Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Biologie, Stefan-Meier Str. 19, 79104 Freiburg, Germany1
Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK2
Author for correspondence: A. Bechthold. Tel: +49 761 2038371. Fax: +49 761 8383. e-mail: andreas.bechthold{at}uni-freiburg.de
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ABSTRACT |
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Keywords: avilamycin A, erythromycin, deoxysugar biosynthetic genes, methyltransferase
Abbreviations: ES-MS, electrospray mass spectrometry
The GenBank accession number for the sequence reported in this paper is AF333038.
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INTRODUCTION |
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In contrast to avilamycin A, in which a long sugar side chain is connected to a small polyketide moiety, erythromycin is a macrolide polyketide, to which only two separate sugar residues (L-mycarose and D-desosamine) are transferred (Fig. 1). The biosynthesis of erythromycin is well understood and many mutants containing deletions in single biosynthetic genes are available (Paulus et al., 1990
; Haydock et al., 1991
; Weber et al., 1991
; Stassi et al., 1993
; Lambalot et al., 1995
; Summers et al., 1997
; Salah-Bey et al., 1998
; Gaisser et al., 1998
). eryB genes have been shown to be involved in dTDP-mycarose biosynthesis. One of these genes, eryBIII, encodes a C-methyltransferase involved in methylation at position C3 during dTDP-L-mycarose biosynthesis (Gaisser et al., 1998
).
For better understanding of the biosynthesis of avilamycin A we have initiated the characterization of another gene (aviG1) of the avilamycin biosynthetic gene cluster located upstream of the avilamycin polyketide synthase biosynthetic gene. The deduced amino acid sequence of aviG1 was very similar to EryBIII. The involvement of AviG1 in the biosynthesis of avilamycin A was demonstrated by targeted gene disruption and complementation of an eryBIII mutant of Saccharopolyspora erythraea.
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METHODS |
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General genetic manipulation.
Isolation of E. coli plasmid DNA, digestion of DNA with restriction endonucleases and Southern hybridization were carried out according to the directions of the suppliers of kits, enzymes and reagents (Amersham Pharmacia). Southern hybridization was performed with Hybond-N nylon membranes (Amersham Pharmacia). Probes were labelled with digoxigenin (DIG) by using a DIG labelling and detection kit (Roche). Restriction mapping and other routine molecular biology methods were performed as described by Sambrook et al. (1989) . Protoplast formation, transformation and regeneration of protoplasts from Str. viridochromogenes Tü57 were carried out by standard procedures (Hopwood et al., 1985
). PCR was carried out using a Perkin Elmer GeneAmp 2400 thermal cycler. The conditions were as described by Bechthold & Floss (1994)
.
DNA sequencing and computer-assisted sequence analysis.
DNA was sequenced by the dideoxynucleotide chain-termination method using thermosequenase (Amersham Pharmacia). Universal and reverse primers (Amersham Pharmacia) were used. Sequencing reactions were performed on an automated sequencer (Vistra 725) from Molecular Dynamics and on an ABI sequencer from 4-Base Lab. DNA sequences were analysed using the DNASIS software package (version 2, 1995; Hitachi Software Engineering). BLASTX analysis (Altschul et al., 1997 ) was used to search the GenBank CDC translations + PDB + SWISS-PROT + Spupdate + PIR, release 2.0 for matching sequences.
Generation of a chromosomal aviG1 mutant of Str. viridochromogenes Tü57.
For generation of a chromosomal aviG1 mutant of Str. viridochromogenes Tü57 by homologous recombination, plasmid pMIK1 was constructed. A 7·7 kb BamHI fragment containing the entire methyltransferase gene was cloned into pBluescript SK- to create plasmid B7. B7 was digested with BglII and SpeI (the SpeI site is located within the polylinker of pBluescript SK-), treated with Klenow fragment and religated to generate pMIK1a. A 2·5 kb SacI fragment of pMIK1a, containing aviG1, was ligated into the corresponding sites of pSP1, generating pSP-MIK (Fig. 2). pSP-MIK was digested with NcoI to delete a 285 bp fragment within aviG1, resulting in plasmid pMIK1. pMIK1 was used to transform protoplasts of Str. viridochromogenes Tü57. Selection of primary transformants was performed on erythromycin-containing plates. For characterization of transformants by Southern hybridization, an internal 1·7 kb SmaI fragment of the ermE gene of pSP1 and the internal 2·2 kb SacI fragment of pMIK1 were used as probes. After screening for erythromycin-sensitivity, a double cross-over mutant, named Str. viridochromogenes GW1, was obtained. Chromosomal DNA from this mutant was analysed by Southern hybridization using the 2·2 kb SacI fragment as probe. The result of Southern hybridization was confirmed by PCR using oligonucleotide primers HaloF (5'-GCCGAGCAAAGCTTGGAGAATCAT-3') and HaloR (5'-TGGTGGCATGCGATGTCACCTCC-3').
