School of Pharmacy1 and Department of Bacteriology2, University of Wisconsin, 425 N. Charter St, Madison, WI 53706, USA
Bioprocess Research, Central Research Division, Pfizer Inc., Groton, CT 06340, USA3
Department of Medicinal Chemistry, School of Pharmacy and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA4
Author for correspondence: C. Richard Hutchinson. Tel: +1 608 262 7582. Fax: +1 608 262 3134. e-mail: crhutchi{at}facstaff.wisc.edu
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ABSTRACT |
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Keywords: avermectin, branched-chain amino acids, isotope labelling
Abbreviations: AcdH, the specific acyl-CoA dehydrogenases produced by S. avermitilis and S. coelicolor; AD, any other acyl-CoA dehydrogenase; PMS, phenazine methosulfate
The GenBank accession numbers for the sequences described in this paper are AF142581 (Streptomyces coelicolor) and AF143210 (Streptomyces avermitilis).
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INTRODUCTION |
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METHODS |
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Escherichia coli DH5 (Sambrook et al., 1989
) was obtained from D. MacNeil (Merck & Co. Research Laboratories), S. coelicolor J802 (dagA1 agaA7) (Hodgson & Chater, 1981
) from David Hodgson (University of Warwick, UK) and Streptomyces lividans TK64 (Hopwood et al., 1985
) from David Hopwood (John Innes Centre, Norwich, UK). Plasmid pT7-7 (Tabor, 1990
) was obtained from Stan Tabor (Harvard University, USA). E. coli DH5
and BL21(DE3) (Novagen) were grown at 37 °C on LuriaBertani (LB) medium (Sambrook et al., 1989
). S. coelicolor J802 was grown on R2YE agar (Hopwood et al., 1985
) at 30 °C for general use. S. avermitilis ATCC 31272 (Burg et al., 1979
) was grown as described previously (Denoya et al., 1994
, 1996
). Solid minimal medium was as described by Hopwood et al. (1985
), except that SeaKem ME agarose (FMC BioProducts) replaced agar, and the glucose in the medium was replaced with one or more carbon sources at a final concentration of 2·5 g l-1 each.
Oligodeoxynucleotides for PCR and sequencing primers were synthesized with a Perkin-Elmer ABI model 3948 DNA synthesizer/purifier, or purchased from Genosys. EasyStart 100 (PCR Mix-in-a-Tube, Molecular Bio-Products) was used for PCR reactions. Taq polymerase was purchased from Sigma. PCR reactions were carried out in a Perkin-Elmer Cetus model 480 DNA thermal cycler. DNA sequencing was carried out in a Perkin-Elmer ABI Prism 377 DNA Sequencer. All primer syntheses and DNA sequencing related to cloning of the S. coelicolor acdH were done at the Biotechnology Center, University of Wisconsin-Madison; S. avermitilis DNA was sequenced by Lark Technologies.
Preparation of methacrylyl-CoA.
Methacrylyl-CoA was synthesized using acetyl-CoA synthetase as described by Patel & Walt (1987 ). The reaction mixture was modified as follows: CoA (6·5 mmol), ATP (25 mmol), MgCl2 (100 mmol), acetyl-CoA synthetase (3 units) and methacrylic acid (pH 7·2, 500 mmol) were mixed and the reaction was incubated at 37 °C for 2 h.
Methacrylyl-CoA was purified by HPLC on a Nova-Pak C18 column using 20% methanol in 0·05 M phosphate buffer, pH 5·7, at a flow rate of 2·0 ml min-1 with UV detection at 254 nm; the peak with a retention time of 6·7 min was collected. A Sephadex G-10 column with deionized H2O as the mobile phase was used for desalting; the peak absorbing at 254 nm was collected and vacuum-dried. Methacrylyl-CoA was characterized by proton NMR in 2H2O, using the NMR spectrum of isobutyryl-CoA for comparison. NMR spectra of methacrylic acid and the isobutyric acid reference standard were also measured in 2H2O. The NMR data showed two vinylic protons ( 5·7 p.p.m. and
6·05 p.p.m.) and one
C(CH3)=CH2 group (
1·9 p.p.m.) in the product; in contrast, the spectrum of isobutyryl-CoA had one methine proton (
2·85 p.p.m.) and a
CH(CH3)2 group (
1·15 p.p.m.).
