1 Institut für Kristallographie, Takustrasse 6 and 2 Institut für Biochemie, Thielallee 63, Freie Universität Berlin, D-14195 Berlin, Germany
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Abstract |
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Keywords: antibiotics/peptide synthetase/protein design
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Introduction |
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NRPSs and the related PKSs have been analysed recently by extensive engineering approaches referred to as combinatorial biosynthesis in order to generate the production of new antimicrobial substances (Cane et al., 1998; Rodriguez and McDaniel, 2001
; Staunton and Wilkinson, 2001
). A promising approach to altering the substrate specificity of peptide synthetases has been the directed mutagenesis of putative substrate binding pockets in selected A-domains (Stachelhaus et al., 1999
; von Döhren et al., 1999
). The amino acid sequence of non-ribosomally produced peptides was further altered by the addition, deletion or exchange of enzymatic domains or even complete amino acid incorporating modules as implicated by the modular structure of NRPSs (Stachelhaus et al., 1995
; Schneider et al., 1998
; Doekel and Marahiel, 2000
; Mootz et al., 2000
, Schauwecker et al., 2000
). Sequence comparisons, mutagenesis studies and limited proteolysis indicated that one module consists of
1000 amino acid residues. However, only limited structural information of distinct NRPS domains is available so far (Conti et al., 1997
; Weber et al., 2000
). In addition, interactions between the different domains are also mostly unknown. The manipulation of complex proteins by insertion, exchange or deletion of large internal regions could have a major impact on the overall structural conformation and folding pathway. Choosing the correct site for recombination of peptide synthetase genes should therefore be important for the activity of the resulting enzyme. Approaches have been initiated to analyse recombinant NRPSs generated by using fusion sites located in variable surface accessible putative linker regions (Stachelhaus et al., 1995
; Schneider et al., 1998
; Doekel and Marahiel, 2000
; Mootz et al., 2000
). However, linkers may have a crucial role in controling communication and inter-modular proteinprotein contacts between different modules or domains of multi-modular proteins and they are important to direct correlated movements of the various domains (Gokhale and Khosla, 2000
). Linkers between the CA-domains of NRPSs have been shown to be important and the CA-domain couple may form a catalytic unit with a specific interface (Belshaw et al., 1999
). The proposed editing function of the C-domains might further contribute to the failure of rearrangements in that regions or to the very low activities of the resulting hybrid enzymes (Stachelhaus et al., 1995
; Schneider et al., 1998
). Furthermore, linkers have been shown to be specific and essential for the inter-modular product transfer upon engineering of PKSs (Gokhale et al., 1999
; Ranganathan et al., 1999
). We therefore attempted to identify permissive sites suitable for the rearrangement of NRPSs within conserved motifs. As a model system, we chose the surfactin synthetase complex consisting of the two three-modular enzymes SrfAA and SrfAB and the monomodular enzyme SrfAC. The internal L-leucine activating module of SrfAA was deleted by genetic engineering and the two terminal modules were recombined at different sequence motifs located in the A-, T- and C-domains, resulting in various SrfAA derivatives lacking the second L-leucine-incorporating module. This strategy ensured that no changes were introduced in variable linker regions which might be involved in directing domain interactions.
We demonstrate here that the conserved sequence motif HHIIxDGVS located in the C-domains of peptide synthetase modules is suitable as a recombination site for the genetic engineering of NRPSs. In our conserved motif fusion approach, only a recombinant surfactin synthetase subunit fused at this region showed high activities in all enzymatic assays and resulted in the production of the expected lipohexapeptide in B.subtilis. We further present evidence for an altered profile of antimicrobial activity of the newly synthesized surfactin derivative.
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Materials and methods |
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Strains and plasmids used in this study are listed in Table I. The B.subtilis strain ATCC 21332 was used as a source of surfactin synthetase genes and for the engineering of surfactin synthetase and strain DH5a was used for cloning procedures and propagation of plasmids. Bacterial cells were cultivated in Luria broth (LB) or in Landy medium (Landy et al., 1948
) supplemented with 0.1% yeast extract and 2 mg/l phenylalanine (Vollenbroich et al., 1993), at temperatures of 28 or 37°C. If appropriate, ampicillin was added to a final concentration of 100 mg/ml. NRPSs were overproduced in Escherichia coli strain M15 (x pREP4) with an N-terminal poly(His)6-tag using the vector pQE30. The construction of the plasmids psrfCDM-M12/3, psrfTD-M1/23 and psrfADH-M1/23 for the overproduction of recombinant SrfAA proteins was described previously (Symmank et al., 1999
). For marker exchange mutagenesis of B.subtilis, DNA fragments of the constructed srfAA derivatives including the fusion sites were cloned into the suicide vector pMMN13 (Nakano and Zuber, 1989
). DNA techniques such as purification and recombination of DNA followed standard protocols (Sambrook et al., 1989
). PCR was performed with Vent-polymerase (New England Biolabs). Transformation of B.subtilis ATCC 21332 was essentially done as described (Cutting and Vander Horn, 1990
).