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Complementation of the erythromycin eryBIII mutant 335.
For complementation of the eryBIII mutant 355, a HindIII restriction site 5' to the ribosome-binding site and an SphI restriction site 3' to the termination codon were introduced into aviG1 by using PCR. The fragment was cloned into the HindIII and SphI sites of pUC18 to generate pUCB, which was digested with HindIII, followed by a fill-in reaction (Sambrook et al., 1989 ). After BamHI digestion, the fragment harbouring aviG1 was ligated into pSG142, previously digested with NdeI, followed by treatment with Klenow polymerase and BglII digestion. The cloning region around the former NdeI site in the resulting plasmid, pSGaviG1, was sequenced, revealing the deletion of 9 nt at the NdeI/HindIII fusion site. Plasmid pSGaviG1 was used to transform the Sac. erythraea eryBIII mutant 335 as described by Gaisser et al. (1998)
. The presence of the eryBIII mutation was confirmed using chromosomal DNA of a Sac. erythraea eryBIII mutant and the transformed strain containing aviG1 as template. PCR reactions were carried out as described previously (Gaisser et al., 1998
) and the sequence of the PCR fragments was confirmed. The PCR fragment obtained with chromosomal DNA of Sac. erythraea eryBIII mutant as template was also used as a probe in Southern hybridization experiments with BamHI-digested genomic DNA of Sac. erythraea NRRL2338, eryBIII mutant 355 and eryBIII mutant 355 harbouring aviG1. No difference in the pattern of the labelled DNA bands was detected. These results indicated that the eryBIII gene was not involved in a recombination event.
Analysis of avilamycin and erythromycin derivatives.
For avilamycin production, Str. viridochromogenes Tü57 and mutant Str. viridochromogenes GW1 were grown as described above. Cultures were extracted with an equal volume of ethyl acetate. After evaporation of the solvent the dried extracts were redissolved in methanol. TLC analysis was carried out on silica gel plates (silica gel 60 F254, Merck) with CH2Cl2/CH3OH (9:1, v/v) as solvent. Avilamycin derivatives could be detected after treatment with anisaldehyde/H2SO4 (Braun, 1995 ).
HPLC analysis was performed on a Waters Alliance HT Liquid Chromatograph with a diode-array detector and a ProC18, 50x4 mm column. The detection wavelength was 220 nm. The solvent system was as follows: Solvent A, acetonitrile/[H2O/H3PO4 (99·9:0·1)], 5:95 (v/v); solvent B: acetonitrile/[H2O/H3PO4 (99·9:0·1)], 42:58 (v/v); non-linear gradient, 0100% solvent B over 16 min at a flow rate of 1·2 ml min-1. Avilamycin A was identified by comparison with an authentic sample. For erythromycin A production, culture supernatants were treated as described previously and the extracts were analysed using electrospray MS (ES-MS; Gaisser et al., 1998 ).
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RESULTS AND DISCUSSION |
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Complementation of the erythromycin eryBIII mutant 335
To demonstrate the complementation of the Sac. erythraea eryBIII mutant 335, plasmid pSGaviG1 was isolated which places the expression of aviG1 under the control of the activator ActII-ORF4 (Rowe et al., 1998 ). Extracts of culture supernatants of the wild-type strain, eryBIII mutant 355 and eryBIII mutant 355 containing aviG1 were analysed by ES-MS. Extracts of the wild-type strain gave the expected signal for erythromycin A at m/z 734·7. Mutant 355 gave a signal at m/z 720·7, which indicates the formation of 3'-C-desmethyl erythromycin A (Gaisser et al., 1998
). The MS data obtained with the eryBIII mutant 355 containing aviG1 showed peaks at m/z 720·7 and 734·7, indicating that erythromycin A as well as 3'-C-desmethyl erythromycin A are formed (Fig. 5
). To confirm that methylation occurred at 3'-C of L-mycarose, MS/MS analyses of the peaks at m/z 734·7 and m/z 720·7 were performed. In both cases, a daughter ion at m/z 576·5 was detected unambiguously, indicating the loss of the 3'-C sugar (data not shown). From these data we can conclude that the expression of aviG1 did allow complementation of the eryBIII mutation. AviG1 seems to be able to participate in L-mycarose biosynthesis, providing further support for the proposal that AviG1 encodes a C- methyltransferase capable of acting on position C3 of an activated sugar. Since L-mycarose and 2-deoxy-D-evalose differ mainly in their stereochemistry at position C5, AviG1 is therefore most likely involved in the synthesis of 2-deoxy-D-evalose. However, results using bioassays revealed that the expression of aviG1 restored erythromycin production just to a very small extent (data not shown). No reproducible difference could be detected between the size of the haloes of Sac. erythraea 335 and Sac. erythraea 335 expressing aviG1 (data not shown).
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Received 18 May 2001;
revised 13 September 2001;
accepted 15 October 2001.