). The genes are organized into a cluster. In A. chrysogenum, in addition to acvA and ipnA, a second cluster contains the genes encoding enzymes that catalyze the reactions of the later steps of the cephalosporin pathway (cefEF and cefG). Within the last few years, several studies have indicated that the fungal
-lactam biosynthesis genes are controlled by a complex regulatory network, e.g., by the ambient pH, carbon source, and amino acids. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of
-lactams and their physiological meaning for the producing fungi, and they can be expected to have a major impact on rational strain improvement programs. The knowledge of biosynthesis genes has already been used to produce new compounds.
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.
GENERAL ASPECTS OF
-LACTAM COMPOUNDS
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-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).
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BIOSYNTHESIS OF PENICILLINS AND CEPHALOSPORINS: AN OUTLINE
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To give a brief overview of the complete biosynthetic pathways, references for fungal enzymes and genes have been omitted in the text and are given in the tables and the following sections.
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).
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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).
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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.
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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.
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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).
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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).
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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).
GENETIC NOMENCLATURE
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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.
CLUSTERING OF BIOSYNTHESIS GENES
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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).
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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).
Early studies with cell-free systems of P. chrysogenum (185) and A. chrysogenum (30) demonstrated the existence of ACVS activity. It was suggested that two separate enzymes may be involved in the formation of ACV. However, based on the observation that less ACV is formed from the dipeptide AC and L-valine than from the free amino acids, Banko et al. (31) proposed that a single multifunctional enzyme may be responsible for ACV synthesis. This proposal was supported by the first isolation of an ACVS protein by van Liempt et al. (333), who purified ACVS of A. nidulans 118-fold. Their results suggested that ACVS consists of a single polypeptide chain. Since then, ACVS enzymes have been purified from different organisms, including S. clavuligerus (25, 26, 157), A. chrysogenum (25, 26, 345), and Nocardia lactamdurans (74). Attempts to purify ACVS from P. chrysogenum have thus far been unsuccessful because the enzyme seems to be rapidly degraded during chromatographic purification (7). Although not entirely clarified, it is believed that ACVS multienzymes are monomers with molecular masses of around 420 kDa and exhibit different catalytic activities, such as the specific recognition of the three amino acid precursors and their activation, peptide bond formation, isomerization of the L-valine moiety to the D form, and release of the peptide. As in ribosomal peptide biosynthesis, the carboxyl function of the amino acid is activated by the formation of a mixed anhydride with the
-phosphate of ATP,
resulting in the release of pyrophosphate (PPi). This has been used to develop assays based on amino acid-dependent exchange of
32P between PPi and ATP (333).
After activation of an amino acid, the aminoacyl adenylate formed is
cleaved by the action of an enzyme thiol, resulting in the formation of
a thioester bond between the enzyme (at an appropriate location on the
enzyme) and the amino acid and in the release of AMP. These
thioesterified amino acids play the same role as the tRNA-bound amino
acids in the ribosomal peptide biosynthesis. They are high-energy
intermediates, which are the targets for nucleophilic attack by the
amino group of a second amino acid, resulting in the formation of a
peptide bond. As in the ribosome, the nascent peptide grows from the
amino terminus to the carboxy terminus and the intermediate peptides
remain bound (as thioesters) to the enzyme (169). The
substrate specificity is less strict than in protein synthesis, since a
variety of tripeptide analogs are known (28).
By assuming three independent activation sites, the dissociation
constants for the S. clavuligerus ACVS have been estimated to be 1.25 and 1.5 mM for cysteine and ATP, respectively, and 2.4 and
0.25 mM for valine and ATP, respectively. No
L-
-AAA-dependent ATP-PPi exchange was
detected with the enzyme preparation used, although the amino acid
binding to the enzyme was dependent on ATP (292). The reason
for the lack of detection of L-
-AAA-dependent ATP-PPi exchange remains obscure, because fungal ACVS (from
both A. nidulans and A. chrysogenum) drove
radioactivity exchange that was dependent on all three amino acids
(292, 333). Dissociation constants for aminoacyl-tRNA
synthetases are much lower than those of S. clavuligerus
ACVS, usually below 100 µM for their respective amino acids (64,
150, 161). This may be a way of guaranteeing the supply of amino
acids to the primary metabolism and avoiding the depletion of vital
cellular components by secondary metabolism (292).
L-Valine is apparently epimerized to the D form
at the tripeptide stage, since no D-valine intermediate has
been detected (292).