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Overproduction and purification of proteins from E.coli were carried our as already described (Symmank et al., 1999). B.subtilis cells were suspended in lysis buffer containing a final concentration of 0.1% lysozyme and lysed upon repeated freezethaw cycles at 20°C. The cells were then centrifuged at 30 000 g for 2 h or subsequently disrupted with a French press at a maximum pressure of 700 psi. The three-modular enzymes SrfAA and SrfAB were purified by loading the bacterial extract on an Ultrogel AcA 34 column (100x4 cm i.d.) and separated at a flow-rate of 0.5 ml/min in 50 mM TrisHCl, pH 8.0, 100 mM NaCl and 5 mM DTT. Bimodular enzymes were purified with a Sephacryl S-200 HR column (60x3.5 cm i.d.) at a flow-rate of 1 ml/min in the same buffer. The peptide synthetases eluted in the void volume. Proteins were routinely analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) (Laemmli, 1970) and quantified with the Bradford assay (Bradford, 1976
). If not used directly for enzymatic assays, the proteins were stored in 5% (v/v) glycerol at 70°C.
ATPPPi exchange assay
The amino acid adenylation activity was determined with the ATPPPi exchange assay (Symmank et al., 1999). The assay was performed in a reaction volume of 200 µl containing 2 mM substrate amino acid, 2.5 mM MgCl2, 0.5 mM ATP, 0.1 mM Na4P2O7, 50 mM MESHEPES, pH 6.5 and 0.11 µCi (
240 000 c.p.m.) 32P-labelled PPi. The reaction was started by addition of 350 pmol enzyme and incubated at 37°C. The reaction was stopped by addition of 500 µl of cold stop solution (1% acid-washed pulverized charcoal (Fluka, ultrapure) in 0.1 M Na4P2O7 and 14% HClO4) and incubation for 10 min on ice. The charcoal was filtered through a GF92 glass filter (Schleicher & Schuell), washed with water and the amount of bound [32P]ATP was determined with a liquid scintillation counter.
In vitro peptide formation
The in vitro peptide formation by recombinant surfactin synthetase subunits was analysed by incubating 50 pmol of purified enzyme with 20 mM amino acid substrates, 0.5 µM ATP, 10 mM MgCl2, 500 mM MESHEPES pH 6.5 and 2.7 µCi 14C-labelled amino acid (200 mCi/mmol). To initiate the reaction, each sample contained 160 µM 3-hydroxytetradecanoyl-coenzyme A. The reaction was performed in a total volume of 100 µl and incubated for 60 min at 37°C. After phenol extraction, the reaction mixture was separated by thin-layer chromatography (TLC) on a silica gel 60 plate (Merck) with chloroformmethanolH2O (65:25:4, v/v/v) as the mobile phase. After separation, the TLC plates were dried and exposed to X-ray film for 3 weeks.
Isolation of lipopeptides
Lipopeptides were isolated according to Ohno et al. (Ohno et al., 1992), with slight modifications. Landy medium was inoculated with a fresh bacterial preculture 1:100 and incubated at 30°C and 120 r.p.m. in a shaker for 3 days. After incubation, the culture was adjusted to pH 2.0 with concentrated HCl and the lipopeptides were precipitated for 1 h at 4°C (Ohno et al., 1992
). The precipitate was pelleted for 20 min at 4°C at 8000 g and extracted with methanol for 2 h at room temperature on a shaker at 250 r.p.m. The extract was centrifuged for 10 min at 9000 g at room temperature and the supernatant was concentrated by rotary evaporation. The residue obtained was dissolved in a suitable volume of methanol, discolored with charcoal and used for further analysis.
Lipopeptides were analysed by TLC on silica gel 60 plates (Merck) with chloroformmethanolH2O (65:25:4, v/v/v) as the mobile phase. After separation, the plates were air dried and developed by spraying with water and heating slightly.
