Keto Acid Dehydrogenase Gene Cluster

JOURNAL OF BACTERIOLOGY, June 1995, p. 3504–3511 Vol. 177, No. 12 0021-9193/95/$04.00ϩ0 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. MCARTHUR, MARGARET R. MORGENSTERN, DEBORAH D. SKINNER, KIM STUTZMAN-ENGWALL, RICHARD G. WAX, AND WILLIAM C. WERNAU Bioprocess Research, Central Research Division, Pﬁzer Inc., Groton, Connecticut 06340

Received 22 February 1995/Accepted 10 April 1995

A second cluster of genes encoding the E1␣,E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1␣), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1␤) is located 83 bp 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 the 5؅ end of bkdF is also described. The mutant exhibited a typical Bkd؊ phenotype: it lacked E1 BCDH 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, filamentous soil after chemical mutagenesis (17). The mutant could synthe- microorganism which produces eight distinct but closely re- size natural avermectins only when the ␣-branched-chain lated polyketide compounds named avermectins that have po- fatty acids or a precursor bearing the isopropyl or sec-butyl tent anthelmintic activity (8). The avermectin polyketide back- (S-form) group was added to the fermentation medium (Fig. bone is derived from seven acetate and five propionate 1, bottom), while supplementation with a wide variety of extender units added to an ␣-branched-chain fatty acid starter, alternative fatty acids results in the formation of a corre- which is either S(ϩ)-␣-methylbutyric acid or isobutyric acid sponding series of novel avermectins (15). These results are (32). The C-25 position of naturally occurring avermectins has possible since short-chain fatty acids are readily taken up by two possible substituents: a sec-butyl residue derived from the S. avermitilis from the fermentation medium in a process incorporation of S(ϩ)-␣-methylbutyryl coenzyme A [S(ϩ)- that has been postulated to require at least a fatty acid ␣-methylbutyryl-CoA] (‘‘a’’ avermectins) or an isopropyl binding and transport protein (probably similar to the Esch- residue derived from the incorporation of isobutyryl-CoA erichia coli FadL protein) and a membrane-associated acyl- (‘‘b’’ avermectins). One metabolic route to the acyl-CoA CoA synthetase (probably similar to the E. coli FadD pro- forms of the ␣-branched-chain fatty acid starter units is from tein) (17). the ␣-branched-chain amino acids isoleucine and valine The BCDH complex is a multienzyme complex composed through a branched-chain amino acid transaminase reaction of four functional components: a BCDH and decarboxylase followed by a branched-chain ␣-keto acid dehydrogenase (E1␣ and E1␤), a dihydrolipoamide acyltransferase (E2), and (BCDH) reaction. Alternatively, ␣-branched-chain fatty a dihydrolipoamide dehydrogenase (E3) (33). The BCDH acyl-CoA derivatives can arise from branched-chain ␣-keto complex catalyzes the oxidative decarboxylations of ␣-keto- acids produced by de novo synthesis (25). These metabolic isovalerate, ␣-keto-␤-methylvalerate, and ␣-ketoisocaproate (the pathways are depicted in Fig. 1. A mutant of S. avermitilis deamination products of the branched-chain amino acids va- with no detectable BCDH activity was previously isolated line, isoleucine, and leucine, respectively), releasing CO2 and generating the corresponding acyl-CoA analogs and NADH (25). The genes encoding the components of the BCDH com- * Corresponding author. Mailing address: Bioprocess Research, plex of Pseudomonas putida (44) and the pyruvate dehydro- Pfizer Inc., Eastern Point Rd., Groton, CT 06340. Phone: (203) 441- genase (PDH) and BCDH dual-purpose complex of Bacillus 4791. Fax: (203) 441-3198. subtilis (19) and Bacillus stearothermophilus (18) have been

3504 VOL. 177, 1995 S. AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3505

FIG. 1. Pathways of valine and isoleucine catabolism and their postulated relationship to avermectin biosynthesis. cloned and found to be clustered in the following sequence: served (42a), suggesting that these genes were silent or that gene encoding E1␣, gene encoding E1␤, gene encoding E2, their functions could be accomplished by other genes. Here we and gene encoding E3. Recently, the genes for the branched- report the cloning and characterization of a gene cluster en- chain fatty acid-speciﬁc BCDH from B. subtilis were cloned coding another BCDH in S. avermitilis, bkdFGH. These genes, and sequenced (48). This operon consisted of only the three designated bkd genes by analogy with the nomenclature intro- genes encoding the E1␣,E1␤, and E2 components. Addition- duced to describe similar genotypes of P. putida (44), are ally, the sequences of several eukaryotic genes encoding either located approximately 12 kb downstream of the bkdABC gene E1␣ or E1␤ BCDH subunits have been reported (16, 21, 29, 30, cluster. We also describe an S. avermitilis bkd mutant con- 51–53). structed by deletion of a genomic region comprising the 5Ј end of To understand further the importance of the BCDH-cata- bkdF. The mutant, which lacks BCDH activity, cannot make the lyzed reaction as a source of precursors for natural avermectin natural avermectins in a medium lacking both S(ϩ)-␣-methyl- production and to manipulate the production of these antibi- butyrate and isobutyrate. However, supplementation with otics, we decided to clone and analyze the genes encoding S(ϩ)-␣-methylbutyrate restores production of the correspond- BCDH from S. avermitilis. Recently, we reported the cloning ing ‘‘a’’ avermectins, while supplementation with cyclohexan- and analysis of a cluster of genes encoding the E1␣,E1␤, and ecarboxylic acid results in the formation of a novel cyclohexyl E2 components of a BCDH complex of S. avermitilis (bkdA, avermectin without the coexpression of the natural analogs. bkdB, and bkdC) (Fig. 2) (39). When bkdA and bkdB, encoding the E1␣ and E1␤ BCDH subunits, were coexpressed in E. coli, MATERIALS AND METHODS a functional E1(␣␤) BCDH activity was detected (39). How- ever, when the genomic copies of these genes were inactivated Microorganisms and growth conditions. Streptomyces lividans TK64 (from D. by gene disruption, no obvious phenotypic changes were ob- A. Hopwood, John Innes Centre, Norwich, United Kingdom) (20), S. avermitilis 3506 DENOYA ET AL. J. BACTERIOL.