Each ACVS is encoded by a single structural gene (designated
acvA) of more than 11 kb (Table 1). The clustering of
penicillin biosynthesis genes (Fig. 3) was first shown for A. nidulans (201, 305) and P. chrysogenum
(305). The identification of the gene cluster was based on
the assumption that biosynthesis genes for antibiotics are clustered,
and information had accumulated about ipnA genes from
several organisms. Hence, by cross-hybridization with ipnA
genes as the probe, the acvA genes from both organisms were
detected upstream from the ipnA genes, separated by about 1 kb (Fig. 3). In A. nidulans, the presence of an open reading frame (ORF) upstream of the ipnA gene was confirmed by
disruption of the upstream region, which led to a
non-penicillin-producing phenotype of the transformants (201,
305). Furthermore, MacCabe et al. (201) had purified
A. nidulans ACVS and used it to generate partial amino acid
sequence data, from which oligonucleotides were deduced and
synthesized. They were used for Southern analysis, which showed that
the ACVS-encoding gene is in fact upstream of the ipnA gene.
In addition, the molecular mapping data obtained predicted a size of
more than 11 kb for the acvA gene (201, 305). This was supported by MacCabe et al. (201), who first showed by Northern blot analysis that the acvA transcript is indeed
larger than 9.5 kb. This finding was confirmed by the sequencing of the acvA genes of A. nidulans (203),
P. chrysogenum (91, 307) and A. chrysogenum (130) and by a Northern blot analysis of
the P. chrysogenum acvA gene (91) (Table 1).
Subsequently, the corresponding acvA genes were also cloned
and sequenced from bacterial cephamycin producers such as S. clavuligerus (Fig. 3).
Even in fungi, the acvA genes form a single ORF, which does
not seem to be interrupted by introns, although this assumption has not
been proved yet by sequencing of the respective cDNAs. The properties
of genes and their deduced enzymes are summarized in Table 1. Fungal
acvA genes are divergently oriented with respect to the
ipnA genes (Fig. 3). The sizes of the intergenic regions between the genes vary slightly among the different fungi and are about
1 kb long (Fig. 3). In both A. nidulans and P. chrysogenum, the acvA mRNA starts within the intergenic
region between acvA and ipnA (Table 1; Fig. 3).
acvA expression is driven by a rather weak promoter that is
probably located entirely in the intergenic region between
acvA and ipnA (see "Expression of biosynthesis genes under standard fermentation conditions" below).
Amino acid sequences of ACVS proteins of all fungal and bacterial
species so far identified contain three homologous regions of about 600 amino acids. These contain repeated domains that have extensive amino
acid sequence similarities to each other, to the corresponding regions
of the ACVS protein of other fungi and bacteria, and to the repeated
domains identified for Bacillus brevis gramicidin S
synthetases 1 and 2 and tyrocidine synthetase I (169, 329).
Since all of these enzymes specifically recognize amino acids and form
adenylates, it is most likely that the respective adenylate-forming
domains (for the nomenclature of ACVS domains, see reference
169) recognize and adenylate one of the constituent amino acids. The order of the biosynthesis of the
-(L-
-AAA)-L-Cys-D-Val tripeptide is believed to reflect the linear organization of the ACVS
in AAA-, Cys- and Val-activating domains (reviewed in reference 169). A surprising result, however, was the
observation of the formation of
O-methyl-seryl-D,L-valine by ACVS
upon replacement of cysteine by O-methylserine
(297). This finding suggested that the second peptide bond
is initially formed. Consequently, an order of peptide formation
starting with Cys-Val and continuing with addition of
L-
-AAA would thus be conceivable. To get more information about the order of peptide formation, Kallow et al. (162) investigated enzyme-bound intermediates by omitting
the last amino acid, L-Val, in an in vitro reaction.
Depending on the reaction mode, this would lead to the accumulation of
either L-cysteinyl- or
L-
-AAA-L-cysteinyl intermediates bound to
the A. nidulans ACVS enzyme. In fact, the formation of the
AC thioester in the absence of L-Val was observed. It was
concluded that the first peptide bond is formed between the
-carboxyl of L-
-AAA and L-Cys and that
this is followed by addition of the dipeptidyl intermediate to
L-Val. The formation of
O-methylseryl-D,L-valine by ACVS
previously observed (297) was suggested to be due either to
a side reaction initiating peptide synthesis in position 2 of ACVS or
to an N-terminal cleavage of the N-terminal aminoadipyl side chain of
the tripeptide formed (162). This conclusion is also
consistent with the result that L-Cys-D-Val is
not a substrate for ACV biosynthesis (297) while
-(L-AAA)-L-Cys is accepted as a substrate
for adenylation and biosynthesis (28, 31; reviewed in reference 7).
