1995 bkdFGH gene mutant suitable for the production of novel antiparasitic avermectin

J OURNAL OF B ACTERIOLOGY,June1995,p.3504–3511Vol.177,No.12 0021-9193/95/$04.00?0

Copyright?1995,American Society for Microbiology

A Second Branched-Chain?-Keto Acid Dehydrogenase

Gene Cluster(bkdFGH)from Streptomyces avermitilis:

Its Relationship to Avermectin Biosynthesis and the

Construction of a bkdF Mutant Suitable for the

Production of Novel Antiparasitic Avermectins

CLAUDIO D.DENOYA,*RONALD W.FEDECHKO,EDMUND W.HAFNER,

HAMISH A.I.M C ARTHUR,MARGARET R.MORGENSTERN,

DEBORAH D.SKINNER,KIM STUTZMAN-ENGWALL,

RICHARD G.WAX,AND WILLIAM C.WERNAU

Bioprocess Research,Central Research Division,P?zer Inc.,Groton,Connecticut06340

Received22February1995/Accepted10April1995

A second cluster of genes encoding the E1?,E1?,and E2subunits of branched-chain?-keto acid dehydro-

genase(BCDH),bkdFGH,has been cloned and characterized from Streptomyces avermitilis,the soil microor-

ganism which produces anthelmintic avermectins.Open reading frame1(ORF1)(bkdF,encoding E1?),would

encode a polypeptide of44,394Da(406amino acids).The putative start codon of the incompletely sequenced

ORF2(bkdG,encoding E1?)is located83bp downstream from the end of ORF1.The deduced amino acid

sequence of bkdF resembled the corresponding E1?subunit of several prokaryotic and eukaryotic BCDH

complexes.An S.avermitilis bkd mutant constructed by deletion of a genomic region comprising the5?end of

bkdF is also described.The mutant exhibited a typical Bkd?phenotype:it lacked E1BCDH activity and had

lost the ability to grow on solid minimal medium containing isoleucine,leucine,and valine as sole carbon

sources.Since BCDH provides an?-branched-chain fatty acid starter unit,either S(?)-?-methylbutyryl

coenzyme A or isobutyryl coenzyme A,which is essential to initiate the synthesis of the avermectin polyketide

backbone in S.avermitilis,the disrupted mutant cannot make the natural avermectins in a medium lacking

both S(?)-?-methylbutyrate and isobutyrate.Supplementation with either one of these compounds restores

production of the corresponding natural avermectins,while supplementation of the medium with alternative

fatty acids results in the formation of novel avermectins.These results verify that the BCDH-catalyzed reaction

of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for

antibiotic biosynthesis in S.avermitilis.

Streptomyces avermitilis is a gram-positive,?lamentous soil microorganism which produces eight distinct but closely re-

lated polyketide compounds named avermectins that have po-

tent anthelmintic activity(8).The avermectin polyketide back-

bone is derived from seven acetate and?ve propionate

extender units added to an?-branched-chain fatty acid starter,

which is either S(?)-?-methylbutyric acid or isobutyric acid

(32).The C-25position of naturally occurring avermectins has

two possible substituents:a sec-butyl residue derived from the

incorporation of S(?)-?-methylbutyryl coenzyme A[S(?)-?-methylbutyryl-CoA](‘‘a’’avermectins)or an isopropyl residue derived from the incorporation of isobutyryl-CoA

(‘‘b’’avermectins).One metabolic route to the acyl-CoA

forms of the?-branched-chain fatty acid starter units is from

the?-branched-chain amino acids isoleucine and valine

through a branched-chain amino acid transaminase reaction

followed by a branched-chain?-keto acid dehydrogenase

(BCDH)reaction.Alternatively,?-branched-chain fatty

acyl-CoA derivatives can arise from branched-chain?-keto

acids produced by de novo synthesis(25).These metabolic

pathways are depicted in Fig.1.A mutant of S.avermitilis

with no detectable BCDH activity was previously isolated after chemical mutagenesis(17).The mutant could synthe-size natural avermectins only when the?-branched-chain fatty acids or a precursor bearing the isopropyl or sec-butyl (S-form)group was added to the fermentation medium(Fig. 1,bottom),while supplementation with a wide variety of alternative fatty acids results in the formation of a corre-sponding series of novel avermectins(15).These results are possible since short-chain fatty acids are readily taken up by S.avermitilis from the fermentation medium in a process that has been postulated to require at least a fatty acid binding and transport protein(probably similar to the Esch-erichia coli FadL protein)and a membrane-associated acyl-CoA synthetase(probably similar to the E.coli FadD pro-tein)(17).