FIG. 2. Restriction map of the region of the S. avermitilis genome containing the genes encoding the components of the BCDH complex. Abbreviations for restriction sites: B, BamHI; Bg, BglII; P, PstI; and S, SphI. The original cloned PCR product containing a portion of the bkdF gene is indicated by a small solid box and an asterisk. The arrows and boxes below the map indicate the location and orientation of the ORFs (solid boxes represent completely sequenced ORFs and shaded boxes represent partially sequenced ORFs). The letters below the boxes refer to bkd genes (bkdABC and bkdFGH gene clusters) discussed in this work. pGEM-3Z subclones are also shown.

ATCC 31272 (8), and S. avermitilis ATCC 53567 (a bkd-deficient mutant, deriv- from M. J. Bibb (John Innes Centre), was used as a source of the Saccharopoly- ative of strain ATCC 31272, isolated after chemical mutagenesis) (17) were spora erythraea 1.6-kb BglII fragment carrying the ermE marker (which confers grown as described previously (11, 17, 20). E. coli DH5␣ competent cells were resistance to erythromycin). Shuttle vector pCD262 is a chimeric construct be- purchased from Life Technologies (Gaithersburg, Md.). General culture condi- tween pGEM-3Z and pMT660 (6). When a culture of S. avermitilis that has been tions for Streptomyces species and E. coli were as described previously (20, 36). transformed with this vector is subjected to stress conditions, such as high The antibiotics used were ampicillin (50 ␮g/ml), erythromycin (4 ␮g/ml), and temperature or protoplast formation and regeneration, numerous plasmid-free thiostrepton (4 ␮g/ml). The concentrations of antibiotic were the same for both colonies can be recovered in the absence of antibiotic selection (11a). We have solid and liquid cultures. S. avermitilis avermectin fermentations were carried out taken advantage of this characteristic to use pCD262 as a vector for gene with a minimal defined medium (to which various additions of fatty acids could replacements in S. avermitilis. The gene replacement vector pCD768, a derivative be made) as previously described (17), except that hydrolyzed starch (114 g/liter) of pCD262, was constructed (see Fig. 5). pCD768 was prepared from trans- was used instead of thinned and potato soluble starches, leucine and NaCl were formed S. lividans TK64 cells for use in transforming S. avermitilis protoplasts. omitted, and glycine (2 g/liter) was added. In some fermentations, either S(ϩ)- Preparation of S. avermitilis protoplasts and transformation were performed as ␣-methylbutyric acid or cyclohexanecarboxylic acid was added to the medium described previously (20), following modifications as described previously (26). (0.04% [wt/vol] final concentration). Solid minimal medium was as previously E1 BCDH and PDH assays. E1 BCDH and PDH activities were determined by 14 14 described (20), except that agarose (SeaKem ME agarose; FMC BioProducts, measuring the production of CO2 from [1- C]-␣-ketoisocaproate or from Rockland, Maine) replaced agar, and the medium was supplemented with iso- [1-14C]pyruvate, respectively, as described previously (17, 31, 39). [1-14C]-␣- leucine, leucine, and valine (2.5 g/liter each). ketoisocaproate (55 mCi/mmol, 50 ␮Ci/ml) and [1-14C]pyruvate (28 mCi/mmol, DNA isolation, amplification, and cloning. General DNA manipulations were 81 ␮Ci/ml) were purchased from Amersham, Arlington Heights, Ill. Protein performed as described previously (36). Total chromosomal DNA fromS. avermitilis contents were determined by the method of Bradford (7). was prepared by cesium chloride gradient centrifugation (20). Plasmids were isolated Nucleotide sequence accession number. The nucleotide sequence reported from E. coli and Streptomyces species as described previously (12, 20). S. avermitilis here has been deposited at GenBank under accession number U26308. genomic DNA was amplified essentially as described previously (38, 39). The DNA primers (Genosys, The Woodlands, Tex.) were 5Ј-AAGAATTCGAGCTCGGCG ACGGCGCCACCTCCGAGGGCGAC-3Ј (rightward) and 5Ј-AAGGATCCT RESULTS AND DISCUSSION CTAGAGGTSSWGTGGKGGCCGATSCGGWA-3Ј (leftward). The Interna- tional Union of Biochemistry rules of nomenclature for nucleotides were used to Cloning and sequencing of a second S. avermitilis gene en- describe DNA sequences. Redundancies were identified according to the Interna- coding an E1␣ subunit of