Using a microbiological assay for detection of pantothenic acid,
Baldwin et al. (25, 26) observed that 1 mol of pantothenic acid was liberated per mol of purified A. chrysogenum ACVS.
This implied the presence of one phosphopantetheine molecule per ACVS molecule. It was therefore thought that ACVS follows the classical thiotemplate mechanism; i.e., after activation as aminoacyl adenylates, the intermediates bound as thioesters are assembled by one central swinging arm, the cofactor 4'-phosphopantetheine. Sequencing of the
ACVS structural genes (Table 1) revealed, however, that in the three
repeated regions of each ACVS, some similarity to
4'-phosphopantetheine attachment sites described for polyketide
synthases (i.e., Asp-Ser-Leu) is evident (203). This may
reflect the attachment of multiple cofactors to ACVS. Because a single
phosphopantetheine arm is sufficient for the activity of fatty acid
synthases, the finding of several phosphopantetheine attachment sites
suggests a modified mechanism for the thiotemplate pathway to
polypeptides (multiple-cofactor model) (203, 291, 313, 314).
Although the relevance of all three pantetheine attachment sites of
ACVS enzymes has not been proved experimentally, it is believed that
peptide assembly is accomplished by the transfer of acyl intermediates
between adjacent cofactors (313, 314). In the
carboxyl-terminal region of the ACVS enzymes, sequence similarities to
the thioesterase active-site region, GXSXG, have been found which
would be required to release the generated tripeptide from the enzyme
(203).
The current view of the thiotemplate mechanism of ACVS catalysis is
summarized in detail by Zhang and Demain (347) and by Kleinkauf and von Döhren (169).
ACVS enzymes are especially interesting since they represent a route
for peptide bond formation independent of the ribosome and allow the
incorporation of many nonproteinogenic amino acids (28,
168). Furthermore, since different parts of peptide synthetases are specific for certain amino acids, this can be used to engineer genetically new peptide synthetases, and hence to produce new compounds, possibly with new pharmacological activities. This approach
has been successfully used by Stachelhaus et al. (312) and
is summarized in "Applications" below.
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).
IPNS activity was first detected in cell extracts of A. chrysogenum (107, 174, 248). The IPNS reaction requires ferrous iron, molecular oxygen as the cosubstrate, and ascorbate as the electron donor to form the
-lactam and thiazolidine ring of IPN (151, 288, 340; reviewed in reference
245). It was shown that P. chrysogenum
IPNS is strongly inhibited by glutathione and is also sensitive to
cobalt inhibition (267). IPNS was purified to homogeneity
from A. chrysogenum (27, 142, 251) and has subsequently been obtained from P. chrysogenum
(267), A. nidulans (339), several
actinomycetes such as S. clavuligerus (153), and
the gram-negative bacterium Flavobacterium sp.
(250). It was shown that two interconvertable forms of the
enzyme exist, an oxidized state with a disulfide linkage and a reduced
state (27).
Only the free thiol form of the tripeptide ACV serves as a substrate;
the bis-disulfide dimer which is spontaneously formed is inactive
(259). S. clavuligerus possesses a disulfide
reductase that recognizes bis-ACV as a substrate (6). In
P. chrysogenum, a broad-range disulfide reductase belonging
to the thioredoxin family of oxidoreductases was found which
efficiently reduced bis-ACV to the thiol monomer. When coupled to IPNS
in vitro, it converted bis-ACV to IPN and was therefore suggested to
play a role in penicillin biosynthesis (72). In enzyme
assays in vitro, the thiol groups of both the ACV tripeptide and the
IPNS enzyme are kept in a reduced state by the addition of ascorbate
and dithiothreitol (see, e.g., reference 46). In
these assays, the appearance of antibiotic activity due to the
formation of IPNS from the antibiotically inactive ACV is measured with
an indicator organism which is sufficiently sensitive (174).
Alternatively, IPN can be monitored by high-pressure liquid
chromatography (154).
