From the Department of Biotechnology, Norwegian
University of Science and Technology, N-7491 Trondheim, Norway and
¶ SINTEF Applied Chemistry, SINTEF, N-7034 Trondheim, Norway
Received for publication, December 11, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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The loading module for the nystatin polyketide
synthase (PKS) in Streptomyces noursei is represented by
the NysA protein composed of a ketosynthase (KSS),
acyltransferase, dehydratase, and an acyl carrier protein. The absolute
requirement of this protein for initiation of nystatin biosynthesis was
demonstrated by the in-frame deletion of the nysA gene in
S. noursei. The role of the NysA KSS domain,
however, remained unclear, since no data on the significance of the
"active site" serine (Ser-170) residue in the loading modules of
type I PKSs were available. Site-specific mutagenesis of Ser-170 both
in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no
effect on nystatin biosynthesis. A second mutation (S413N) of the NysA
KSS domain was discovered that completely abolished the
ability of the hybrids to restore nystatin biosynthesis, presumably by
affecting the ability of the resulting proteins to catalyze the
required substrate decarboxylation. In contrast, NysA and its Ser-170
mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a
starter unit. Together, these data suggest that the KSS
domain of NysA differs from the KSQ domains found in the
loading modules of several PKS type I systems in that the active
site residue is not significant for its activity.
Modular (type I) polyketide synthases
(PKSs)1 are multifunctional
proteins responsible for the biosynthesis of structurally diverse
natural products, macrolides, with a wide range of pharmacological applications. PKSs catalyze decarboxylative condensations of simple carboxylic acids into the growing polyketide chain by a mechanism similar to the fatty-acid synthases. PKSs, however, are more diverse in
their catalytic reactions, including the use of different primer and
extender molecules. Each PKS module is a collection of domains with
distinct catalytic functions during PKS catalysis, one module being
responsible for one condensation step in the biosynthetic pathway. The
minimal domains necessary for condensation are ketosynthase (KS),
acyltransferase (AT), and acyl carrier protein (ACP). In addition, the
domains ketoreductase, dehydratase (DH), and enoyl reductase
responsible for different degrees of the reduction state of every
A distinctive feature of modular PKS is the presence of a loading
module for chain initiation, which is usually fused to the first
extender module. The loading module of the 6-deoxyerythronolide B
synthase catalyzing the biosynthesis of the erythromycin macrolactone ring in Saccharopolyspora erythraea is composed of the AT
and ACP domains. Although several models for chain initiation have been
proposed for this system (2, 3), evidence accumulated so far indicates
that monocarboxylic acyl-CoA species are utilized as substrates.
However, recent characterization of the new modular PKS proteins has
shown that multidomain loading modules are more common. Such loading
modules typically possess, in addition to AT and ACP, a domain
designated KSQ, which is similar to the chain length factor
(CLF) of type II PKSs (4). Both CLF and KSQ are similar to
the KS domains, while having a glutamine residue substituting cysteine
in the active site. In the actinorhodin type II PKS CLF forms a
heterodimer with KS, and by mutational studies it has been demonstrated
that both domains are competent to catalyze substrate decarboxylation,
as long as at least one of the domains has a glutamine residue
positioned in its active site (4). A similar property has been
suggested for the KSQ domains of some modular PKS, such as
monensin PKS loading module, where a Q177A substitution in the
KSQ domain resulted in a 10-fold reduction of the
decarboxylase activity in vitro (4). In addition,
KSQ is typically accompanied by a distinct AT domain, with
proposed high specificity for dicarboxylic substrates compared with
monocarboxylic substrates (3, 5). According to a model suggested by
others (4), AT loads a dicarboxylic acid onto the ACP, whereas the KSQ domain governs decarboxylation in a way resembling the
catalysis performed by extender modules. However, the residue in an
"active site" position of the KS might be not the only one crucial
for decarboxylation. Mutational analysis of the actinorhodin PKS, for
example, demonstrated that additional KS residues might be critical,
indicating that the precise mechanism for the KS-dependent decarboxylation still remains unclear (6).
