Novartis Pharma AG, Research, Core Technology Area, CH-4002 Basel , Switzerland1
Author for correspondence: Thomas Schupp. Tel: +41 61 32 47903. Fax: +41 61 32 43279. e-mail: thomas.schupp{at}pharma.novartis.com
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ABSTRACT |
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Keywords: antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering
Abbreviations: PKS, polyketide synthase; AHBA, 3-amino-5-hydroxybenzoic acid
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INTRODUCTION |
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The identification and sequencing of the rifamycin polyketide synthase (PKS) gene cluster by our group (Schupp et al., 1998 ) and by August et al. (1998)
disclosed five genes, rifArifE, responsible for the synthesis of the rifamycin polyketide chain. The encoded multifunctional enzymes contain a total of ten PKS modules, which use 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit to catalyse successive rounds of polyketide chain elongation to build the polyketide backbone of the rifamycin molecule.
Our interest in further studying rifamycin B biosynthesis led us to undertake the inactivation of the rifF gene, situated directly downstram of the PKS genes, and to analyse the effect of this mutation on rifamycin biosynthesis. The rifF gene product has been characterized as rifamycin amide synthase by sequence homologies to different arylamine N-acetyltransferases (August et al. , 1998 ) and the putative function of the RifF protein in rifamycin biosynthesis was suggested to be a cyclase, catalysing the formation of an intramolecular amide bond between the carboxyl end of the synthesized linear polyketide chain and the amino group of the AHBA starter unit (Chen et al., 1999
).
In this paper we describe the inactivation of the rifF gene by introducing a 9 bp deletion into the chromosomal rifF gene of the rifamycin B high-producing strain Amycolatopsis mediterranei N/813 (Ghisalba et al., 1984 ). We were able to isolate and characterize a series of linear intermediates of rifamycin B biosynthesis. Our results confirm the putative function of RifF postulated from sequence homology, and give new insights into the succession of biosynthetic steps during the rifamycin B biosynthesis.
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METHODS |
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Escherichia coli DH5 (Hanahan, 1983
) was used for propagation of plasmids and cloning of DNA. E. coli ET12567 (dam- dcm- hsdM: MacNeil et al., 1992
) was used for preparation of plasmid DNA for transfer into A. mediterranei by electroporation. E. coli was grown in LB medium (Miller, 1972
). For plasmid maintenance, tetracycline (25 µg ml-1), chloramphenicol (50 µg ml-1) or ampicillin (50 µg ml-1) were added where appropriate. Cultivation temperatures were 37 °C for E. coli and 28 °C for A. mediterranei.
Plasmids and construction of new cloning vectors.
The E. coli cloning vector pUCBM21 (Boehringer Mannheim) was used for the construction of suicide plasmids for gene disruption and gene replacement in A. mediterranei. To remove the single PstI site in the multiple-cloning site of pUCBM21, the plasmid was cleaved with PstI, treated with Klenow enzyme (Boehringer Mannheim) and religated, giving plasmid pBM21*. The aacC4 cassette (Blondelet-Rouault et al., 1997 ), which confers apramycin resistance in E. coli and A. mediterranei , was introduced into the HindIII site of pBM21* as a 1·7 kb HindIII DNA fragment resulting in plasmid pASN3.
Electroporation of A. mediterranei N/813.
A. mediterranei N/813 cells were harvested after 2 d growth in 40 ml liquid SOC medium and washed three times in 10% glycerol at room temperature. The cells were resuspended in 4 ml 10% glycerol containing 40 µg lysozyme ml-1 and incubated for 20 min with gentle shaking (50 r.p.m.). After one washing step with 4 ml 10% glycerol at 4 °C the cells were resuspended in 1·6 ml ice-cold 10% glycerol and used immediately for electroporation. Plasmid DNA (0·51·0 µg) was heat denatured (5 min at 95 °C) before it was mixed with 200 µl cell suspension on ice. The mixture was immediately transferred into a 0·2 cm electrocuvette and a single electric pulse (Gene Pulser II; Pulse Controller Plus; Bio-Rad) was applied (1·52·0 kV; 1000 ; 25 µF). The electroporated mycelium was immediately diluted with 0·8 ml YMG medium, transferred to 10 ml YMG medium and incubated for 1416 h at 28 °C with vigorous shaking (250 r.p.m.). The whole culture was then plated on a YMG agar plate containing 50 µg apramycin ml-1 .
Genetic procedures.
Chromosomal DNA isolation was carried out according to Pospiech & Neumann (1995) . DNA fragments were recovered from agarose gels using the Qiaex Kit (Qiagen) and DNA labelling was performed with [32P]dCTP using a Nick Translation Kit (Boehringer Mannheim). Southern hybridization experiments were performed using Hybond-N+ nylon membranes (Amersham) following the manufacturers alkali blotting protocol. Genetic procedures involving E. coli, and in vitro DNA manipulations were performed according to standard protocols (Sambrook et al. , 1989
).
DNA sequencing.
DNA sequencing was by the dideoxynucleotide chain-termination method with an automated laser fluorescence sequencer (Applied Biosystems, ABI PRISM377). Sequencing reactions were done with the standard reverse primer using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Perkin Elmer) according to the suppliers instructions.