The BCDH complex is a multienzyme complex composed of four functional components:a BCDH and decarboxylase (E1?and E1?),a dihydrolipoamide acyltransferase(E2),and a dihydrolipoamide dehydrogenase(E3)(33).The BCDH complex catalyzes the oxidative decarboxylations of?-keto-isovalerate,?-keto-?-methylvalerate,and?-ketoisocaproate(the deamination products of the branched-chain amino acids va-line,isoleucine,and leucine,respectively),releasing CO

2

and generating the corresponding acyl-CoA analogs and NADH (25).The genes encoding the components of the BCDH com-plex of Pseudomonas putida(44)and the pyruvate dehydro-genase(PDH)and BCDH dual-purpose complex of Bacillus subtilis(19)and Bacillus stearothermophilus(18)have been

*Corresponding author.Mailing address:Bioprocess Research,

P?zer Inc.,Eastern Point Rd.,Groton,CT06340.Phone:(203)441-4791.Fax:(203)441-3198.

3504

cloned and found to be clustered in the following sequence: gene encoding E1?,gene encoding E1?,gene encoding E2, and gene encoding E3.Recently,the genes for the branched-chain fatty acid-speci?c BCDH from B.subtilis were cloned and sequenced(48).This operon consisted of only the three genes encoding the E1?,E1?,and E2components.Addition-ally,the sequences of several eukaryotic genes encoding either E1?or E1?BCDH subunits have been reported(16,21,29,30, 51–53).

To understand further the importance of the BCDH-cata-lyzed reaction as a source of precursors for natural avermectin production and to manipulate the production of these antibi-otics,we decided to clone and analyze the genes encoding BCDH from S.avermitilis.Recently,we reported the cloning and analysis of a cluster of genes encoding the E1?,E1?,and E2components of a BCDH complex of S.avermitilis(bkdA, bkdB,and bkdC)(Fig.2)(39).When bkdA and bkdB,encoding the E1?and E1?BCDH subunits,were coexpressed in E.coli, a functional E1(??)BCDH activity was detected(39).How-ever,when the genomic copies of these genes were inactivated by gene disruption,no obvious phenotypic changes were ob-served(42a),suggesting that these genes were silent or that their functions could be accomplished by other genes.Here we report the cloning and characterization of a gene cluster en-coding another BCDH in S.avermitilis,bkdFGH.These genes, designated bkd genes by analogy with the nomenclature intro-duced to describe similar genotypes of P.putida(44),are located approximately12kb downstream of the bkdABC gene cluster.We also describe an S.avermitilis bkd mutant con-structed by deletion of a genomic region comprising the5?end of bkdF.The mutant,which lacks BCDH activity,cannot make the natural avermectins in a medium lacking both S(?)-?-methyl-butyrate and isobutyrate.However,supplementation with S(?)-?-methylbutyrate restores production of the correspond-ing‘‘a’’avermectins,while supplementation with cyclohexan-ecarboxylic acid results in the formation of a novel cyclohexyl avermectin without the coexpression of the natural analogs.

MATERIALS AND METHODS Microorganisms and growth conditions.Streptomyces lividans TK64(from D.

A.Hopwood,John Innes Centre,Norwich,United Kingdom)(20),S.

avermitilis

ATCC31272(8),and S.avermitilis ATCC53567(a bkd-de?cient mutant,deriv-

ative of strain ATCC31272,isolated after chemical mutagenesis)(17)were

grown as described previously(11,17,20).E.coli DH5?competent cells were

purchased from Life Technologies(Gaithersburg,Md.).General culture condi-

tions for Streptomyces species and E.coli were as described previously(20,36).