the BCDH complex by using a tional Union of Biochemistry Group Codes, as follows: K ϭ G ϩ T, S ϭ G ϩ C, and W ϭ A ϩ T. Restriction recognition sequences were incorporated at the 5Ј end of homology probing approach. The finding that disruption of a the rightward (EcoRI and SacI) and leftward (BamHI and XbaI) primers to facilitate cluster of genes, bkdABC, encoding a BCDH complex failed to cloning. A 550-bp PCR-amplified fragment was recovered by electroelution from an produce any evident phenotypic changes (11a) prompted us to agarose gel, digested with both EcoRI and XbaI, and cloned between the EcoRI and pursue the cloning of additional bkd genes in S. avermitilis. XbaI sites of pGEM-3Z (Promega, Madison, Wis.) to produce pCD613. DNA sequencing and computer analyses. A combination of transposon mini Three E1␣ BCDH peptide sequences from humans (16), rats ␥␦-1 insertions (5) and primer walking was used for DNA sequencing of double- (53), and P. putida (9) and the E1␣ subunit of the dual PDH- stranded DNA templates. Alkali denaturation procedures were performed as BCDH complex from B. stearothermophilus (18) were aligned described in the TaqTrack Sequencing Systems technical manual (Promega). to identify conserved regions that could serve as candidate The nucleotide sequence was determined by the dideoxy sequencing method (37) as described previously (11). The Genetics Computer Group (Madison, Wis.) sequences for the design of corresponding PCR primers. Sev- software (version 7.3) (13) was used for sequence analysis. eral regions of extended resemblance (49) were chosen, Strep- Colony and Southern hybridizations and subclone constructions. DNA probes tomyces 32 gene codon assignments (50) were used, and minimal were prepared by nick translation (36) by using [␣- P]dCTP as described pre- homology requirements for PCR primer design were followed viously (11, 46). A cosmid library of S. avermitilis chromosomal DNA was prepared with total genomic DNA that was partially digested with Sau3A and was as previously discussed (40). One PCR primer was based on a ligated into the BamHI site in pKC505 (34). The genomic library was screened region encompassing amino acids 212 to 220 of the P. putida by colony hybridization as described previously (11) with an E1␣ bkd-specific 32 E1␣ BCDH protein (9), which was used as a representative P-labeled 0.35-kb SalI fragment derived from pCD613. Ten positively hybrid- model of an E1␣ BCDH subunit. These amino acids are lo- izing cosmid clones (of Ͼ2,200 screened) were found to contain overlapping sequences by restriction mapping and Southern blot hybridizations (41). The cated within the thiamine PPi binding motif (49). The second following genomic fragments were subcloned from the cosmid clones into primer was based on a region encompassing amino acids 307 to pGEM-3Z: 2.3-kb BamHI (pCD740), 1.4-kb BamHI (pCD854), 4.1-kb BamHI 314 of the same E1␣ BCDH subunit. The latter amino acid (pCD713), and 5-kb BamHI (pCD747) (Fig. 2). sequence comprises the end of the subunit interaction site and 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 the beginning of the phosphorylation site conserved regions procedures described by Kieser and Hopwood (23). Plasmid pIJ4026, obtained (49). A DNA product of approximately 550 bp was amplified VOL. 177, 1995 S. AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3507

FIG. 3. Nucleotide sequence of the 1.4-kb S. avermitilis genomic region containing the E1␣ ORF and its surrounding regions. The deduced amino acid sequence is given in the single-letter code below the nucleotide sequence. Presumed Shine-Dalgarno ribosome binding sites (3, 42) are underlined, and the potential translation terminator codon is indicated by an asterisk. Selected restriction sites are listed above their recognition sequences (boldface). The nucleotide sequence of the rightward primer (including the restriction recognition sequences present at the 5Ј end of the primer [see Materials and Methods]) is given above its originally anticipated target site (nt 627 to 653) and above its corresponding experimental hybridization site (nt 375 to 401), and the complementary nucleotide sequence of the leftward primer is given above its corresponding target site (nt 906 to 929). Since the leftward primer was actually a degenerate oligonucleotide mixture, only the best matched sequence is