We have reported previously (7) cloning and analysis of the complete
gene cluster governing biosynthesis of the polyene antibiotic nystatin
in Streptomyces noursei, and we showed that the deduced
nys-PKS loading module (NysA) possesses some unusual features. NysA is
a single module polypeptide composed of four catalytic domains (see
Fig. 1) and thus resembles a typical
extender module devoid of ketoreductase domain. It contains a KS-like
domain with a serine residue (KSS) instead of a cysteine in
the proposed active site position 170. The DH domain does not seem to
have any function in NysA, except probably for maintenance of a protein
structural integrity, and most likely represents an evolutionary
remnant. The primary sequence of the AT domain (AT0) displays an
acetate-specific signature (8) and contains the conserved Arg-117
residue (Escherichia coli AT numbering) typical for such
domains with high selectivity for CoA esters of dicarboxylic acids (3).
It has been shown recently (9, 10) that two of these features, the
KSS domain and AT domain with the conserved Arg-117
residue, are shared by other polyene type I PKSs. However, in view of
unclear significance of the KSS domain for the NysA
function, it remained unknown whether malonyl-CoA or acetyl-CoA is used
as primer for nystatin synthesis (Fig. 1, A and
B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbon in the polyketide chain may be present in various combinations. It follows that the chemical structure of the final product is largely reflected in the modular organization of the PKS
proteins (1).
View larger version (14K):
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Fig. 1.
Models for initiation of nystatin
biosynthesis in S. noursei ATCC 11455. A, classical model. Malonate is transacylated by AT0 from
its CoA ester to the phosphopantetheine arm of ACP. The in
situ decarboxylation, presumably catalyzed by the KSS,
takes place before the resulting acetyl unit is transferred to NysB for
condensation with the first propionate extender. B,
alternative model. An acetyl unit is recruited directly from
acetyl-CoA, and no substrate decarboxylation is required before
condensation.
In the present study we have analyzed the in vivo role of
NysA for the initiation of nystatin synthesis in S. noursei.
In particular, the significance of the KSS and the AT0
domains was investigated by site-directed mutagenesis and construction
of the hybrid loading modules. Our data indicate that the
KSS domain is important, but not essential, for the
initiation of nystatin biosynthesis.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Media, and Growth Conditions
Bacterial strains, plasmids, and recombinant phages used in this study are listed in Table I. S. noursei strains were maintained on ISP2 agar medium (Difco), and E. coli strains were grown in L broth or L agar (11). Conjugal plasmid transfer from E. coli ET 12567 (pUZ8002) and the gene replacement procedure were performed as described previously (12, 13). Transformation of E. coli was performed as described elsewhere (14). Agar and liquid media, when appropriate, were supplemented with antibiotics using the following concentrations: chloramphenicol, 20 µg/ml; kanamycin, 50 µg/ml; ampicillin, 100 µg/ml; apramycin, 50 µg/ml. Analysis of the nystatin production by S. noursei strains was performed in SAO-23 liquid medium as reported previously (13, 15).
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DNA Manipulations, DNA Sequencing, and PCR
General DNA manipulations were performed as described previously (11). DNA fragments from agarose gels were purified using the Qiaex II kit (Qiagen, Germany). Southern blot analysis was performed with the digoxigenin-11-dUTP High Prime labeling kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Oligonucleotide primers were purchased from Amersham Biosciences, and DNA sequencing was performed using the ABI Prism Big Dye Terminator Cycle sequencing kit and the Genetic Analyzer 3100 (Applied Biosystems, Inc.). The PCRs were performed with the Expand High Fidelity PCR System (Roche Molecular Biochemicals) using the Eppendorf Mastercycler (Eppendorf, Germany), as described previously (15).
Construction of Vectors for In-frame Deletion and Chromosomal Integration of nysA
nysA In-frame Deletion Vector--
The 11.0-kb BamHI
fragment from phage N76 was cloned into pGEM3Zf(), and from this
construct (pL76B11) the 5.83-kb PstI-SacI DNA
fragment encompassing the coding region of the nysA gene and surrounding sequences was ligated into the corresponding sites of
plasmid pGEM3Zf(
). From the resulting plasmid (pL76PS5.8) the 1.68-kb
MscI-PstI and the 2.79-kb
SacI-SmaI DNA fragments were excised and ligated
into the PstI/SacI-digested pGEM3Zf(
). From
this construct, designated pNAD1, the 4.49-kb
EcoRI-HindIII insert was ligated together with
the 3.0-kb EcoRI-HindIII fragment from plasmid
pSOK201, resulting in the nysA replacement vector pNAD2.