The antibiotics used were ampicillin(50?g/ml),erythromycin(4?g/ml),and

thiostrepton(4?g/ml).The concentrations of antibiotic were the same for both

solid and liquid cultures.S.avermitilis avermectin fermentations were carried out

with a minimal de?ned medium(to which various additions of fatty acids could

be made)as previously described(17),except that hydrolyzed starch(114g/liter)

was used instead of thinned and potato soluble starches,leucine and NaCl were

omitted,and glycine(2g/liter)was added.In some fermentations,either S(?)-?-methylbutyric acid or cyclohexanecarboxylic acid was added to the medium (0.04%[wt/vol]?nal concentration).Solid minimal medium was as previously

described(20),except that agarose(SeaKem ME agarose;FMC BioProducts,

Rockland,Maine)replaced agar,and the medium was supplemented with iso-

leucine,leucine,and valine(2.5g/liter each).

DNA isolation,ampli?cation,and cloning.General DNA manipulations were performed as described previously(36).Total chromosomal DNA from S.avermitilis was prepared by cesium chloride gradient centrifugation(20).Plasmids were isolated from E.coli and Streptomyces species as described previously(12,20).S.avermitilis genomic DNA was ampli?ed essentially as described previously(38,39).The DNA primers(Genosys,The Woodlands,Tex.)were5?-AAGAATTCGAGCTCGGCG ACGGCGCCACCTCCGAGGGCGAC-3?(rightward)and5?-AAGGATCCT CTAGAGGTSSWGTGGKGGCCGATSCGGWA-3?(leftward).The Interna-tional Union of Biochemistry rules of nomenclature for nucleotides were used to describe DNA sequences.Redundancies were identi?ed according to the Interna-tional Union of Biochemistry Group Codes,as follows:K?G?T,S?G?C,and W?A?T.Restriction recognition sequences were incorporated at the5?end of the rightward(Eco RI and Sac I)and leftward(Bam HI and Xba I)primers to facilitate cloning.A550-bp PCR-ampli?ed fragment was recovered by electroelution from an agarose gel,digested with both Eco RI and Xba I,and cloned between the Eco RI and Xba I sites of pGEM-3Z(Promega,Madison,Wis.)to produce pCD613.

DNA sequencing and computer analyses.A combination of transposon mini ??-1insertions(5)and primer walking was used for DNA sequencing of double-stranded DNA templates.Alkali denaturation procedures were performed as described in the TaqTrack Sequencing Systems technical manual(Promega). The nucleotide sequence was determined by the dideoxy sequencing method(37) as described previously(11).The Genetics Computer Group(Madison,Wis.) software(version7.3)(13)was used for sequence analysis.

Colony and Southern hybridizations and subclone constructions.DNA probes were prepared by nick translation(36)by using[?-32P]dCTP as described pre-viously(11,46).A cosmid library of S.avermitilis chromosomal DNA was pre-pared with total genomic DNA that was partially digested with Sau3A and was ligated into the Bam HI site in pKC505(34).The genomic library was screened by colony hybridization as described previously(11)with an E1?bkd-speci?c 32P-labeled0.35-kb Sal I fragment derived from pCD613.Ten positively hybrid-izing cosmid clones(of?2,200screened)were found to contain overlapping sequences by restriction mapping and Southern blot hybridizations(41).The following genomic fragments were subcloned from the cosmid clones into pGEM-3Z:2.3-kb Bam HI(pCD740),1.4-kb Bam HI(pCD854),4.1-kb Bam HI (pCD713),and5-kb Bam HI(pCD747)(Fig.2).