shown. Primer nucleotides identical to either the theoretical target or the experimental priming sites and adjacent regions are in boldface. by using the two primers described above and S. avermitilis Da. The intergenic distance between ORF1 and ORF2 is 83 genomic DNA as a template. Despite the fact that the PCR bp. Figure 3 also shows that there are four homologous nucle- product was larger than the expected size of 0.3 kb, the am- otides at the 3Ј end of the rightward primer (which are impor- pliﬁed fragment was cloned and sequenced. Analysis by the tant for a successful priming [40]), ensuring a better match with Codon Preference program (13) of the corresponding 500-bp its observed hybridization site (nucleotides [nt] 375 to 401) DNA sequence (excluding the primers) revealed an open read- than with its originally anticipated target site (nt 627 to 653). ing frame (ORF) presenting the codon usage and the GϩC The latter observation provides a probable explanation for why third-position bias characteristic of a Streptomyces gene (50). the cloned PCR product was larger than expected. Computer search analysis revealed that the deduced amino Similarity of the deduced gene products to the components acid sequence of the cloned PCR product was highly similar to of the BCDH complex. A computer homology search of trans- those of all the E1␣ BCDH subunits obtained to date (identity, lated nucleotide and peptide sequence databases performed Ͼ50%). The alignment of the amino acid sequences suggested with the deduced amino acid sequence of the S. avermitilis that the leftward primer hybridized to the expected target bkdF gene showed that the best scores (Ͼ31% identity) were region of a typical E1␣ gene, but the rightward primer hybrid- obtained with several E1␣ subunits of prokaryotic (9, 48) and ized considerably upstream of the corresponding target (Fig. eukaryotic (16, 21) BCDH complexes and also with the corre- 3). A 0.35-kb SalI fragment, which is located internally in the sponding E1␣ subunits of the dual BCDH-PDH complexes 0.55-kb insert, was then used as a probe to screen an S. aver- from B. subtilis and B. stearothermophilus (18, 19) and the mitilis chromosomal library (see Materials and Methods). The partially characterized PDH complex from Acholeplasma laid- genomic region containing the E1␣ determinant was cloned, lawii (47). The highest score (38% identity) was obtained with and a restriction map is shown in Fig. 2. A DNA sequence of the deduced primary structure of the S. avermitilis bkdA gene more than 1,400 bp is presented in Fig. 3. The Codon Prefer- (E1␣ subunit) (39). Lower sequence identities (Ͻ29%) to the ence program (13) revealed one complete ORF (ORF1, bkdF) E1␣ subunits of other PDH complexes were detected (4, 22, and the 5Ј end of a second ORF (ORF2, bkdG), both with the 24). A multiple sequence alignment identiﬁed common struc- same orientation (Fig. 2). ORF1 would encode a polypeptide tural motifs well conserved in the E1␣ subunits of all of the of 406 amino acids with an estimated molecular mass of 44,394 ␣-keto acid dehydrogenase complexes examined so far, such as 3508 DENOYA ET AL. J. BACTERIOL.

FIG. 4. Alignment of the predicted amino acid sequence of the S. avermitilis BkdF protein (B-SavF) with those of S. avermitilis BkdA (B-SavA) (39); P. putida BkdA1 (B-Ppu) (9); B. subtilis E1␣ BCDH (B-Bsu) (48); B. subtilis (19) and B. stearothermophilus (18) E1␣ dual PDH-BCDHs (PB-Bsu and PB-Bst, respectively); A. laidlawii partially characterized E1␣ PDH (P?-Ala) (47); bovine (21) and human (16) E1␣ BCDHs (B-Bta and B-Hsa, respectively); and Saccharomyces cerevisiae (4), Ascaris suum (22), human (24), and rat (10a) E1␣ PDHs (P-Sce, P-Asu, P-Hsa, and P-Rra, respectively). Conserved positions are indicated as follows: boldface, residues identical to those in the S. avermitilis bkdF gene product; F, residues identical in all sequences; E, residues either identical or conservatively substituted in the S. avermitilis BkdF protein and at least three other proteins. Three regions of homology in the ␣ subunit of the E1 component of the BCDH and PDH complexes, as identiﬁed by Wexler et al. (49), are indicated above the alignment. Alignments were obtained by using the Pileup program and default parameters of the Genetics Computer Group software (version 7.3) (13). Dots indicate gaps introduced to maximize alignment.