nysA Integration Vector--
The 4.63-kb
EcoRI-SpeI fragment from plasmid pL76B11
(including the coding region of nysA and 157-bp upstream
sequences) was ligated into the EcoRI/XbaI sites
of pGEM3Zf(), as well as into the EcoRI-SpeI
sites of pLITMUS28, resulting in plasmids pL76ES4.6 and pLIT76ES4.6,
respectively. From pL76ES4.6 the 4.63-kb
EcoRI-HindIII insert was excised and ligated into
the EcoRI/HindIII sites of vector pSOK804,
resulting in the nysA integration vector
pKSSAT0. Plasmid pSOK804 is a mobilizable vector capable of
site-specific integration into the S. noursei chromosome
recently constructed in or
laboratory.2
Site-directed Mutagenesis of the nysA KSS Coding Region
To prepare a suitable template for site-directed mutagenesis,
the 2.95-kb ApaI fragment from plasmid pL76PS5.8 was ligated into the corresponding site of plasmid pGEM11Zf(). From this construct, pL76A2.95, the 2.81-kb SpeI-SacI
fragment including the nysA KSS coding region
was ligated into the XbaI/SacI sites of
pLITMUS28, resulting in plasmid pLIT76SS2.8. This plasmid was used as a
template for mutagenesis using the QuickChange Site-directed
mutagenesis kit (Stratagene). The putative active site serine residue
in position 170 (Ser-170) of the nysA gene product was
substituted with cysteine (S170C), glutamine (S170Q), and glycine
(S170G) by using the following mutagenic primer pairs, respectively:
S170C-F,
5'-CGTCACCGTCGACACCACGTGCAGCTCGTCGCTGGTCGCGCTGC-3' (sense), and S170C-R,
5'-GCAGCGCGACCAGCGACGAGCTGCACGTGGTGTCGACGGTGACG-3' (antisense); S170Q-F,
5'-CGTCACCGTCGACACCACGCAGAGCTCGTCGCTGGTCGCGCTGC-3', (sense), and S170Q-R,
5'-GCAGCGCGACCAGCGACGAGCTCTGCGTGGTGTCGACGGTGACG-3' (antisense); S170G-F,
5'-CTCACCGTCGACACCACGGGATCCTCGTCGCTGG-TCGCGC-3' (sense), and S170G-R,
5'-GCGCGACCAGCGACGAGGATCCCGTGGTGTCGACGGTGAC-3' (antisense). Mutated nucleotides are shown in boldface, and all mutations were verified by DNA sequencing. Underlined in all primers are new restrictions sites (AluI for mutations S170C and
S170Q and BamHI for mutation S170G) introduced for
simplified identification of positive clones. After mutagenesis, the
1.02-kb MluI-SpeI fragments including the newly
introduced mutations were excised and ligated together with the 3.60-kb
EcoRI-MluI fragment from pLIT76ES4.6 into the
EcoRI/XbaI sites of vector pGEM3Zf(
). From the
resulting constructs, the 4.63-kb EcoRI-HindIII
inserts were ligated into the corresponding sites of vector pSOK804,
resulting in plasmids pKSCAT0, pKSQAT0, and
pKSGAT0, respectively. All three plasmids are analogous to
the integration vector pKSSAT0, except for the desired
point mutations affecting Ser-170 in the NysA KSS domain.
PCR Amplification of DNA Regions Encoding Individual PKS Domains
The nys-PKS regions encoding individual domains were PCR-amplified and subcloned. Primers for the introduction of unique restriction sites were typically used to facilitate further assembly of the resulting fragments into hybrid genes. The new restriction sites introduced and used for the assembly procedure were in all cases positioned in the inter-domain linker regions to avoid any unwarranted structural effects for the resulting hybrid proteins. All the resulting constructs were verified by DNA sequencing.