DNA preparation and amplification.
Small-scale preparations of E. coli plasmid DNA were made as described by Denoya et al. (1985 ) and Morelle (1988
). Individual DNA fragments and PCR products were separated on agarose gels and purified with a Qiagen QIAEX II gel extraction kit according to the manufacturers instructions. Streptomycete genomic DNA was isolated by the SDS method of Hopwood et al. (1985
). Oligodeoxynucleotides for PCR primers were purified according to the manufacturers protocols.
Several AD sequences available from the databases were aligned to identify conserved regions for the design of PCR primers targeting ADs related to branched-chain amino acid catabolism. The leftwards primers described below (P2 and P134) were based on a C-terminal region conserved in a number of short-chain/branched-chain ADs (Willard et al., 1996 ), starting with a Glu residue that has been postulated to serve as the catalytic residue (Rozen et al., 1994
). Thus, the three nucleotides located at the 3' end of these primers, which have been described as the most important nucleotides for a successful PCR priming in homology cloning (Sommer & Tautz, 1989
), represent the codon corresponding to Glu381 of the mature human short/branched-chain AD. The sequences of the two primers used to clone the acdH of S. coelicolor were: P1, 5'-AAGAATTCAACGGCACCAAGGCCTGGATCACCAAC-3'; and P2, 5'-AATCTAGAGCGCTGGATCTCGGAGGTGCCCTC-3'. The primers used to clone the acdH from S. avermitilis were: P131, 5'-AAGAATTCATATGAACGGCACCAAGGCCTGGATCACCAAC-3'; and P134, 5'-AAGGATCCTCTAGAGCGCTGGATCTCGGAGGTGCCCTC-3'.
PCR reactions were carried out with S. coelicolor DNA according to the EasyStart 100 protocol. The reaction was boiled for 5 min, cooled to 70 °C and 5 U Taq polymerase was added. Amplification was achieved with 25 cycles of denaturation at 97 °C for 50 s followed by annealing at 6070 °C for 30 s followed by extension at 70 °C for 1/22 min. S. avermitilis genomic DNA was amplified essentially as described previously (Skinner & Denoya, 1993 ). The resulting PCR products were purified as described above and analysed by restriction enzyme digestion and/or DNA sequencing.
DNA hybridization, cloning and sequencing.
Southern blot hybridization and colony hybridization were performed with Hybond-N membranes (Amersham) by standard techniques (Sambrook et al., 1989 ). Digoxigenin-AP labelling, hybridization and detection were done with the Genius kit (Boehringer Mannheim), according to the manufacturers protocols. Hybridization was performed at 42 °C overnight, and the blot was washed twice with 1xSSC (0·15 M NaCl and 0·015 M sodium citrate)/0·1% SDS (Sambrook et al., 1989
) for 5 min at room temperature and twice in 0·1xSSC/0·1% SDS for 15 min at room temperature.
Minilibraries of S. coelicolor J802 genomic DNA were constructed for cloning acdH. Genomic DNA was digested with BamHI and size-fractionated (612 kb) by electrophoresis in a 1% agarose gel. Fragments excised from the gel were cloned in pUC18 (Yanisch-Perron et al., 1985 ). The genomic DNA minilibrary was screened by colony hybridization using the PCR product (Fig. 2a
) of acdH as a probe to obtain clone pWHM1301, which was shown to contain an approximately 10 kb BamHI DNA fragment. A 3·5 kb PstI fragment that contained the entire acdH gene was subcloned from pWHM1301 into the PstI site of pGEM-3Zf(-) (Promega) to give pWHM1302 (Fig. 2a
). A cosmid library of S. avermitilis chromosomal DNA (Skinner et al., 1995
) was screened by colony hybridization as described previously (Denoya et al., 1994
) with the S. avermitilis acdH-specific 32P-labelled PCR product. Two positively hybridizing cosmid clones (of >2000 screened) contained overlapping sequences by restriction mapping and Southern blot hybridizations. Genomic DNA restriction fragments carrying acdH were separated and identified by PCR using a sterile toothpick to lift DNA by poking into each gel band as described previously (Kadokami & Lewis, 1994
). The following genomic fragments were subcloned from the cosmid clones into pGEM-3Zf(-): 2·2 kb BamHI (pCD1106), 7·5 kb BamHI (pCD1119) and 4·5 kb SphI (pCD1468) (Fig. 2b
).