HPLC analysis.
For HPLC analysis of metabolites, A. mediterranei culture broths were mixed 1:1 with butanol and shaken for 4 h at room temperature (150 r.p.m.). After centrifugation (10 min, 5000 g) the butanol phase was separated and 1020 µl was analysed by HPLC (Merck model L-6200) using a 4·6x100 mm Symmetry C18, 3·5 mm column (Waters) with gradient elution (5 min linear gradient of 050% acetonitrile, hold for 2 min, 5 min linear gradient of 50100% acetonitrile, hold for 3 min, 2 min linear gradient of 1000% acetonitrile) using a phosphate (0·55% orthophosphoric acid, pH adjusted to 4·5 with triethylamine)/acetonitrile buffer system. The region from 200 to 500 nm was scanned with a DAD-System L-4500 (Merck) and the spectra were compared with a rifamycin B standard.
Isolation of AHBA and rifamycin-biosynthesis intermediates.
The culture broth of a 1 l fermentation of mutant 3/1 in FBR42 medium was extracted twice with ethyl acetate (1 l each) and the crude extract was defatted with hexane (3x500 ml). The complex mixture (5·4 g) was submitted to gel filtration on Sephadex LH20 with methanol. Fractions 3 and 4 (1·87 g), containing UV-active compounds, were further separated by reversed- phase chromatography on LiChrospher RP18 12 µm (200 g) using a gradient composed of watertriethylammoniumformiate buffer (A) and acetonitrile (B) from 060% B. Final purification was performed by preparative TLC using the systems CH2Cl 2/MeOH/H2O 80:17·5:2 and 88:11:1 by vol.
The yields were 2·3 g AHBA, 90 mg compound 1, 18 mg 2, 7 mg 4 (lactone), 39 mg 5, 21 mg 6 (lactone), 11 mg 7 and 9 mg 8. From a cultivation on a 5 l scale which was worked up analogously, 44 mg compound 3 was isolated.
Spectroscopic measurements.
The UV-absorption spectra were recorded in methanol with a Perkin Elmer Lambda 9 spectrophotometer. The IR spectra were taken in KBr pellets on a FT-IR spectrometer BRUKER IFS 66. 1H/13 C NMR spectra were recorded in d6-DMSO on a Bruker Avance DMX-500 spectrometer at 500 MHz with tetramethylsilane as the internal standard. Electrospray mass spectra were measured with a Finnigan SSQ-7000 MS spectrometer.
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RESULTS |
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To enable the second crossover, the selected A. mediterranei transformant was cultivated four times in liquid NL148 medium without apramycin (5% inoculation and 3 d cultivation each time). Apramycin-sensitive mutants were then detected by replica plating single colonies, derived from the last NL148 culture, on YMG agar with and without apramycin. From about 1000 colonies tested, two apramycin- sensitive mutants were detected and chosen for further analysis.
Characterization of two double-crossover mutants
Southern-blot analysis of PstI-digested chromosomal DNA using the 5·1 kb KpnIEcoRI DNA fragment containing rifF as the probe clearly demonstrated the elimination of the single PstI site in the rifF gene in the chromosome of mutant 3/1. The hybridization pattern of this mutant showed only one band, 6·2 kb in size. In contrast to this, the parental strain N/813 and the other mutant, 4/1, gave two hybridizing PstI fragments, 3·7 kb and 2·5 kb in size (Fig. 2 ). These hybridization data are in agreement with the restriction pattern expected for replacement of the DNA fragment containing the non-mutated rifF gene with the one lacking the internal PstI site in mutant 3/1 and the reconstitution of the parental rifF gene in mutant 4/1. This conclusion was confirmed by HPLC analysis of rifamycin B production by the two mutants. Mutant 3/1 does not produce rifamycin B in the production medium FBR42 whereas mutant 4/1 produces about the same amount as the parental strain N/813.
The deletion in the rifF gene of mutant 3/1 was analysed by DNA sequencing. To obtain this information, the internal 1·1 kb BglIISmaI DNA fragment out of the 5·1 kb KpnIEcoRI fragment of pASN4.1 was cloned into the BamHISmaI sites of pUCBM21 in E. coli. The resulting plasmid was named pASN4.2 and the sequence of the cloned DNA fragment was determined. As shown in Fig. 3, mutant 3/1 has an unexpected 9 bp deletion in the rifF gene, derived from 3' exonuclease activity of the Klenow enzyme. This mutation results in a loss of three amino acids (Q, K and S) and a change from L to R at position 29 of the RifF protein (Fig. 3
).
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The culture broth of the mutant 3/1 was extracted with ethyl acetate and by chromatographical separations the new rifamycin-biosynthesis intermediates 2 to 8 released from modules 4 to 10 (Fig. 4 ) as well as the starter unit AHBA and the known early intermediate P8/1-OG (Ghisalba et al., 1981
) (1) were obtained in pure form. P8/1-OG (intermediate 1) is produced as the main intermediate and was identified by comparison of its spectroscopic properties with published data. The presence of the free acid form 1 was determined by IR spectroscopy (band at 2650 cm -1). The physico-chemical and chromatographic parameters of the new intermediates are shown in Table 1
.