PCR Amplification and Subcloning of DNA Regions Encoding
Variant KS Domains--
The 1.75-kb DNA fragments covering the coding
region of the variant NysA KSQ, KSC, and
KSS domains, including the nysA promoter region,
were PCR-amplified from the plasmid pLIT76SS2.8 and its mutated
derivatives (see above) DNA using the following primer pair: NAKS-F,
5'-TACGACTCACTAGTCTTCGGCGCGCG-3' (sense), and NAKS-R,
5'-GGAGGATCCTGGTGGGCCGCACCGC-3' (antisense). The latter
primer contains a BamHI recognition site
(underlined) used for the hybrid assembly procedure (see
below). The resulting PCR fragments were blunt-ligated into the
SmaI site of pUC18, and from the resulting constructs the
1.75-kb EcoRI-BamHI inserts were excised and
ligated into the corresponding sites of vector pGEM3Zf(), resulting
in the constructs pGEM-KSQ, pGEM-KSC, and
pGEM-KSS, respectively (see Table I). In addition, a
derivative of pGEM-KSC harboring a second point mutation
(S413N) of the KS coding region was identified and denoted
pGEM-KSCN. This mutation most probably resulted from an
erroneous PCR amplification. The 1.0-kb MluI-SpeI
fragment from plasmid pGEM-KSCN (including the S413N but
not the S170C mutation) was used to substitute the corresponding
fragments in plasmids pGEM-KSQ and pGEM-KSS,
resulting in plasmids pGEM-KSQN and pGEM-KSSN,
respectively (see Table I).
PCR Amplification and Subcloning of the nysA DH + ACP Coding
Region--
The 1.63-kb DNA fragment encompassing the continuous
coding region of the NysA DH + ACP domains was PCR-amplified from the plasmid pL76B11 DNA, using the following primer pair: NADH-F, 5'-GGAGCATGCCTTCTACGCCGGCAC-3' (sense), and NADH-R,
5'-GCGAAGCTTGGTCAGTCCTCGTCATC-3' (antisense). Underlined in
the primers are the restriction sites (HindIII and
SphI, respectively) used for end digestion and cloning of
the PCR fragment into the corresponding sites of pGEM3Zf(), resulting
in plasmid pGEM-DHACP.
PCR Amplification and Subcloning of the NysB AT1 and the NysC AT3
Coding Regions--
The 0.99-kb DNA fragment including the coding
region of the Nys-PKS module 1 AT domain (AT1) was PCR-amplified from
the pL76B11 DNA using the following primer pair: NBAT1-F,
5'-GGAGGATCCGACGGTGTTCGTGTTCC-3' (sense), and NBAT1-R,
5'-GCAGCATGCCAGTCCACGTCCACAC-3' (antisense). Similarly, the
0.99-kb DNA fragment covering the coding region of the Nys-PKS module 3 AT domain (AT3) was PCR-amplified from the phage N14 DNA using the
following primer pair: NCAT3-F,
5'-GCAGGATCCCTGCGCCGTCCTCTTC-3' (sense), and NCAT3-R,
5'-GCAGCATGCCAGCGGACCGTGACGC-3' (antisense). Both the
resulting PCR products were end-digested with BamHI and SphI (recognition sites underlined in each primer) and
ligated into the corresponding sites of pGEM3Zf(), resulting in the
constructs pGEM-AT1 and pGEM-AT3, respectively.
Assembly of DNA Fragments Encoding Individual PKS Domains into Integration Vectors Expressing Hybrid-loading Modules
One of the variant 1.75-kb EcoRI-BamHI KS-encoding fragments from the construct pGEM-KSC, pGEM-KSQ, pGEM-KSS, pGEM-KSCN, and pGEM-KSQN, or pGEM-KSSN, the 1.6-kb SphI-HindIII DH + ACP insert from pGEM-DHACP, and one of the variant 0.99-kb BamHI-SphI AT inserts from pGEM-AT1 or pGEM-AT3 were ligated into the EcoRI/HindIII sites of the vector pSOK804. By using this strategy the following nine different integration plasmids were obtained: pKSSAT1, pKSCAT1, pKSQAT1, pKSSAT3, pKSCAT3, pKSQAT3, pKSSNAT3, pKSCNAT3, and pKSQNAT3 (see Tables I and II).