the thiamine PPi binding motif, a putative subunit interaction pCD713) carrying the end of bkdF and the rest of the bkd site, and the phosphorylation sites (I and II) of the E1␣ chains cluster and the 1.6-kb BglII fragment (from pIJ4026) carrying of the mammalian BCDH and PDH complexes (49) (Fig. 4). the ermE marker were cloned into the unique BglII site of the Evidence for a second bkd gene cluster in S. avermitilis. E. coli-Streptomyces shuttle vector pCD262 to create pCD761. Codon Preference analysis of fragmented sequence data from Second, pCD761 was linearized with BglII and ligated to the several transposon m␥␦-1 insertions (5) located downstream of 2.3-kb BamHI S. avermitilis fragment (from pCD740) to create bkdF, plus the data presented in Fig. 3, suggested the presence pCD768. This construct, which is expected to produce a 1.4-kb of two additional ORFs. The deduced products of the incom- deletion in the host genome upon recombination, contains 2.3 plete ORFs 2 and 3 (bkdG and bkdH, respectively [Fig. 2]) and 4.1 kb of homologous DNA flanking the target region to showed significant similarity to several prokaryotic and eukary- the left and right, respectively. The ermE marker lies in the otic E1␤ and dihydrolipoamide acyltransferase (E2) compo- orientation opposite that of the ORFs to avoid any possible nents of the BCDH complex (data not shown) (29, 35). These deleterious effect caused by overexpression of downstream results suggest that ORF1 and the putative ORFs 2 and 3 genes. pCD768 was introduced into protoplasts of S. avermitilis would encode three components of a BCDH complex in S. ATCC 31272 by transformation, and many primary transfor- avermitilis. This contention was substantiated by the experi- mants of S. avermitilis that were resistant to both thiostrepton ments discussed below. and erythromycin were obtained. One of these, named CD783, Disruption of the bkdF gene region. To test the idea that the was selected for propagation and for protoplast formation to bkdFGH gene cluster indeed encodes a BCDH complex and eliminate the plasmid. After the transformant was plated on also to confirm the role of the branched-chain amino acid regeneration medium containing erythromycin, five colonies catabolic pathway as a provider of branched-chain fatty acid were selected, one being resistant to both erythromycin and precursors for avermectin biosynthesis, a 1.4-kb BamHI thiostrepton (CD783-pp14) and the others being resistant only genomic region, containing more than 450 bp of the 5Ј end of to erythromycin. Chromosomal DNA was isolated from one of the bkdF gene (E1␣) and approximately 900 bp located in the the thiostrepton-sensitive, erythromycin-resistant colonies region upstream of this gene (Fig. 2), was deleted from the S. (CD783-pp15), and the parental strain (ATCC 31272) and the avermitilis chromosome. The latter upstream region does not DNAs were used to analyze the region around the bkdF gene contain any obvious ORF, as demonstrated by Codon Prefer- by Southern analysis. Southern hybridization confirmed that ence analysis of DNA sequencing data collected from a group the 1.4-kb BamHI fragment was deleted and replaced by the of mini ␥␦-1 insertions randomly distributed in the 1.4-kb frag- ermE marker through the expected double crossover (data not ment (data not shown). As shown in Fig. 5, the construction of shown). the gene replacement vector was carried out in two steps. First, Phenotype of the disruptants. Table 1 summarizes the re- both the 4.1-kb BamHI S. avermitilis genomic fragment (from sults of gene disruption. The disrupted strain (CD783-pp15) VOL. 177, 1995 S. AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3509

biosynthesize avermectins only when supplemented with ap- propriate branched-chain fatty acid precursors in the fermentation 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)inS. avermitilis which has an organization similar to that of the bkdABC cluster and which consists of the genes encoding the E1␣,E1␤, and E2 components in the same orientation. Since the two bkd gene clusters are closely located (only 12 kb 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 5Ј end of the bkdF gene caused complete loss of the E1 BCDH 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 E1 PDH activity level was normal in the disrupted culture, the possibil- ity of the bkdFGH genes encoding a dual BCDH-PDH complex, 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- FIG. 5. Disruption of bkdF in S. avermitilis ATCC 31272. Sizes of relevant mitilis, further investigation will be needed to determine if the restriction fragments are indicated in kilobases. Abbreviations: amp, ampicillin chromosomal disruption reported here has also affected the resistance gene; tsr, thiostrepton resistance gene; erm, erythromycin resistance gene; Bg, BglII; B, BamHI; P, PstI. Vectors are not drawn to scale. expression of a regulatory gene. Recently, Tang and Hutchinson (45) reported that inactiva- tion of the valine dehydrogenase gene (vdh) reduced the production of tylosin in Streptomyces fradiae and spiramycin in 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 TABLE 1. Phenotypes of S. avermitilis bkdF-disrupted mutant and lost the ability to grow on solid minimal medium plates con- control strains taining isoleucine, leucine, and valine as the sole carbon sources and lacked, as predicted from the DNA sequencing Produc- results, E1 BCDH activity (Table 1). In contrast, E1 PDH Growth tion of S. avermitilis Resistance E1 BCDH aver- assays demonstrated a level of activity in CD783-pp15 similar a b on d e strain phenotype c sp act mectins to that of the parental or other control strains (data not ILV shown). In addition, when the mutant culture was grown in US unsupplemented defined fermentation medium (in which ATCC 31272 Erms Tsrs ϩ 69.2 ϩϩ branched-chain and straight-chain fatty acids and their poten- (parent) tial metabolic precursors [e.g., fats and oils] were omitted [see ATCC 53567 Erms Tsrs ϪϽ0.3 Ϫϩ Materials and Methods]), no avermectins were produced (Ta- (bkd-11) ble 1). Short ␣-branched-chain fatty acids are readily taken up CD783 Ermr Tsrr ϩ 66.7 ϩϩ by S. avermitilis cells from the environment and presumably are CD783-pp14 Ermr Tsrr ϩ 65.1 ϩϩ r s activated to their thioesters via an acyl-CoA synthetase (17). CD783-pp15 Erm Tsr ϪϽ0.2 Ϫϩ Therefore, when S(ϩ)-␣-methylbutyrate was added to the de- aSee the text for strain descriptions. fined medium, natural ‘‘a’’ forms of avermectins were pro- b Erms, sensitive to erythromycin; Tsrs, sensitive to thiostrepton; Ermr, resistant to erythromycin; Tsrr, resistant to thiostrepton. duced (Table 1). Similarly, addition of an unnatural branched- c chain fatty acid, such as cyclohexanecarboxylic acid to the ϩ or Ϫ, ability or inability to grow on minimal medium supplemented with isoleucine, leucine, and valine (ILV) as sole carbon sources after 14 days at 29ЊC. medium led to the formation of novel cyclohexanecarboxylic d The specific activity of the E1 component of the BCDH in picomoles of CO2 acid-derived, C-25-substituted avermectins (15) (data not evolved per minute per milligram of protein. The results are the means of duplicate determinations. shown). In addition, the mutant strain maintained growth ca- e pabilities essentially identical to those of the parental strain in ϩ and Ϫ, production and nonproduction, respectively, of natural avermectins by fermentation in defined fermentation medium which was unsupplemented a variety of media. The results showed that the mutant culture (U) or supplemented (S) with ␣-methylbutyrate (0.04% [wt/vol] final concentra- was incapable of producing avermectins per se. It was able to tion). 3510 DENOYA ET AL. J. BACTERIOL.