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Expression of S. coelicolor and S. avermitilis acdH in E. coli.
An NdeI site was introduced into the S. coelicolor acdH translational start codon. The primers used for PCR were: OP1, 5'-AACCATATGGACCACAAACTTTCCCCGGAACTGGAAGAA-3'; and OP2, 5'-GGGACCCGGACTCGGCG-3'. The PCR was carried out as described above with pWHM1302 as a template. The entire acdH gene was assembled by a three-piece ligation of the 0·68 kb NdeIAatII fragment of the PCR product, the 0·55 kb AatIIBglII fragment of pWHM1302 and the NdeIBamHI fragment of pT7-7 to give pWHM1303. pWHM1302 was prepared by subcloning the 3·5 kb PstI fragment containing the acdH from pWHM1301 into the PstI site of pGEM-3Zf(-). Similarly, an NdeI site was created at the S. avermitilis acdH translational start codon by PCR using pCD1468 (see above) as template. DNA primers were: P239, 5'-AAGAATTCATATGGACCACCGTCTCACCCCCGA-3' (rightward); and P238, 5' - AAAAGCTTCTGCAGGCCGGACAAGTTCGAAGGTAGGT-3' (leftward). The PCR-amplified DNA fragment was electroeluted from a 0·8% agarose gel, digested with NdeI and PstI, and ligated into pT7-7 to give pCD1513. The new constructs were confirmed by restriction enzyme digestion and the PCR fragment regions were sequenced.
Plasmids pWHM1303 and pCD1513 were introduced by transformation into E. coli BL21(DE3) for acdH expression. The E. coli host was also transformed with pT7-7 to generate a negative control culture. Transformants were grown at 37 °C in LB broth until the OD600 reached 0·6, then induced by adding IPTG to a final concentration of 0·4 mM. Incubation was continued for another 3 h, and whole cells were analysed for protein expression with 10% SDS-PAGE Ready Gels (Bio-Rad). Overexpressed proteins from the acdH and pT7-7 control strains were extracted by sonication; the cell extracts were fractionated by adding ammonium sulfate to 3555% saturation. The fractionated proteins were analysed by 10% SDS-PAGE Ready Gels and used for the isobutyryl-CoA, n-butyryl-CoA, n-valeryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA oxidation reactions. The protein concentration was determined by the method of Bradford (1976 ).
Characterization of the acdH gene product.
Reaction conditions for the oxidation of isobutyryl-CoA, n-butyryl-CoA, n-valeryl-CoA, isovaleryl-CoA and cyclohexenylcarbonyl-CoA described by Ikeda et al. (1983 ) were modified as follows. The reaction mixture contained 100 mM phosphate buffer (pH 8·0), 6 mM PMS, 0·4 mM FAD, 1 mM substrate and 40 µg protein. The total volume was 0·5 ml. The mixture was incubated at 37 °C for 30 min (when the methacrylyl-CoA product was isolated, the mixture was incubated for 18 h). Proteins used in the reaction were fractionated by ammonium sulfate precipitation as described above. For better resolution in HPLC analysis, PMS, FAD and FADH2 were removed from the reaction mixture using a Waters Accell Plus QMA cartridge conditioned with 10 ml deionized H2O. Reaction mixture (0·5 ml) was loaded and the cartridges were washed with 2 ml 100 mM phosphate buffer (pH 8·0); substrate and product were eluted with 2 ml 500 mM phosphate buffer (pH 8·0) and collected as 0·25 ml fractions. Fractions that contained substrate (usually in the 3rd and 4th fractions) were analysed by HPLC using the conditions described above for methacrylyl-CoA purification. When isobutyryl-CoA was the substrate, a peak at retention time 6·7 min was collected and characterized by NMR as described above.