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Introduction of the S413N Mutation into the Wild-type NysA and Its Mutants
The 1.58-kb SpeI-AarI fragments from
plasmids pGEM-KSSN and pGEM-KSCN, or
pGEM-KSQN (encompassing the nysA codons for the
residues in positions 170 and 413), and the 3.05-kb
AarI-EcoRI fragment from plasmid pLIT76ES4.6 were
ligated into the XbaI/EcoRI sites of pGEM3Zf().
From the resulting constructs the 4.65-kb
EcoRI-HindIII fragments were ligated into the
corresponding sites of pSOK804, resulting in the integration vectors
pKSSNAT0, pKSCNAT0, and pKSQNAT0,
respectively. These vectors are similar to the nysA
expression vector pKSSAT0 (see above), except for the point
mutations affecting Ser-170 and Ser-413 in the NysA
KSS domain.
Sequence Alignment and Phylogenetic Analysis
Alignment of the amino acid sequences was done using the ClustalW 1.82 software provided on-line by the European Bioinformatics Institute (www.ebi.ac.uk/clustalw). The alignment was visualized using the BoxShade 3.21 on-line software from the Swiss node of EMBnet (www.ch.embnet.org/software/BOX_form.html).
The phylogenetic tree was drawn by the TreeView 1.6.6 software
developed by R. D. M. Page at the University of Glasgow
(taxonomy.zoology.gla.ac.uk/rod/treeview.html) using the distances
calculated after the ClustalW alignment at the EBI server (see above).
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RESULTS |
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In-frame Deletion and Complementation of nysA--
In a previous
report (7) we described the nysA gene disruption abolishing
all nystatin synthesis in S. noursei. However, to rule out
the possibility of any polar effects that might have been imposed by
this mutation, we constructed a nysA in-frame deletion
mutant NDA59 by double homologous recombination, using the gene
replacement vector pNAD2. The 1.36-kb in-frame deletion affecting the
nysA gene within this plasmid eliminated the coding sequence
for the C-terminal part of KSS and most of the AT0 domain
(see Fig. 2). Analysis of NDA59 showed that it produces no nystatin (Fig. 2B), thus confirming that
the NysA loading module is essential for initiation of nystatin
synthesis in S. noursei. In an attempt to complement this
mutant, plasmid pKSSAT0 was chromosomally integrated in the
NDA59 strain. This plasmid harbors a DNA fragment encompassing the
nysA gene and a 157-bp sequence upstream of its coding
region, presumably containing the nysA promoter. Nystatin
synthesis was restored to wild-type levels (Fig. 2C) in the
NDA59 (pKSSAT0) strain, demonstrating that the
nysA gene expressed in trans from its endogenous
promoter can efficiently complement the nysA deficiency in
the NDA59 mutant. We next integrated plasmid pKSSAT0 into
the wild-type strain in order to test whether expression of an
additional copy of nysA may stimulate nystatin production. This manipulation had no detectable effect on the nystatin synthesis (data not shown) suggesting that the NysA expression level is not a
limiting factor for the production of this antibiotic under the
conditions tested.
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Replacement of the Proposed Active Site Serine (Ser-170) in the
NysA KSS Domain Has No Effect on Nystatin Synthesis in
Vivo--
It is generally accepted that the initial condensation step
during the PKS-catalyzed polyketide synthesis takes place on the KS
domain of the first extender module. Loading modules usually either
lack the KS domains completely or have KSQ domains with a
glutamine instead of a cysteine in the active site position. These
KSQ domains in the loading modules are believed to be
responsible for decarboxylation of the starter dicarboxylic acid CoA
esters prior to condensation. The KSS domain of NysA has a
serine residue in this position (Fig.
3A), and the role of this
domain for initiation of nystatin synthesis was unknown. We proposed
that if AT0 recruits malonyl-CoA, then KSS should possess a
decarboxylase activity, whereas no such activity is needed if
acetyl-CoA is used (see Fig. 1). To test whether the Ser-170 in
KSS is important for the NysA activity, this residue was
substituted with glutamine (S170Q), cysteine (S170C), and the
catalytically "silent" glycine (S170G). Integration vectors
expressing the resulting mutant nysA genes (plasmids
pKSQAT0, pKSCAT0, and pKSGAT0,
respectively) were introduced into the S. noursei mutant NDA59, and the resulting recombinant strains were analyzed for nystatin
production. Surprisingly, similar nystatin production by the NDA59
(pKSSAT0) and NDA59-based recombinant strains expressing
NysA with mutagenized KS domain was observed (Table II), suggesting
that the Ser-170 residue is not important for NysA-mediated initiation of the nystatin biosynthesis under these conditions.