Mössbauer, electron paramagnetic resonance, and optical
spectroscopy suggested that ACV binds directly to the active-site iron
of IPNS via the cysteinyl thiol of ACV (65). A
six-coordinate metal center at the active site was proposed, with two
or three endogenous histidine ligands, an aspartate, and sites for the thiolate of ACV, oxygen, and solvent (232). It was shown
that the ACV sulfur atom binds to the active-site iron of the enzyme (247, 270). The crystal structure of the A. nidulans IPNS was recently solved at a resolution of 2.5 and 1.3 Å complexed with manganese (279) and with Fe2+
and substrate (280), respectively. The secondary structure
of IPNS was found to consist of 10 helices and 16
-strands. Eight of
the
-strands fold to give a "jelly-roll" motif. The active-site structure shows the manganese ion attached to four protein ligands (His
214, Asp 216, His 270, and Gln 330) and bears two water molecules occupying coordination sites directed into a hydrophobic cavity within
the protein (279). The Fe(II)-ACV-IPNS structure has one protein molecule with ferrous ion and ACV bound at the active site. The
side chain of Gln 330, which coordinates the metal in the absence of
substrate, is replaced by the ACV thiolate (280). In the
substrate complex, three of the five coordination sites are occupied by
protein ligands: His 214, His 270, and Asp 216 (43). The
remaining two sites are occupied by a water molecule (at position 298)
and the ACV thiolate (280). Such a structural characteristic
(an iron-binding site within an unreactive hydrophobic substrate-binding cavity) is probably a requirement for this class of
enzyme, since it results in the isolation of the reactive complex and
subsequent intermediates from the external environment. Thus, the
reaction can be channelled along a single path, avoiding the many side
reactions potentially open to the highly reactive species resulting
from the reduction of dioxygen at the metal (279). The role
of enzymes in such processes has been designated negative catalysis
(275). IPNS catalyzes a unique enzyme reaction with no
precedent in chemistry (279).
All intact IPNS enzymes whose genes have been cloned to date have
proline at position 285 in a highly conserved region
(269; reviewed in reference 152).
This Pro residue seems to be essential for activity because a mutant
(N2) of A. chrysogenum (298) which did not
produce cephalosporin encodes a defective ipnA gene,
probably due to the mutation of a single base pair that results in a
change from Pro (amino acid 285) to Leu (amino acid 269).
Baldwin and Wan (29) proposed a catalytic mechanism for IPNS
which involves the formation of an intermediate carbon radical of the
LLD form of ACV, but complete details of the reaction have yet to be
determined. Additional data on the mechanism of the IPNS reaction
suggests that initial formation of the
-lactam ring is followed by
closure of the thiazolidine ring (24). A model was proposed
by Roach et al. (279, 280).
The IPNS shows a broad substrate specificity, in particular with
alterations in the L-
-AAA moiety and the valine residue of ACV. This finding has been ingeniously used to create novel penicillins from ACV analogs in vitro, although cyclization of unnatural tripeptides occurs at lower efficiency (342).
Nevertheless, many new penicillins have been produced biosynthetically
via this route (23), which is thus very promising for the
generation of new
-lactam compounds in vivo.
The genes encoding IPNS enzymes are designated ipnA
(pcbC). The ipnA gene from A. chrysogenum was the first gene encoding an enzyme of
-lactam
biosynthesis to be cloned and sequenced (283). This was
achieved by purification of the enzyme, determination of its N-terminal
amino acid sequence, and subsequent cloning of the gene by reverse
genetics. The gene was overexpressed in Escherichia coli.
ipnA genes have since been cloned and sequenced from several
different fungi and bacteria. Their features are summarized in Table 2.
The ipnA and acvA genes lie close together on the
chromosome. In contrast to bacteria, in fungi ipnA and
acvA are bidirectionally oriented (Fig. 3). For the fungal
genes, it has been demonstrated that the ipnA transcripts
start in the corresponding intergenic regions (Fig. 3). The fungal IPNS
genes identified thus far do not possess introns (reviewed in reference
49).
Gene Specific for Penicillin Biosynthesis
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).