Analysis of peptides by mass spectrometry
Mass spectra of methanol extracts of the isolated lipopeptides were recorded using a Bruker Reflex MALDI-TOF instrument with delayed extraction containing a 337 nm nitrogen laser for desorption and ionization. Sample aliquots were mixed with a saturated solution of -cyano-4-hydroxycinnamic acid in 30% aqueous acetonitrile containing 0.1% (v/v) trifluoroacetic acid and air dried. The acceleration and reflector voltages were 20 and 23 kV, respectively. Post-source decay (PSD) mass spectra were used for confirming the novel lipopeptides. Data were evaluated with Bruker FAST software.
Synthesis of 3-hydroxytetradecanoyl-coenzyme A
The synthesis of 3-hydroxytetradecanoyl-coenzyme A (ß-HA-CoA) was performed in a two-step reaction. First, from DL-ß-hydroxymyristic acid (ß-HA), N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCCI), ß-HA-NHS was synthesized. Second, ß-HA-CoA was synthesized from ß-HA-NHS and coenzyme A-SH (Blecher, 1981).
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Results |
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In a systematic screening for permissive fusion sites within highly conserved regions of NRPSs, recombinations at the sequence motifs FF(E/D)LGG(H/D)SL present in T-domains, HHIIxDGVS present in C-domains and at the hinge region found in A-domains resulted in the amino acid activating hybrid bimodular enzymes SrfTD-M1/23, SrfCDM-M12/3 and SrfADH-M1/23 (Table II). To find out if the constructs are suitable for the synthesis of the desired lipohexapeptide in vivo, we first analysed the in vitro formation of peptide products by the recombinant bimodular synthetases. The purified enzymes were incubated with reaction mixtures containing 14C-labelled L-glutamic acid and the reaction was started by adding ß-hydroxytetradecanoyl-coenzme A. The samples were analysed by TLC and we could observe two putative product profiles. No clear product formation was detectable with the proteins SrfTD-M1/23 and SrfADH-M1/23. The thioester formation of the enzyme SrfADH-M1/23 with L-glutamic acid was previously found to be drastically reduced (Table II
) and this could contribute to the failure to observe a product formation. However, the result was unexpected for the enzyme SrfTD-M1/23 as it showed a high rate of adenylation and thioacylation of its cognate amino acid substrates (Table II
) (Symmank et al., 1999
). The hybrid T-domain in the enzyme SrfTD-M1/23 might therefore be unable to communicate efficiently with the other enzymatic domains. The second profile included the product pattern of the recombinant enzyme SrfCDM-M12/3 and that of the wild-type enzyme SrfAA. A similar complex pattern of 89 separated bands were detected with both enzymes (data not shown). This gave the first evidence that only the two latter analysed enzymes were able to produce some products in our assay. However, owing to the lack of labelled references, we could not further identify the observed products separated by TLC.
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We now started to construct a B.subtilis mutant in order to analyse the usefulness of the selected fusion sites in vivo. Using a combined homologous recombinationmarker exchange approach, we intended to delete the second L-leucine incorporating module of the surfactin synthetase AA by exchanging the chromosomal srfAA gene with the recombinant genes expressing the two most promising hybrid enzymes SrfCDM-M12/3 and SrfTD-M1/23 (Figure 1). If the constructed recombinant proteins retain all enzymatic activities, the resulting mutants should be able to produce the lipohexapeptide surfactin ADL2, a surfactin A derivative carrying a deletion of the second L-leucine residue (Figure 1A
). Approximately 1.8 kb (srfCDM-M12/3) and 2.2 kb (srfTD-M1/23) DNA fragments containing the corresponding fusion sites were isolated from the plasmids psrfCDM-M12/3 and psrfTD-M1/23 and cloned into the suicide vector pMMN13, resulting in the plasmids pIPsrfA-DLeuTD and pIPsrfA-DLeuCDM. The suicide plasmids were then transformed into B.subtilis ATCC 21332 and plasmid integration was selected by growth on chloramphenicol. The subsequent excision of the integrated plasmids by a second homologous recombination was identified by the loss of chloramphenicol resistance. The correct recombinations in the resulting mutants B.subtilis R13TD and R13CDM (Figure 1B
) were verified by PCR and restriction analysis.