Gene replacement in S. avermitilis.
Plasmid pIJ4026, obtained from M. J. Bibb (John Innes Centre, Norwich, UK), was used as a source of the Saccharopolyspora erythraea 1·6 kb BglII fragment carrying ermE (which confers resistance to erythromycin; Bibb et al., 1994 ). Shuttle vector pCD262 is a chimeric construct between pGEM-3Z and pMT660 (Birch & Cullum, 1985
). When a culture of S. avermitilis transformed with this vector is subjected to stress, such as high temperature or protoplast formation and regeneration in the absence of antibiotic selection, numerous plasmid-free colonies can be recovered (Denoya et al., 1995
). We have taken advantage of this to use pCD262 as a vector for gene replacements in S. avermitilis. The gene replacement vector pCD1137, a derivative of pCD262, was constructed (see Fig. 6
); it was used to transform S. lividans TK64 and reisolated for use in transforming S. avermitilis protoplasts. The latter were prepared and transformed as described previously (Denoya et al., 1995
).
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Final purification was achieved with a preparative Waters HPLC instrument as above. The material eluted from the 40M KP-SIL column was solubilized in a minimal volume of methanol (~3 ml) and the avermectins were separated on a Waters C18 symmetry column (19x300 mm; elution with 70% acetonitrile/30% water at 15 ml min-1). Fractions containing avermectin B1b were collected, the acetonitrile was removed under vacuum, and the aqueous solution was extracted with three equal volumes of ethyl acetate. The ethyl acetate was then removed under vacuum, yielding 22·4 mg B1b from culture CD1018 and 17·5 mg from CD1173. Each sample was solubilized in 0·5 ml deuterated chloroform for analytical NMR.
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RESULTS |
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Overexpression of S. coelicolor and S. avermitilis acdH in E. coli
For characterization of the S. coelicolor acdH product, we constructed a pT7-7 derivative, pWHM1303, containing the complete acdH coding region under the control of an IPTG-inducible gene for T7 RNA polymerase. E. coli BL21(DE3) containing pWHM1303 was analysed for acdH expression by SDS-PAGE. A protein of 42 kDa, the approximate mass of the predicted S. coelicolor acdH product (41·7 kDa), was detected in the IPTG-induced extracts from E. coli BL21(DE3)(pWHM1303), but not from the pT7-7 control strain (Fig. 4a). Proteins extracted from these strains were fractionated by addition of ammonium sulfate (Fig. 4b
) to give enzymically active samples with protein concentrations of 4·6 mg ml-1 and 6·0 mg ml-1 for the pWHM1303 overexpression and pT7-7 control strains, respectively. These protein extracts were used for the oxidation of isobutyryl-CoA, n-butyryl-CoA, n-valeryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA in vitro. Similarly, upon heat induction of the S. avermitilis acdH in E. coli BL21(DE3)(pCD1513), a band of about 42 kDa, which is the approximate mass of the predicted S. avermitilis AcdH protein, was detected in protein extracts.
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Disruption of the acdH gene in S. avermitilis
To confirm that acdH encodes a functional AD in S. avermitilis and to define further its role in the catabolism of branched-chain amino acids, the gene was inactivated by insertion of the Sac. erythraea ermE marker that confers resistance to erythromycin (Bibb et al., 1994 ). The replacement vector was constructed in two steps (Fig. 6
). First, both the 2·2 kb BamHI S. avermitilis genomic fragment (from pCD1106) carrying the 5'-half of acdH and the 1·6 kb BglII fragment (from pIJ4026) carrying the ermE marker were cloned into the unique BglII site of the E. coli/Streptomyces shuttle vector pCD262 to create pCD1126. Second, pCD1126 was linearized with BglII and ligated to the 7·5 kb BamHI S. avermitilis fragment (from pCD1119) to create pCD1137. This construct, which was expected to produce an insertional inactivation of the 980 nt ORF in the host genome upon recombination, contains 2·2 and 7·5 kb of homologous DNA to the left and right, respectively, of the target region. The ermE marker lies in the opposite orientation to that of the ORF.