Gene replacement.Genomic replacement was achieved by following the ap-proaches of Anzai et al.(2)and Stutzman-Engwall et al.(43)and the general procedures described by Kieser and Hopwood(23).Plasmid pIJ4026,obtained from M.J.Bibb(John Innes Centre),was used as a source of the Saccharopoly-spora erythraea1.6-kb Bgl II fragment carrying the ermE marker(which confers resistance to erythromycin).Shuttle vector pCD262is a chimeric construct be-tween pGEM-3Z and pMT660(6).When a culture of S.avermitilis that has been transformed with this vector is subjected to stress conditions,such as high temperature or protoplast formation and regeneration,numerous plasmid-free colonies can be recovered in the absence of antibiotic selection(11a).We have taken advantage of this characteristic to use pCD262as a vector for gene replacements in S.avermitilis.The gene replacement vector pCD768,a derivative of pCD262,was constructed(see Fig.5).pCD768was prepared from trans-formed S.lividans TK64cells for use in transforming S.avermitilis protoplasts. Preparation of S.avermitilis protoplasts and transformation were performed as described previously(20),following modi?cations as described previously(26). E1BCDH and PDH assays.E1BCDH and PDH activities were determined by measuring the production of14CO2from[1-14C]-?-ketoisocaproate or from [1-14C]pyruvate,respectively,as described previously(17,31,39).[1-14C]-?-ketoisocaproate(55mCi/mmol,50?Ci/ml)and[1-14C]pyruvate(28mCi/mmol, 81?Ci/ml)were purchased from Amersham,Arlington Heights,Ill.Protein contents were determined by the method of Bradford(7).

Nucleotide sequence accession number.The nucleotide sequence reported here has been deposited at GenBank under accession number U26308.

RESULTS AND DISCUSSION

Cloning and sequencing of a second S.avermitilis gene en-coding an E1?subunit of the BCDH complex by using a homology probing approach.The?nding that disruption of a cluster of genes,bkdABC,encoding a BCDH complex failed to produce any evident phenotypic changes(11a)prompted us to pursue the cloning of additional bkd genes in S.avermitilis. Three E1?BCDH peptide sequences from humans(16),rats (53),and P.putida(9)and the E1?subunit of the dual PDH-BCDH complex from B.stearothermophilus(18)were aligned to identify conserved regions that could serve as candidate sequences for the design of corresponding PCR primers.Sev-eral regions of extended resemblance(49)were chosen,Strep-tomyces gene codon assignments(50)were used,and minimal homology requirements for PCR primer design were followed as previously discussed(40).One PCR primer was based on a region encompassing amino acids212to220of the P.putida E1?BCDH protein(9),which was used as a representative model of an E1?BCDH subunit.These amino acids are lo-cated within the thiamine PP

i

binding motif(49).The second primer was based on a region encompassing amino acids307to 314of the same E1?BCDH subunit.The latter amino acid sequence comprises the end of the subunit interaction site and the beginning of the phosphorylation site conserved regions (49).A DNA product of approximately550bp was

ampli?ed

by using the two primers described above and S.avermitilis

genomic DNA as a template.Despite the fact that the PCR

product was larger than the expected size of0.3kb,the am-

pli?ed fragment was cloned and sequenced.Analysis by the

Codon Preference program(13)of the corresponding500-bp

DNA sequence(excluding the primers)revealed an open read-

ing frame(ORF)presenting the codon usage and the G?C

third-position bias characteristic of a Streptomyces gene(50).

Computer search analysis revealed that the deduced amino

acid sequence of the cloned PCR product was highly similar to

those of all the E1?BCDH subunits obtained to date(identity,?50%).The alignment of the amino acid sequences suggested that the leftward primer hybridized to the expected target

region of a typical E1?gene,but the rightward primer hybrid-

ized considerably upstream of the corresponding target(Fig.