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Construction and Expression of Hybrid Loading Modules with Mutant KS and Alternative AT Domains in the NDA59 Mutant-- If one assumes that NysA is using malonyl-CoA as a starter, then the results described above would imply that the supposed KS-mediated decarboxylation of malonyl-CoA is independent of the type of residue in the active site position 170. Alternatively, the NysA might utilize acetyl-CoA as a starter, suggesting that decarboxylation is not at all required in the initiation process, and the KSS domain has no catalytic role (Fig. 1). To resolve these issues, we constructed hybrid NysA derivatives where the AT0 domain was substituted with the AT domains from the downstream PKS modules that must have high specificity for the dicarboxylic acid substrates.
Based on the known nystatin structure and sequence analysis of its biosynthetic genes, the AT domains of the nys-PKS modules 1 (AT1) and 3 (AT3) are clearly specific for methylmalonyl- and malonyl-CoA, respectively (7). Both these domains were individually used to substitute for AT0 in NysA and its KS mutants. The purpose of these experiments was to unravel whether NysA can utilize dicarboxylic acid substrates in vivo, and whether this ability is dependent on the type of residue in position 170 of the loading module. Care was taken to keep the polypeptide length, the interdomain linkers, and the domain order of each hybrid similar to the native NysA protein. The C-terminal part of NysA encompassing DH and ACP domains was kept intact in all cases. The resulting vectors were introduced into S. noursei mutant NDA59, and the recombinant strains were analyzed for nystatin production.
None of the integration vectors expressing the AT1 hybrids (pKSSAT1, pKSCAT1, and pKSQAT1) could restore synthesis of either nystatin or related polyenes when introduced into the NDA59 mutant (Table II). However, the integration vectors expressing the AT3 hybrids (pKSSAT3, pKSCAT3, and pKSQAT3) were able to restore nystatin production in NDA59, although not as efficiently as the pKSSAT0 expressing the wild-type nysA (Table II). These results indicate that NysA is capable of using malonyl-CoA as a starter unit in a manner that seems to be independent of the type of residue in position 170. In this way our data contradict previous reports (3, 4) stating that the type of residue in this particular position is critical for decarboxylase activity and suggest that other residues within NysA may be essential. The lower production levels obtained with the AT3 hybrids (between 11 and 17%) compared with those obtained with the wild-type NysA may be due to several reasons, the improper folding of the hybrid proteins being most likely. This result may also in part be explained if the hybrids have narrower substrate specificity, i.e. if they are capable of accepting only malonyl-CoA, whereas both acetyl-CoA and malonyl-CoA may be used by the wild-type NysA.
A Novel KS Mutation (S413N) Abolishes the in Vivo Activity
Exclusively in the AT3 Hybrids--
During the hybrid construction
procedure (see "Experimental Procedures"), an unwarranted mutation
was discovered, resulting in substitution of the highly conserved
serine residue in position 413 of the NysA KS domain with an asparagine
(S413N). First, this mutation was introduced into the integration
vectors expressing the AT3 hybrids, and the resulting plasmids
(pKSSNAT3, pKSCNAT3, and pKSQNAT3)
were used to complement NDA59. Surprisingly, none of the resulting
recombinant strains was able to produce nystatin (Table II), suggesting
that Ser-413 was critical for the in vivo activity of the
AT3 hybrids. Sequence comparisons (Fig. 3A) revealed that this particular residue is highly conserved in the KSs of single polypeptide loading modules as well as extender module KSs. We reasoned
that if this mutation somehow affects the decarboxylase activity linked
to KSS, and wild-type NysA can use both acetyl- and
malonyl-CoA as starter units, the effect of such a mutation would be
less dramatic on the AT0 background. Therefore, the S413N mutation was
introduced into the integration vectors expressing NysA and its Ser-170
mutants, and the resulting plasmids (pKSSNAT0,
pKSCNAT0, and pKSQNAT0) were introduced into
the NDA59 mutant. Remarkably, the nystatin synthesis in the resulting
recombinant strains was restored to 19-36% compared with the NDA59
(pKSSAT0) (Table II). Together, these results suggest that
the observed effect of the S413N mutation on the ability of the loading
modules to initiate nystatin biosynthesis depends on the type of AT
domain present (see "Discussion").