Streptomyces ambofaciens, suggesting that valine catabolism is 15. Dutton, C. J., S. P. Gibson, A. C. Goudie, K. S. Holdom, M. S. Pacey, J. C. an important but not exclusive source of fatty acid precursors Ruddock, J. D. Bu’Lock, and M. K. Richards. 1991. Novel avermectins produced by mutational biosynthesis. J. Antibiot. 44:357–365. for macrolide biosynthesis (under defined growth conditions) 16. Fisher, C. W., J. L. Chuang, T. A. Griffin, K. S. Lau, R. P. Cox, and D. T. in these microorganisms. The S. avermitilis bkd strain reported Chuang. 1989. Molecular phenotypes in cultured maple syrup urine disease here demonstrated a direct correlation between the deletion of cells: complete E1-alpha cDNA sequence and mRNA and subunit contents a genomic region comprising the 5Ј end of the bkdF gene and of the human branched chain alpha-keto acid dehydrogenase complex. J. Biol. Chem. 264:3448–3453. a complete loss of the ability to synthesize avermectins when 17. Hafner, E. W., B. W. Holley, K. S. Holdom, S. E. Lee, R. G. Wax, D. Beck, grown in a defined (unsupplemented) medium. These results H. A. I. McArthur, and W. C. Wernau. 1991. Branched-chain fatty acid showed that the BCDH-catalyzed reaction of branched-chain requirement for avermectin production by a mutant of Streptomyces aver- amino acid catabolism constitutes a crucial step in providing mitilis lacking branched-chain 2-oxo acid dehydrogenase activity. J. Antibiot. 44:349–356. fatty acid precursors for antibiotic biosynthesis in S. avermitilis. 18. Hawkins, C. F., A. Borges, and R. N. Perham. 1990. Cloning and sequence The bkdF-disrupted strain is stable: we have not observed analysis of the genes encoding the ␣ and ␤ subunits of the E1 component of any reversion or genomic rearrangements, even after a large the pyruvate dehydrogenase multienzyme complex of Bacillus stearother- number of passages (data not shown), and the strain has main- mophilus. Eur. J. Biochem. 191:337–346. 19. Hemila¨, H., A. Palva, L. Paulin, S. Arvidson, and I. Palva. 1990. Secretory S tained all the characteristic phenotypic traits of a bkd mutant. complex of Bacillus subtilis: sequence analysis and identity to pyruvate de- Most importantly, the genomic disruption discussed here can hydrogenase. J. Bacteriol. 172:5052–5063. be reproduced in any other avermectin production strain by 20. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. using essentially the gene replacement vector and procedures Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. 1985. Genetic manipulation of Streptomyces—a laboratory manual. The John Innes delineated in Fig. 5. The latter greatly facilitates the develop- Foundation, Norwich, England. ment of a larger selection of S. avermitilis bkd strains that can 21. Hu, C.-W. C., K. S. Lau, T. A. Griffin, J. L. Chuang, C. W. Fisher, R. P. Cox, be used to generate valuable novel (unnatural) C-25-substi- and D. T. Chuang. 1988. Isolation and sequencing of a cDNA encoding the tuted antiparasitic avermectins (15). decarboxylase (E1)-␣ precursor of bovine branched-chain ␣-keto acid dehydrogenase complex: expression of E1-␣ mRNA and subunit in maple-syrup- urine-disease and 3T3-L1 cells. J. Biol. Chem. 263:9007–9014. ACKNOWLEDGMENTS 22. Johnson, K. R., R. Komuniecki, Y. Sun, and M. J. Wheelock. 1992. Charac- terization of cDNA clones for the alpha subunit of pyruvate dehydrogenase We thank David A. Hopwood for critical reading of the manuscript from Ascaris suum. Mol. Biochem. Parasitol. 51:37–48. and Karen De Benedictis for expert legal assistance in this project. 23. Kieser, T., and D. A. Hopwood. 1991. Genetic manipulation of Streptomyces: integrating vectors and gene replacement. Methods Enzymol. 204:430–458. REFERENCES 24. Koike, K., Y. Urata, S. Matsuo, and M. Koike. 1990. Characterization and 1. Anderson, R. P., and J. R. Roth. 1977. Tandem genetic duplications in phage nucleotide sequence of the gene encoding the human pyruvate dehydroge- and bacteria. Annu. Rev. Microbiol. 31:473–506. nase alpha-subunit. Gene 93:307–311. 2. Anzai, H., Y. Kumada, O. Hara, T. Murakami, R. Itoh, E. Takano, S. Imai, 25. Lehninger, A. L. 1975. Oxidative degradation of amino acids, p. 559–586. In A. Satoh, and K. Nagaoka. 