3).A0.35-kb Sal I fragment,which is located internally in the

0.55-kb insert,was then used as a probe to screen an S.aver-

mitilis chromosomal library(see Materials and Methods).The genomic region containing the E1?determinant was cloned, and a restriction map is shown in Fig.2.A DNA sequence of more than1,400bp is presented in Fig.3.The Codon Prefer-ence program(13)revealed one complete ORF(ORF1,bkdF) and the5?end of a second ORF(ORF2,bkdG),both with the same orientation(Fig.2).ORF1would encode a polypeptide of406amino acids with an estimated molecular mass of44,394Da.The intergenic distance between ORF1and ORF2is83 bp.Figure3also shows that there are four homologous nucle-otides at the3?end of the rightward primer(which are impor-tant for a successful priming[40]),ensuring a better match with its observed hybridization site(nucleotides[nt]375to401) than with its originally anticipated target site(nt627to653). The latter observation provides a probable explanation for why the cloned PCR product was larger than expected. Similarity of the deduced gene products to the components of the BCDH complex.A computer homology search of trans-lated nucleotide and peptide sequence databases performed with the deduced amino acid sequence of the S.avermitilis bkdF gene showed that the best scores(?31%identity)were obtained with several E1?subunits of prokaryotic(9,48)and eukaryotic(16,21)BCDH complexes and also with the corre-sponding E1?subunits of the dual BCDH-PDH complexes from B.subtilis and B.stearothermophilus(18,19)and the partially characterized PDH complex from Acholeplasma laid-lawii(47).The highest score(38%identity)was obtained with the deduced primary structure of the S.avermitilis bkdA gene (E1?subunit)(39).Lower sequence identities(?29%)to the E1?subunits of other PDH complexes were detected(4,22, 24).A multiple sequence alignment identi?ed common struc-tural motifs well conserved in the E1?subunits of all of the ?-keto acid dehydrogenase complexes examined so far,such

as

the thiamine PP

i binding motif,a putative subunit interaction

site,and the phosphorylation sites(I and II)of the E1?chains of the mammalian BCDH and PDH complexes(49)(Fig.4). Evidence for a second bkd gene cluster in S.avermitilis. Codon Preference analysis of fragmented sequence data from several transposon m??-1insertions(5)located downstream of bkdF,plus the data presented in Fig.3,suggested the presence of two additional ORFs.The deduced products of the incom-plete ORFs2and3(bkdG and bkdH,respectively[Fig.2]) showed signi?cant similarity to several prokaryotic and eukary-otic E1?and dihydrolipoamide acyltransferase(E2)compo-nents of the BCDH complex(data not shown)(29,35).These results suggest that ORF1and the putative ORFs2and3 would encode three components of a BCDH complex in S. avermitilis.This contention was substantiated by the experi-ments discussed below.

Disruption of the bkdF gene region.To test the idea that the bkdFGH gene cluster indeed encodes a BCDH complex and also to con?rm the role of the branched-chain amino acid catabolic pathway as a provider of branched-chain fatty acid precursors for avermectin biosynthesis,a 1.4-kb Bam HI genomic region,containing more than450bp of the5?end of the bkdF gene(E1?)and approximately900bp located in the region upstream of this gene(Fig.2),was deleted from the S. avermitilis chromosome.The latter upstream region does not contain any obvious ORF,as demonstrated by Codon Prefer-ence analysis of DNA sequencing data collected from a group of mini??-1insertions randomly distributed in the1.4-kb frag-ment(data not shown).As shown in Fig.5,the construction of the gene replacement vector was carried out in two steps.First, both the4.1-kb Bam HI S.avermitilis genomic fragment(from pCD713)carrying the end of bkdF and the rest of the bkd cluster and the1.6-kb Bgl II fragment(from pIJ4026)carrying the ermE marker were cloned into the unique Bgl II site of the E.coli-Streptomyces shuttle vector pCD262to create pCD761. Second,pCD761was linearized with Bgl II and ligated to the 2.3-kb Bam HI S.avermitilis fragment(from pCD740)to create pCD768.This construct,which is expected to produce a1.4-kb deletion in the host genome upon recombination,contains2.3 and4.1kb of homologous DNA?anking the target region to the left and right,respectively.The ermE marker lies in the orientation opposite that of the ORFs to avoid any possible deleterious effect caused by overexpression of downstream genes.pCD768was introduced into protoplasts of S.avermitilis ATCC31272by transformation,and many primary transfor-mants of S.avermitilis that were resistant to both thiostrepton and erythromycin were obtained.One of these,named CD783, was selected for propagation and for protoplast formation to eliminate the plasmid.After the transformant was plated on regeneration medium containing erythromycin,?ve colonies were selected,one being resistant to both erythromycin and thiostrepton(CD783-pp14)and the others being resistant only to erythromycin.Chromosomal DNA was isolated from one of the thiostrepton-sensitive,erythromycin-resistant colonies (CD783-pp15),and the parental strain(ATCC31272)and the DNAs were used to analyze the region around the bkdF gene by Southern analysis.Southern hybridization con?rmed that the1.4-kb Bam HI fragment was deleted and replaced by the ermE marker through the expected double crossover(data not shown).