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The colony type of the wild-type strain B.subtilis ATCC 21332 and the mutant strains R13TD and R13CDM showed striking differences. In contrast to the rough and wrinkled wild-type colonies, the morphology of the mutant R13TD was smooth. The colony surface of the mutant R13CDM showed an intermediate appearance. The mutant R13TD showed furthermore an increased autolysis, which was complete after 10 days of incubation on LB agar at room temperature. This phenotype was not observed either with the wild-type strain or with mutant R13CDM. In addition, we observed a retarded growth of the mutant R13TD in LB or Landy medium (Figure 2
). While the growth of the wild-type strain and mutant R13CDM was similar, the final cell density of mutant R13TD was reduced to
60% if compared with the two former strains.
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We compared the lipopeptide biosynthesis of the mutant R13CDM with that of the wild-type strain ATCC 21332. In both strains, the lipopeptide production started in the late exponential growth phase and continued after further incubation in the stationary phase (data not shown). Lipopeptides were extracted with methanol from the culture supernatant after 3 days of growth in Landy medium and were analysed by TLC. As expected, the wild-type strain B.subtilis ATCC 21332 produced the lipoheptapeptide surfactin A which was visible as a white spot after development of the TLC plate (Figure 4). The Rf value of 0.51 agrees with the literature (Ullrich et al., 1991
; Menkhaus et al., 1993
). With the extract of the supernatant of the mutant R12CDM, a predominant spot with an Rf value of 0.39 was obtained, presumably representing the lipohexapeptide surfactin ADL2 lacking one L-leucine residue. Considering the amounts of the samples analysed and assuming a similar sensitivity for the detection of the two lipopeptides in the TLC assay, the production rate of surfactin ADL2 in the strain R13CDM was estimated to account for only
5% of the production rate of surfactin A in the wild-type strain B.subtilis ATCC 21332. This would correspond to
25 mg of surfactin ADL2 per litre of Landy medium.
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The lipopeptide surfactin A causes hemolysis and has an inhibiting activity against a broad range of microorganisms. Commercial blood agar plates were inoculated with the strains B.subtilis ATCC 21332 and R13CDM and incubated for 24 h at 37°C. The strain B.subtilis ATCC 21332 produced a clear hemolytic zone surrounding the bacterial colonies (Figure 7A). In contrast, no hemolysis was visible with the mutant R13CDM. Similar results were obtained by using methanol extracts of culture supernatants from the two strains grown in Landy medium.
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Discussion |
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The T-domains have a symmetric structure and consist of 75 amino acid residues with a structural core spanning 37 amino acid residues in both directions from an invariant serine in the conserved sequence motif D/(D/N)FFxLGGHS(L/I), which serves as a 4'-phosphopantetheine binding site (Weber et al., 2000
). Our results localize the proposed interface between A- and T-domains within amino acid residues located N-terminal to the invariant serine residue. Interactions between the A- and T-domains obviously remained intact after recombination within this motif and resulted in a fully active thioester forming hybrid T-domain.
The lack of any detectable products synthesized from SrfTD-M1/23 in vitro could point to an essential interaction between T- and C-domains, which then also might involve the N-terminal half site of the T-domain as the hybrid T-domain preserving the C-terminal part was obviously not sufficient to communicate with its cognate C-domain. This result agrees with the observation that the homologous ACPKS couple in PKSs needs to be preserved during engineering (Gokhale et al., 1999; Ranaganathan et al., 1999). However, the TC linker connecting two distinct elongation modules has been successfully used as a fusion site to recombine modules from the tyrocidine synthetase and the expected peptide formation in vitro could be shown (Mootz et al., 2000
). Taken together with the above-mentioned somewhat contradictory results using the AT linker as a fusion site, these data suggest that interdomain or intermodular communications might be different within the surfactin synthetase and the tyrocidine synthetase.
A peptide production from strain R13TD could not be expected as no production of surfactin synthetases was observed. This could indicate some changes in general regulation networks. Effects of mutations in the surfactin synthetase operon on efficient sporulation and competence development have been reported and regulatory genes such as comS have been found to be inserted in that region (Nakano et al., 1991; Hamoen et al., 1995
). The deletions in the SrfAA coding region introduced upon construction of the mutant R13TD might have affected the expression of genes essential for the regulation of sporulation and surfactin biosynthesis.