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Intermediate 8 released from module 10. In NMR ROESY (rotated frame nuclear Overhauser enhancement spectroscopy) experiments, a strong nuclear Overhauser enhancement (NOE) is measured between the protons of the methyl group at C-16 and the olefinic proton at C-14, thus indicating the geometry of the terminal carbon double bond as trans .
Determination of the relative stereochemistry
As intermediates 4 and 6 released from modules 6 and 8 contain a 6- membered lactone ring, the relative stereochemistry for the two carbon sequences C-5, C-6, C-7, C-8 and C-9, C-10, C-11, C-12, respectively, could be deduced from measurements of coupling constants in proton NMR spectra and from NOE experiments (Fig. 7 ). The overlapping carbon sequence C-7, C-8, C-9, C-10 is covered by the lactone form of the intermediate released from module 7, which was found in addition to the corresponding linear intermediate 5. The results, therefore, revealed the complete relative configurations for the methine carbons of the side chain (except for C-4 which is achiral in the final rifamycins); the determined relative stereochemistry corresponds to the known absolute chirality of the ansa ring in the rifamycins (Brufani et al., 1964
).
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DISCUSSION |
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The RifF protein shows a high degree of similarity to different arylamine N-acetyltransferases, e.g. of Mycobacterium smegmatis (36·5% identity over 178 aa), of E. coli (28% identity over 230 aa). These sequence similarities together with our results demonstrate the function of the rifF gene product as a cyclase (rifamycin amide synthase) performing the intramolecular ring closure by amide bond formation between the carboxyl group at one end and the amino group at the opposite end of the mature linear polyketide molecule synthesized by the rifamycin PKS. The role of the RifF protein in rifamycin B biosynthesis is therefore the cyclization and simultaneous release of the polyketide chain from the acyl carrier protein of module 10 of the rifamycin PKS.
Mutant strain 3/1 produced a series of linear intermediates of the rifamycin B biosynthesis and not, as expected, only the full-length linear intermediate produced by module 10. To explain this result, the activity of a second enzyme is needed that is able to catalyse the release of the linear intermediates from the acyl carrier proteins of modules 3 to 10. The thioesterase encoded by orf12 (August et al., 1998 ), almost 30 kb downstream of rifF , may be the protein having this function. In addition, the obstruction of the normal release of the full-length acyl chain from the rifamycin PKS probably leads to an accumulation of linear intermediates docked at the acyl carrier proteins of the different modules, which are then released from the PKS by the thioesterase, thereby preventing blockage of the enzyme complex. Such an editing activity was recently discussed for the thioesterase TylO in the tylosin biosynthesis (Butler et al., 1999
).
Noncyclized polyketide intermediates of 16-membered macrolides were also isolated from the culture broth of the mycinamicin producer Micromonospora griseorubida sp. nov. (tetraketide and pentaketide) and the corresponding triketide in a nonproducing mutant (Kinoshita et al., 1988 ). Tetra- and pentaketides were observed in nonproducing mutants of the tylosin producer Streptomyces fradiae (Huber et al., 1990
). These observations, together with our results, suggest that the formation of noncyclized polyketide intermediates is perhaps a general phenomenon during either the normal or impaired catalysis by type I PKS.
The introduction of the 9 bp deletion inside the rifF gene results in the loss of three amino acids (Q, K and S) and the exchange of L to R at position 29 (Fig. 8 ). These alterations in the RifF protein caused the inactivation of the enzyme because the cyclization reaction at the end of the biosynthesis of the polyketide backbone is no longer performed. The exact function of the altered or lost amino acids inside the RifF protein is not clear at the moment, but one possible candidate for an essential function for the cyclization reaction is the conserved L at position 29 (Fig. 8
).
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Different rifamycin molecules have been postulated as the earliest macrocyclic intermediates of rifamycin biosynthesis: proansamycin A or B deduced from the early intermediate protorifamycin I and structure comparison with streptovaricin precursors (Ghisalba et al., 1978 , 1979
) and proansamycin X, based on hypothetical intermediates of the rifamycin PKS gene cluster (August et al., 1998
; Tang et al., 1998
). The structure of the observed module 10 intermediate 8 and the deduced function of RifF, performing the ring closure of the linear endproduct 8 of the PKS, now provides strong evidence that proansamycin B is the direct macrocyclic product of the rifamycin PKS (Fig. 9
). The presence of the trans-cis sequence for the conjugated olefinic system in rifamycin B (and its precursor proansamycin B) can be explained by migration of the (13,14)trans-(15,16)trans double bonds via enolysation and subsequent isomerization of the geometry. From the hypothetical proansamycin B, a simple one-step transformation, oxidation at C-4', would lead to protorifamycin I, the earliest macrocyclic precursor of the rifamycins isolated so far (Ghisalba et al., 1978
).
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ACKNOWLEDGEMENTS |
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Received 9 August 1999;
accepted 3 September 1999.