Phenotype of the disruptants.Table1summarizes the re-sults of gene disruption.The disrupted strain

(CD783-pp15)

exhibited a typical bkd mutant phenotype (the other three thiostrepton-sensitive,erythromycin-resistant clones men-tioned above also had a similar phenotype).The mutant had lost the ability to grow on solid minimal medium plates con-taining isoleucine,leucine,and valine as the sole carbon sources and lacked,as predicted from the DNA sequencing results,E1BCDH activity (Table 1).In contrast,E1PDH assays demonstrated a level of activity in CD783-pp15similar to that of the parental or other control strains (data not shown).In addition,when the mutant culture was grown in unsupplemented de?ned fermentation medium (in which branched-chain and straight-chain fatty acids and their poten-tial metabolic precursors [e.g.,fats and oils]were omitted [see Materials and Methods]),no avermectins were produced (Ta-ble 1).Short ?-branched-chain fatty acids are readily taken up by S.avermitilis cells from the environment and presumably are activated to their thioesters via an acyl-CoA synthetase (17).Therefore,when S (?)-?-methylbutyrate was added to the de-?ned medium,natural ‘‘a’’forms of avermectins were pro-duced (Table 1).Similarly,addition of an unnatural branched-chain fatty acid,such as cyclohexanecarboxylic acid to the medium led to the formation of novel cyclohexanecarboxylic acid-derived,C-25-substituted avermectins (15)(data not shown).In addition,the mutant strain maintained growth ca-pabilities essentially identical to those of the parental strain in a variety of media.The results showed that the mutant culture was incapable of producing avermectins per se.It was able to

biosynthesize avermectins only when supplemented with ap-propriate branched-chain fatty acid precursors in the fermen-tation medium.

Concluding remarks.The presence of multiple copies of the same or similar genes in Streptomyces is not unusual (10,14,28).We had previously shown that S.avermitilis has a gene cluster (bkdABC )encoding BCDH and that bkdA and bkdB could be coexpressed in E.coli to produce a functional E1(??)BCDH activity (39).Here we revealed a second bkd gene cluster (bkdFGH )in S.avermitilis which has an organization similar to that of the bkdABC cluster and which consists of the genes encoding the E1?,E1?,and E2components in the same orientation.Since the two bkd gene clusters are closely located (only 12kb apart)and have the same orientation,we speculate that the clusters might have arisen by tandem genetic duplica-tion and that the two copies accumulated,through mutations,enough differences to ensure survival,avoiding recombination-deletion events (1).

We have found that the bkdABC genes could be inactivated by gene replacement without causing obvious phenotypic changes (42a).In contrast,disruption of a genomic region comprising the 5?end of the bkdF gene caused complete loss of the E1BCDH activity in the mutant described here.The latter strongly suggests that only one multienzyme complex is respon-sible for the BCDH activity in S.avermitilis .Since the E1PDH activity level was normal in the disrupted culture,the possibil-ity of the bkdFGH genes encoding a dual BCDH-PDH com-plex,as described for B.subtilis and B.stearothermophilus (18,19),seems improbable.Recently,Madhusudhan et al.(27)reported that chromosomal mutations affecting bkdR ,a gene divergently transcribed from the bkd operon of P.putida and encoding a positive regulator of this operon,resulted in a loss of BCDH activity.If a homologous gene is present in S.aver-mitilis ,further investigation will be needed to determine if the chromosomal disruption reported here has also affected the expression of a regulatory gene.