The C-domains are characterized by the active site motif HHIIxDGVS involved in the catalysis of non-ribosomal peptide bond formation. A catalytic mechanism has been proposed where the second histidine residue of that motif is essential for peptide bond formation (de Crécy-Lagard et al., 1995). C-domains have been differentiated into an unspecific N-terminal donor and a specific C-terminal acceptor pocket (Belshaw et al., 1999
; Ehmann et al., 2000
; Linne and Marahiel, 2000
). The fatty acidL-glutamic acid intermediate in the hybrid SrfCDM-M12/3 instead of a fatty acidL-glutamic acidL-leucine intermediate in the wild-type enzyme SrfAA should therefore be accepted as a substrate by the donor pocket of the C-domain. In fact, out of the analysed fusion sites only a hybrid bimodular enzyme fused at this motif produced in vitro a similar product pattern if compared with the wild-type enzyme SrfAA and the expected peptide was synthesized in vivo. Furthermore, the HHIIxDGVS motif has been successfully used as a fusion site for the module exchange between the highly homologous NRPSs surfactin A synthetase from B.subtilis and lichenysin A synthetase from B. licheniformis (Yakimov et al., 2000
). In this work, the entire amino acid incorporating modules 1 and 5 of the surfactin synthetase have been replaced by the corresponding modules of lichenysin synthetase, showing that this fusion site seems to be generally suitable also for module swaps within the surfactin synthetase.
The identified lipohexapeptide SrfADL2 from strain R13CDM is one of the first rational designed peptide constructed by the directed deletion of a complete internal module of a peptide synthetase. We could further show that the variety of the incorporated ß-hydroxy fatty acids into surfactin SrfADL2 is identical with the pattern of wild-type surfactin A (Peypoux et al., 1991; Leenders et al., 1999
), indicating that the activity of the associated ß-hydroxy fatty acid transferring acyltransferase is not influenced by the rearrangement of the surfactin synthetase. However, the productivity of surfactin SrfADL2 was only estimated at
5% to that of wild-type surfactin A, giving a yield of
2550 mg of SrfADL2 per litre of medium compared with 0.51 g/l for SrfA (Peypoux and Michel, 1992
; de Ferra et al., 1997
). We could show that an altered expression rate of the recombinant surfactin synthetase genes is obviously not responsible for this effect as the enzymes were clearly detectable by SDSPAGE. The reduced chain length of peptide SrfADL2 could severely affect its three-dimensional structure. The wild-type surfactin A has a horse saddle-like conformation with the polar side chains of the L-glutamic acid and L-aspartic acid residues opposing the non-polar ß-hydroxy fatty acid (Bonmatin et al., 1992
, 1994
). The deletion of the second L-leucine residue in surfactin SrfADL2 will most probably alter that conformation with possible effects on the stability of SrfADL2 in the bacterial cell. Rapid in vivo degradation and less efficient export mechanisms might further contribute to the observed relatively low production of SrfADL2. While a suboptimal conformation of the constructed hybrid C-domain might also reduce the specific activity of the recombinant surfactin synthetase, the observed high yields of modified lipoheptapetides after module exchange using the HHIIxDGVS motif (Yakimov et al., 2000
) gave further evidence of some problems in stability or transport of the synthesized lipohexapeptide.
First preliminary bioassays revealed that the constructed surfactin derivative SrfADL2 has clearly altered biological activities compared to the wild-type surfactin SrfA. Most interesting was the lack of any detectable hemolytic activity concomitant with an increase in growth inhibition of bacterial cells. Considering the reduced SrfADL2 production in strain R13CDM compared to the SrfA production in strain ATCC 21332, this difference is even more striking. This result gives first evidence that SrfADL2 or similar surfactin derivatives might exhibit a reduced toxicity against eukaryotic cells, which could improve their therapeutic applications.
The results obtained with the engineering of NRPSs indicate that a general optimal fusion site suitable for rearrangements might not exist. Linker regions which have been successfully used as fusion sites for the recombination of the tyrocidine or bacitracin synthetases seem not to be suitable for the recombination of surfactin synthetase. In contrast, the potential of the HHIIxDGVS motif for combinatorial biosynthesis of further surfactin derivatives has already been demonstrated (Yakimov et al., 2000). It will now be interesting to prove the versatility of this motif for the recombination of other NRPSs.
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Notes |
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4 To whom correspondence should be addressed. E-mail: fbern{at}bpc.uni-frankfurt.de
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Acknowledgments |
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References |
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Received February 20, 2002; revised August 2, 2002; accepted August 26, 2002.