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DISCUSSION |
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Modular PKSs have proven to be exciting targets for the rational genetic engineering with the aim of producing new macrolide antibiotics. Several strategies such as single domain inactivation, domain swapping and insertion, and module rearrangements have led to synthesis of novel polyketides (16). The process of initiation of polyketide synthesis governed by the distinct loading modules may also be a target for genetic manipulations aimed at the synthesis of new products (5, 17). The majority of the modular PKS loading modules can be divided into two classes, depending on their domain organization and, consequently, their mode of initiation of polyketide synthesis. One class is represented by the loading modules having, in addition to the AT and ACP domains, a KSQ domain with suggested decarboxylase activity. These modules, represented by oleandomycin, niddamycin, and pikromycin PKSs, accept dicarboxylic acid-CoA esters such as malonyl-CoA and methylmalonyl-CoA as starters. Such starter molecules must be decarboxylated before they are loaded onto the first extender module, and the KSQ domain seems to be crucial for this process (4). Yet another class of loading modules found, for example, in the erythromycin and avermectin PKSs is characterized by having the AT and ACP domains only. These modules apparently use monocarboxylic acid-CoA esters such as propionyl-CoA and isobutyryl-CoA to load them onto the first extender module of the corresponding PKS. Therefore, no decarboxylation is required during initiation of polyketide synthesis in the latter systems.
The deduced loading module for the nystatin PKS, NysA, is composed of the KSS, AT0, DH, and ACP domains (see Fig. 1). By constructing a nysA in-frame deletion mutant, we have demonstrated that NysA is essential for in vivo production of nystatin in S. noursei. This result suggests that the first extension module of NysB cannot be loaded directly by a starter carboxylic acid, as was apparently the case for erythromycin PKS, where inactivation of the 6-deoxyerythronolide B synthase loading module did not abolish erythromycin production completely (2, 18). In ery-PKS, the direct loading of the KS domain of the first extension module was proposed as an alternative mechanism for chain initiation (3).
During the polyketide chain elongation the KS domains of each extender module supposedly play two roles: (i) they catalyze condensation of the extender unit onto the polyketide chain, and (ii) simultaneously promote the in situ decarboxylation of the recruited dicarboxylic acyl-CoA esters during the condensation process. These two functions seem to be inseparable in the extender KSs, since the condensation process strictly depends on decarboxylation (19, 20). The condensation invariantly requires the conserved cysteine in the KS active site, whereas the mechanism and amino acid residue(s) involved in decarboxylation are still not completely unraveled (4, 6). It is suggested that there exists a cooperation between the KS and AT domains on each module for the discriminative recruitment and concomitant decarboxylation of dicarboxylated substrates (3, 5). In the complex loading modules containing KSQ domains, the active site glutamine has been shown to be important for decarboxylase activity (4). In such loading modules the AT domains are found to be structurally and functionally similar to their counterparts found in the extender modules, with high specificity for the dicarboxylic acyl-CoAs (3). NysA has an extender-type AT domain suggesting that malonyl-CoA might be its preferred substrate. However, this assumption heavily relies on the decarboxylase activity of the KSS, analogous to what has been demonstrated for the KSQ in other systems. At least in the type II PKS system, the CLF with serine residue in the active site position retained most of its decarboxylase activity (6).