Genomic replacement was achieved by following the approaches of Anzai et al. (1988 ) and Stutzman-Engwall et al. (1992
) as previously described (Denoya et al., 1995
). Plasmid pCD1137 was introduced into protoplasts of S. avermitilis ATCC 31272 by transformation, and many primary transformants resistant to both thiostrepton (the vector marker) and erythromycin were obtained. One of these was selected for propagation and for protoplast formation to eliminate the plasmid. After the transformant was plated on regeneration medium containing erythromycin, more than 50% of the colonies were resistant only to erythromycin. Chromosomal DNA was isolated from one of the thiostrepton-sensitive, erythromycin-resistant colonies (S. avermitilis CD1156) and compared with DNA from the parental strain (ATCC 31272). Southern blot hybridization confirmed that the ermE marker had been inserted and had disrupted the target ORF through the expected double crossover (data not shown).
Phenotype of the S. avermitilis disruptants
Table 1 compares the growth of a mutant CD1156 and the wild-type strain. The acdH-disrupted strain had lost the ability to grow on solid minimal medium containing valine, isoleucine and/or leucine as the sole carbon sources. In addition, the mutant grew more weakly than the control strain on acetate or butyrate, but as well as the control on either glucose, caproic acid or caprylic acid as the sole carbon source. To confirm the phenotype of the mutant, a second independent experiment repeated disruption of acdH in S. avermitilis ATCC 31272. Numerous disruptants were recovered and analysed, all showing a similar phenotype to the mutant described above. In addition, analysis of 35 disrupted cultures showed that the mutants grew at normal rates in rich medium and were capable of producing avermectins at least as well as the wild-type.
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Incorporation of [2,3,3'-13C3]isobutyrate into avermectin
To probe the physiological role of acdH further, incorporation of sodium [2,3,3'-13C3]isobutyrate was investigated using a bkd mutant of S. avermitilis that can make avermectin only when supplied with exogenous short-chain fatty acids. Thus high levels of labelling of the isobutyrate-derived starter unit of avermectin directly reflecting the level of labelling of the isobutyrate added were obtained and purification was simplified as the methylbutyrate-derived avermectins were not produced.
13C-NMR analysis of avermectin B1b (22·4 mg) isolated after incorporation of the labelled isobutyrate into a bkd mutant (S. avermitilis CD1018) showed greater than 10-fold enrichment of the signals corresponding to C-26 (multiplet), C-26a (doublet) and C-27 (doublet) (data not shown). These three carbons are derived from C-2, C-3 and C-3' of isobutyrate. The avermectin 13C-NMR spectrum revealed clear enrichment of the carbons derived from acetate (1·25-fold) and propionate (1·6-fold) (Fig. 7). The pattern of enrichment matched that previously determined from incorporation experiments using [1,2-13C]acetate and [2,3-13C2]propionate (data not shown). Thus these results are consistent with the labelled isobutyrate being (a) isomerized in vivo to n-butyryl-CoA (see Fig. 1
) and subsequently degraded to generate [1,2-13C]acetate and [2-13C]acetate, and (b) oxidized directly, presumably via methacrylyl-CoA, to [2,3,3'-13C3]methylmalonyl-CoA (see Fig. 1
).
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DISCUSSION |
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At least six distinct members of this enzyme family have been identified in mammalian cells. Four of them catalyse the first step of the ß-oxidation cycles for straight-chain fatty acids with various chain lengths. These are short (C4C6 CoA), medium (C4C16 CoA), long (C6C22 CoA) and very long chain ADs (Wolf & Akers, 1986 ; Matsubara et al., 1989
; Kelly et al., 1993
; Aoyama et al., 1994
). Two others catalyse the third reaction in the oxidation of branched-chain amino acids. Isovaleryl-CoA dehydrogenase is involved in leucine metabolism and 2-methyl-branched-chain AD is involved in isoleucine/valine metabolism. Recently, a rat short/branched-chain AD able to oxidize 2-methylbutyryl-CoA, isobutyryl-CoA and n-butyryl-CoA efficiently has also been characterized (Willard et al., 1996
). At least in eukaryotic systems, five of the ADs are tetrameric flavoproteins with a subunit size of 4045 kDa. Very long chain AD appears to be a dimer of 71 kDa subunits. All ADs share similar structural and functional features, including a consensus signature pattern in their amino acid sequences.