Recently,Tang and Hutchinson (45)reported that inactiva-tion of the valine dehydrogenase gene (vdh )reduced the pro-duction of tylosin in Streptomyces fradiae and spiramycin

in

TABLE 1.Phenotypes of S.avermitilis bkdF -disrupted mutant and

control strains

S.avermitilis strain a Resistance phenotype b Growth on ILV c

E1BCDH sp act d

Produc-tion of aver-mectins e U

S

ATCC 31272(parent)Erm s Tsr s ?69.2??ATCC 53567(bkd-11)Erm s Tsr s ??0.3??CD783

Erm r Tsr r ?66.7??CD783-pp14Erm r Tsr r ?65.1??CD783-pp15

Erm r Tsr s

?

?0.2

?

?

a See the text for strain descriptions.

b

Erm s ,sensitive to erythromycin;Tsr s ,sensitive to thiostrepton;Erm r ,resis-tant to erythromycin;Tsr r ,resistant to thiostrepton.c

?or ?,ability or inability to grow on minimal medium supplemented with isoleucine,leucine,and valine (ILV)as sole carbon sources after 14days at 29?C.d

The speci?c activity of the E1component of the BCDH in picomoles of CO 2evolved per minute per milligram of protein.The results are the means of duplicate determinations.e

?and ?,production and nonproduction,respectively,of natural avermectins by fermentation in de?ned fermentation medium which was unsupplemented (U)or supplemented (S)with ?-methylbutyrate (0.04%[wt/vol]?nal concentra-tion).

V OL .177,1995S.AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3509

Streptomyces ambofaciens,suggesting that valine catabolism is an important but not exclusive source of fatty acid precursors for macrolide biosynthesis(under de?ned growth conditions) in these microorganisms.The S.avermitilis bkd strain reported here demonstrated a direct correlation between the deletion of a genomic region comprising the5?end of the bkdF gene and a complete loss of the ability to synthesize avermectins when grown in a de?ned(unsupplemented)medium.These results showed that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step in providing fatty acid precursors for antibiotic biosynthesis in S.avermitilis. The bkdF-disrupted strain is stable:we have not observed any reversion or genomic rearrangements,even after a large number of passages(data not shown),and the strain has main-tained all the characteristic phenotypic traits of a bkd mutant. Most importantly,the genomic disruption discussed here can be reproduced in any other avermectin production strain by using essentially the gene replacement vector and procedures delineated in Fig.5.The latter greatly facilitates the develop-ment of a larger selection of S.avermitilis bkd strains that can be used to generate valuable novel(unnatural)C-25-substi-tuted antiparasitic avermectins(15).

ACKNOWLEDGMENTS

We thank David A.Hopwood for critical reading of the manuscript and Karen De Benedictis for expert legal assistance in this project.

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赖世雄-英语语法

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Terminology Translations on lexicology 英语词汇学术语翻译 A acronym 首字母拼音词acronymy 首字母拼音法 addition 增词 adjective compound 复合形容词 affective meaning 感情意义 affix 词缀 affixation 词缀法 Albanian 阿尔巴尼亚语（族）aliens 非同化词alliteration 头韵（法）allomorph 词素（形位）变体ambiguity 歧义 amelioration of meamng 词义的升华analogy 类推 analytic language 分析性语言antithsis 对偶 antonym 反义词 antonymy 反义关系 appreciative term 褒义词 archaic word 古词 archaism 古词语

argot 隐语（黑话）Armenian 亚美尼亚语（族）Associated transfer 联想转移association 联想 associative meanings 关联意义 B back－formation 逆生法 back clipping 词尾截短 Balto－Slavic 波罗斯拉夫语（族）bilinguall 双语的 basic word stock 基本词汇 blend 拼缀词 blending 拼缀法 borrowed word 借词 bound form粘着形式 bound morpheme 粘着语素（形位）bound root 粘着词根 C casual style 随便文体 catchPhrase 时髦语 Celtic 凯尔特语（族） central meaning 中心意义 Clipping 截短法 collocability 搭配能力