One obvious strategy to test whether the KSS is at all required for initiation of nystatin biosynthesis would be to make an in-frame deletion within the KSS-coding region of nysA. However, previous attempts on using such strategies for inactivation of the nys-PKS reduction domains typically abolished all polyene production, presumably by disturbing the folding of the resulting mutant proteins.3 Our mutagenesis results clearly show that the type of residue in the active site position 170 of KSS has no significance for the in vivo activity of NysA. However, the successful complementation of the nysA-deficient mutant NDA59 using the AT3 hybrids indicates that NysA is somehow capable of catalyzing the required decarboxylation of malonyl-CoA. Although the type of residue at position 170 had little effect on the ability of the hybrid proteins to initiate nystatin biosynthesis, the second S413N mutation completely aborted this function in such hybrids. The most probable explanation is that the latter mutation has abolished decarboxylase activity of these proteins preventing proper priming with the malonyl-CoA recruited by the AT3. Alternatively, this substitution might have caused unwarranted structural changes in the AT3 hybrids leading to loss of all in vivo activity. The latter explanation seems unlikely, since introduction of the S413N mutation in the wild-type NysA, as well as in the NysA KSC and KSQ mutants, did not abolish their ability to initiate nystatin synthesis, although it has been diminished by 64-81%. By assuming that the S413N mutation abolishes decarboxylase activity in NysA, the residual in vivo activity observed for NysA and its active site mutants bearing this mutation could be due to the utilization of acetyl-CoA as a starter. This notion is in agreement with a recent report showing that loading module AT of the ery-PKS has, in contrast to the extender AT, a relaxed specificity toward acyl-CoA substrates and can accept both propionyl-CoA, acetyl-CoA, and butyryl-CoA (21). No clues as to which amino acid residues might be responsible for the proposed broad substrate specificity of AT0 in NysA could be found by inspecting the alignment of the AT amino acid sequences (data not shown). Loading modules of the polyene PKSs seem to be rather unique, as they are represented by separate polypeptides, whereas in other modular PKS systems loading modules are fused to the first extender module. This feature may in principle provide a greater degree of freedom in folding of the polyene PKS loading modules that, in turn, might affect the choice of the starter unit by these proteins.
The inability of the AT1 hybrids to complement nystatin synthesis in NDA59 could be due to several reasons. Based on the results obtained with the AT3 hybrids, we suggest that these observations cannot be solely explained by the lack of the decarboxylase activity in the hybrid proteins. A possible explanation may be that the downstream nys-PKS module in NysB cannot use the propionate primer generated by these engineered proteins, and further elongation is aborted. Alternatively, the heterologous AT1 domain might be the cause of the misfolding of the hybrid modules and the subsequent loss of activity. Similar problems are documented by others (22), and in such cases site-directed mutagenesis has successfully been applied as an alternative strategy for changing the substrate specificity of AT domains.
Alignment of the amino acid sequences for the KS-like domains in the
PKS type I loading modules clearly shows that all KSQ
domains contain a glycine at position 413 (NysA numbering), whereas the
single polypeptide loading modules contain highly conserved serine
(Fig. 3A). A threonine residue in the PimS0 loading module is structurally very similar to serine and thus might well serve as its
functional substitute. Interestingly, the alignment of the KS domains
from the extender modules of 13 PKS type I systems (encompassing
together over 100 KS domains) shows that the Ser-413 residue is highly
conserved among these domains, with only one exception; KS in the
module 2 of rifamycin PKS (data not shown). Ser-413 was also found to
be highly conserved in the extender KSs of the PKS type I systems
discovered in such diverse bacterial genera as
Mycobacterium, Nostoc, Stigmatella,
Desulfovibrio, and Planktothrix (data not shown).
Finally, the phylogenetic tree based on the KS sequence alignments
clearly shows that KSS cluster with the extender KS
domains, whereas the KSQ domains represent a separate
branch (Fig. 3B). All these data further suggest that single
polypeptide loading modules differ from their KSQ
counterparts, which could explain the discrepancy in the data obtained
upon the active site residue mutagenesis reported here and by others
(4).
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ACKNOWLEDGEMENTS |
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We are grateful to R. Aune for help with the S. noursei fermentations and to C. R. Hutchinson, S. Valla, and A. R. Strøm for participation in the discussions in the course of this work.
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FOOTNOTES |
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* This work was supported by a grant from the Research Council of Norway.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. Tel.: 47-73-59-69-42; Fax: 47-73-59-12-83; E-mail: trygve.brautaset@biotech.ntnu.no.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M212611200
2 O. Sekurova, personal communication.
3 T. Brautaset and S. B. Zotchev, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PKS, polyketide synthase; KS, ketosynthase; AT, acetyltransferase; ACP, acyl carrier protein; DH, dehydratase; CLF, chain length factor.
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