The blast search showed the closest identity (up to 46%) between the deduced AcdH amino acid sequence and a sub-group of ADs consisting mostly of short-chain specific enzymes. As we expected, the AcdH enzyme not only catalysed conversion of isobutyryl-CoA to methacrylyl-CoA, but also oxidized n-butyryl-CoA, n-valeryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA. Therefore, acdH appears to encode a short/branched-chain AD. An acyl-CoA oxidase purchased from Sigma did not oxidize isobutyryl-CoA to methacrylyl-CoA under conditions that allowed enzymic oxidation of palmitoyl-CoA to its 2,3-unsaturated ester, and clearly has different functions from the AcdHs we describe here.
The properties of the products encoded by acdH in S. avermitilis and S. coelicolor, taken together with the sequence data showing that two upstream ORFs encode products putatively involved in leucine catabolism, support our belief that the acdH product is involved in degradation of branched-chain amino acids. In mammals, two ADs catalyse the third reaction in the oxidation of branched-chain amino acids. These are isovaleryl-CoA dehydrogenase in leucine metabolism and 2-methyl-branched-chain acyl-CoA dehydrogenase in isoleucine/valine metabolism. Both mammalian enzymes have a narrow substrate specificity (Ikeda et al., 1983 ). Since the S. avermitilis acdH-disrupted mutant is unable to grow on valine, isoleucine and/or leucine as sole carbon source(s) one might speculate that Streptomyces spp. have a single branched-chain AD with broader substrate specificity than the mammalian counterparts and a role in the catabolism of all three branched-chain amino acids. However, the [13C]isobutyrate incorporation experiments showed that at least the valine catabolic pathway is still operating in the acdH mutant, though a greater than 50% decrease in 13C at methylmalonate-derived positions of avermectin suggest the catabolic rate is significantly reduced. The latter observation could be explained by assuming that two or more ADs with overlapping specificities contribute to the assimilation of branched-chain amino acids. Inactivation of just one of the overlapping activities, such as the broad substrate range AcdH in the mutant S. avermitilis culture, would be sufficient to prevent growth under the stringent conditions of a minimal medium having branched-chain amino acids as sole carbon source.
The reason why the S. avermitilis acdH mutant grew poorly on acetate and butyrate as a sole carbon source is unclear. It has recently been demonstrated that in addition to the glyoxylate cycle streptomycetes have a novel pathway for growth on acetate as a sole carbon source (Han & Reynolds, 1997 ). Evidence indicates that this pathway passes through a butyryl-CoA intermediate. The inability of the acdH mutant to grow on either acetate or butyrate therefore may implicate the conversion of isobutyryl-CoA to methacrylyl-CoA as a step in the acetate assimilation pathway. Interconversion of butyryl-CoA and isobutyryl-CoA in streptomycetes is well-established (Sherman et al., 1986
; Reynolds et al., 1988
).
Several earlier reports have described a relationship between the catabolism of branched-chain amino acids and macrolide antibiotic production (Omura et al., 1983 ; Tang et al., 1994
), suggesting that under some growth conditions, this catabolic route can provide the n-butyryl-CoA, 2-methylmalonyl-CoA or propionyl-CoA substrates used by the polyketide synthases to build the carbon skeleton of the antibiotic. In the present case the isotopic enrichment of the starter unit and the amount of avermectin produced were not affected when the S. avermitilis acdH mutant was grown in a rich medium, even though about 50% less of the isobutyrate-derived 2-methylmalonate was incorporated into propionate-derived positions of the polyketide. These results confirm that the starter unit for the polyketide portion of avermectin comes from valine and isoleucine catabolism (Denoya et al., 1995
, 1996
). Because 13C-enrichment of the propionate-derived positions of avermectin B1b was less than 10% of that in the starter unit, they also indicate that in a rich growth medium the amount of methylmalonate and propionate derived from isobutyrate catabolism and used to make the avermectins is very low.
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ACKNOWLEDGEMENTS |
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Received 31 December 1999;
revised 22 April 1999;
accepted 27 April 1999.