1988. Replacement of Streptomyces hygroscopicus A. L. Lehninger (ed.), Biochemistry. Worth Publishers, Inc., New York. genomic segments with in vitro altered DNA sequences. J. Antibiot. 41:226– 26. MacNeil, D. J., and L. M. Klapko. 1987. Transformation of Streptomyces 233. avermitilis by plasmid DNA. J. Ind. Microbiol. 2:209–218. 3. Baylis, H. A., and M. J. Bibb. 1987. The nucleotide sequence of a 16S rRNA 27. Madhusudhan, K. T., D. Lorenz, and J. R. Sokatch. 1993. The bkdR gene of gene from Streptomyces coelicolor A3(2). Nucleic Acids Res. 15:7176. Pseudomonas putida is required for expression of the bkd operon and en- 4. Behal, R. H., K. S. Browning, and L. J. Reed. 1989. Nucleotide and deduced codes a protein related to Lrp of Escherichia coli. J. Bacteriol. 175:3934– amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase. 3940. Biochem. Biophys. Res. Commun. 164:941–946. 28. Magdalena, J., M. Forsman, M. V. Lenzini, A. Brans, and J. Dusart. 1992. 5. Berg, C. M., G. Wang, L. D. Strausbaugh, and D. E. Berg. 1993. Transposon- Two different ␤-lactamase genes are present in Streptomyces cacaoi. FEMS facilitated sequencing of DNAs cloned in plasmids. Methods Enzymol. 218: Microbiol. Lett. 99:101–106. 279–306. 29. Matsuda, I., J. Asaka, I. Akaboshi, F. Endo, H. Mitsubuchi, and Y. No- 6. Birch, A. W., and J. Cullum. 1985. Temperature-sensitive mutants of the bukuni. 1990. Maple syrup urine disease: complete primary structure of the Streptomyces plasmid pIJ702. J. Gen. Microbiol. 131:1299–1303. E1-beta subunit of human branched chain alpha-ketoacid dehydrogenase 7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of complex deduced from the nucleotide sequence and a gene analysis of microgram quantities of protein utilizing the principle of protein-dye bind- patients with this disease. J. Clin. Invest. 86:242–247. ing. Anal. Biochem. 72:248–254. 30. McKean, M. C., K. A. Winkeler, and D. J. Danner. 1992. Nucleotide se- 8. Burg, R. W., B. M. Miller, E. E. Baker, J. Birnbaum, S. A. Currie, R. quence of the 5Ј end including the initiation codon of cDNA for the E1␣ Hartman, Y.-L. Kong, R. L. Monaghan, G. Olson, I. Putter, J. B. Tunac, H. subunit of the human branched chain ␣-ketoacid dehydrogenase complex. Wallick, E. O. Stapley, R. O¯ iwa, and S. O¯ mura. 1979. Avermectins, new Biochim. Biophys. Acta 1171:109–112. family of potent anthelmintic agents: producing organism and fermentation. 31. Miran, S. G., J. E. Lawson, and L. J. Reed. 1993. Characterization of PDH Antimicrob. Agents Chemother. 15:361–367. beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from 9. Burns, G., T. Brown, K. Hatter, J. M. Idriss, and J. R. Sokatch. 1988. Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90:1252–1256. Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from 32. O¯ mura, S., and Y. Tanaka. 1984. Biochemistry, regulation, and genetics of Pseudomonas putida to the corresponding subunits of mammalian branched- macrolide production, p. 199–229. In S. O¯ mura (ed.), Macrolide antibiotics. chain-oxoacid and pyruvate dehydrogenases. Eur. J. Biochem. 176:311–317. Chemistry, biology, and practice. Academic Press, New York. 10. Chater, K. F., and D. A. Hopwood. 1993. Streptomyces, p. 83–99. In A. L. 33. Perham, R. N. 1991. Domains, motifs, and linkers in 2-oxo acid dehydroge- Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other nase multienzyme complexes: a paradigm in the design of a functional gram-positive bacteria: biochemistry, physiology, and molecular genetics. protein. Biochemistry 30:8501–8512. American Society for Microbiology, Washington, D.C. 34. Richardson, M. A., S. Kuhstoss, P. Solenberg, N. A. Schaus, and R. N. Rao. 10a.Cullingford, T. E. Unpublished data. EMBL accession no. Z12158. 1987. A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the 11. Denoya, C. D., D. D. Skinner, and M. R. Morgenstern. 1994. A Streptomyces cloning of three different spiramycin-resistance genes from a Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like ambofaciens library. Gene 61:231–241. protein that directs the production of homogentisic acid and an ochronotic 35. Russell, G. C., and J. R. Guest. 1991. Sequence similarities within the family pigment in Escherichia coli. J. Bacteriol. 