,
-heterodimer,
composed of 11-kDa (
) and 29-kDa (
) subunits, derived from a
40-kDa precursor polypeptide by posttranslational cleavage. The
processing event that generated the two subunits of recombinant IAT
from the 40-kDa precursor polypeptide occurred between Gly 102 and Cys
103 (13). Mutation of aatA in the 11-kDa (
)
subunit, resulting in replacement of Thr 105 with Asn, led to inactive
and uncleaved recombinant IAT. However, coexpression of this mutant
aatA with an aatA derivative encoding the
subunit in E. coli produced acyl-CoA:6-APA acyltransferase activity (323). These results suggest that the formation of
recombinant IAT involves cooperative folding events between the
subunits. In vitro transcription and translation were used to determine the origin of the IAT hydrolase activity that cleaved the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro-translated protein provided evidence that IAT
hydrolysis is an autocatalytic event (323).
Site-directed mutagenesis of the aatA gene and expression in
E. coli revealed that Cys 103 is required for IAT proenzyme
cleavage. Whether this requirement reflects a direct participation of
Cys 103 in cleavage or as part of a cleavage recognition site has not
been clarified yet. However, it cannot be entirely excluded yet that
Cys 103 is involved in IAT enzyme activity, because all of these
experiments were based on the detection of enzyme specific activity
(324). The encoded amino acid sequence in the cleavage site
is identical in P. chrysogenum and A. nidulans
(Arg-Asp-Gly...Cys-Thr-Thr) (13, 14, 324).
IAT enzymes seem to be functionally similar to bacterial penicillin and
cephalosporin acylases that catalyze the deacylation of acyl
side chains of penicillins and cephalosporins to yield 6-APA and
7-aminocephalosporanic acid, respectively. The penicillin acylase from
E. coli ATCC 11105 is a periplasmic enzyme which consists of
two nonidentical subunits (40) that are produced by
posttranslational processing from a precursor protein (41). This functional similarity between fungal IATs and bacterial acylases is striking. However, there is only very low sequence similarity (approximately 11% identical amino acids) between fungal IATs and the
E. coli acylase.
Genes Specific for Cephalosporin Biosynthesis
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).
-ketoglutarate-linked dioxygenases.
Ring expansion by DAOC synthetase and the hydroxylation reaction are
both carried out by the same peptide in A. chrysogenum (93, 284, 290). Purification of this enzyme, determination of its N-terminal amino acid sequence, and reverse genetics allowed the
cloning of the structural gene, designated cefEF
(284). Expression of the cloned cefEF gene in
E. coli (284) established unequivocally that in
A. chrysogenum one protein is responsible for the ring expansion of penicillin N to DAOC and the 3' hydroxylation of DAOC to
DAC (Fig. 2; Table 4). In contrast, in S. clavuligerus, the
two enzymatic activities were separated by anion-exchange chromatography (156) and were later found to be encoded by
two genes, cefE and cefF (Table 4; Fig. 2).
The cefEF gene of A. chrysogenum is located on
chromosome II (300). It is closely linked to the
cefG gene but is transcribed in the opposite direction (Fig.
3). The intergenic region of about 1 kb is believed to contain the
promoters for both genes (131). In S. clavuligerus, the cefF gene is closely linked to the
cefD and cefE genes but is transcribed in the
opposite orientation (Fig. 3) (175). In Lysobacter
lactamgenus and Nocardia lactamdurans, different orders
of genes were found (75, 77, 79, 166, 167) (Fig. 3).
The only data on regulation of the A. chrysogenum gene thus
far reported is a Northern blot analysis which showed that a 1.2-kb transcript corresponding to the cefEF gene was detectable in
cells after 48 h of growth in a defined production medium
(131).
Gene Specific for Cephalosporin C Biosynthesis in Fungi
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).
cefG contains two introns, as demonstrated by sequencing of its cDNA (219, 220). It is closely linked to cefEF but is transcribed in the opposite orientation. The size of the separating intergenic region is not clear, because Gutiérrez et al. (131) found 938 bp, Mathison et al. (219) found 1,077 bp, and Matsuda et al. (220) reported a 1,114-bp intergenic region (Fig. 3; Table 5). Northern blot analysis of A. chrysogenum RNA showed a very weak transcript of about 1.4 kb, corresponding to the cefG gene, in cells grown in a defined production medium for 48 and 96 h (131, 219). These findings seem to agree with reports on the late conversion of DAC to cephalosporin C in cephalosporin fermentations (344). cefEF appears to be expressed at an earlier stage of the fermentation, suggesting that cefEF and cefG are expressed differently from their intergenic region (131) (Fig. 3).COMPARTMENTALIZATION OF GENE PRODUCTS
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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).
|
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).
ORIGIN OF
-LACTAM BIOSYNTHESIS GENES IN
FUNGI
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-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 10
9 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