176:5312–5319. of dihydrolipoamide acyltransferases and discovery of a previously uniden- 11a.Denoya, C. D., and K. Stutzman-Engwall. Unpublished results. tified fungal enzyme. Biochim. Biophys. Acta 1076:225–232. 12. Denoya, C. D., A. R. Trevisan, J. Zorzopulos, M. Woloj, and E. Rubeglio. 36. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a 1985. Diversity of plasmid profiles in multiply resistant Klebsiella pneumoniae laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold strains isolated from a single nosocomial environment with a strong antibi- Spring Harbor, N.Y. otic selection pressure. Microbios Lett. 29:87–93. 37. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with 13. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467. sequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395. 38. Skinner, D. D., and C. D. Denoya. 1993. A simple DNA polymerase chain 14. Donadio, S., M. J. Staver, J. B. McAlpine, S. J. Swanson, and L. Katz. 1991. reaction method to locate and define orientation of specific sequences in Modular organization of genes required for complex polyketide biosynthesis. cloned bacterial genomic fragments. Microbios 75:125–129. Science 252:675–679. 39. Skinner, D. D., M. R. Morgenstern, R. W. Fedechko, and C. D. Denoya. 1995. VOL. 177, 1995 S. AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3511

Cloning and sequencing of a cluster of genes encoding branched-chain tiﬁcation and analysis of the genes coding for the putative pyruvate dehy- ␣-keto acid dehydrogenase from Streptomyces avermitilis and the production drogenase enzyme complex in Acholeplasma laidlawii. J. Bacteriol. 174:1388– of a functional E1[␣␤] component in Escherichia coli. J. Bacteriol. 177:183– 1396. 190. 48. Wang, G., T. Kuriki, K. L. Roy, and T. Kaneda. 1993. The primary structure 40. Sommer, R., and D. Tautz. 1989. Minimal homology requirements for PCR of branched-chain ␣-oxo acid dehydrogenase from Bacillus subtilis and its primers. Nucleic Acids Res. 17:6749. similarity to other ␣-oxo acid dehydrogenases. Eur. J. Biochem. 213:1091– 41. Southern, E. M. 1975. Detection of speciﬁc sequences among DNA frag- 1099. ments separated by gel electrophoresis. J. Mol. Biol. 98:503–517. 49. Wexler, I. D., S. G. Hemalatha, and M. S. Patel. 1991. Sequence conserva- 42. Strohl, W. R. 1992. Compilation and analysis of DNA sequences associated tion in the ␣ and ␤ subunits of pyruvate dehydrogenase and its similarity to 20: with apparent streptomycete promoters. Nucleic Acids Res. 961–974. branched-chain ␣-keto acid dehydrogenase. FEBS Lett. 282:209–213. 42a.Stutzman-Engwall, K., and C. D. Denoya. Unpublished results. 50. Wright, F., and M. J. Bibb. 1992. Codon usage in the GϩC-rich Streptomyces 43. Stutzman-Engwall, K. J., S. L. Otten, and C. R. Hutchinson. 1992. Regula- genome. Gene 113:55–65. tion of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J. Bacteriol. 174:144–154. 51. Wynn, R. M., J. L. Chuang, J. R. Davie, C. W. Fisher, M. A. Hale, R. P. Cox, 44. Sykes, P. J., G. Burns, J. Menard, K. Hatter, and J. R. Sokatch. 1987. and D. T. Chuang. 1991. Cloning and expression in E. coli of mature E1␤ Molecular cloning of genes encoding branched-chain keto acid dehydroge- subunit of bovine mitochondrial branched-chain ␣-keto acid dehydrogenase nase of Pseudomonas putida. J. Bacteriol. 169:1619–1625. complex. Mapping of the E1␤-binding region on E2. J. Biol. Chem. 267: 45. Tang, L., and C. R. Hutchinson. 1993. Sequence, transcriptional, and func- 1881–1887. tional analyses of the valine (branched-chain amino acid) dehydrogenase 52. Zhang, B., D. W. Crabb, and R. A. Harris. 1988. Nucleotide and deduced gene from Streptomyces coelicolor. J. Bacteriol. 175:4176–4185. amino acid sequence of the E1␣ subunit of human liver branched-chain 46. Tibbetts, M. W., E. W. Hafner, M. R. Morgenstern, D. D. Skinner, and C. D. ␣-ketoacid dehydrogenase. Gene 69:159–164. Denoya. 1992. Cloning of a DNA fragment involved in pigment production 53. Zhang, B., M. J. Kuntz, G. W. Goodwin, R. A. Harris, and D. W. Crabb. 1987. in Streptomyces avermitilis. FEMS Microbiol. Lett. 91:9–14. Molecular cloning of a cDNA for the E1-alpha subunit of rat liver branched 47. Wallbrandt, P., V. Tegman, B.-H. Jonsson, and Å. Wieslander. 1992. Iden- chain alpha-ketoacid dehydrogenase. J. Biol. Chem. 262:15220–15224.